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To ensure the best quality of data report, raw data used in this trial are available upon request (<[email protected]>). Introduction {#sec006} ============ Calcineurin inhibitors (CNIs), such as Cyclosporine A (CsA) or tacrolimus, are the most commonly used maintenance immunosuppressive drugs after kidney transplantation \[[@pone.0153635.ref001]\] even if CNIs could lead to nephrotoxicity \[[@pone.0153635.ref002]\]. The mechanisms underlying CsA-induced nephrotoxicity (CIN) remain not fully elucidated \[[@pone.0153635.ref003]\]. Renal hemodynamic plays a central role during acute CIN: renal vasoconstriction has been reported as an initial event linked to CIN \[[@pone.0153635.ref003]\]. CsA is associated with renal afferent arteriolar vasoconstriction in rats and tubular injury during acute CsA nephrotoxicity \[[@pone.0153635.ref004]\]. The pharmacological antagonism of Mineralocorticoid Receptor (MR) reduces both cardiovascular and all-cause morbidity and/or mortality during chronic related (or not) heart failure \[[@pone.0153635.ref005], [@pone.0153635.ref006]\]. The MR expressed in endothelium and smooth muscle cells participates to the control of vascular tone: both endothelial and vascular smooth muscle MR modulate the responses to vasodilators and vasoconstrictors \[[@pone.0153635.ref007], [@pone.0153635.ref008]\]. Pharmacological antagonism of MR by both spironolactone \[[@pone.0153635.ref009], [@pone.0153635.ref010]\] and eplerenone \[[@pone.0153635.ref011], [@pone.0153635.ref012]\] is highly efficient to blunt CIN in experimental models. The vascular smooth muscle MR has been recently shown to play a key role during acute CIN in mice by preventing increased renal vascular resistance in acute CIN \[[@pone.0153635.ref013]\]: this could explain, at least partially, the beneficial effects of MR antagonism in CIN. Chronic renal impairment could limit the use of MR antagonists (MRAs): even if hyperkalemia is feared, spironolactone and eplerenone could be safely used if a close monitoring of kalemia and renal function is ensured \[[@pone.0153635.ref014], [@pone.0153635.ref015]\]. However, the higher frequency of polypharmacy in chronic kidney disease patients could lead to drug-drug interactions and limit MRAs use, especially during kidney transplantation when immunosuppressive drugs metabolized by the P450 cytochrome (like CsA) are necessary. MRAs are not commonly used in this population despite the potential benefits to reduce cardiovascular risk and CIN after renal transplantation. Gonzalez Monte *et al*. reported the benefits of adding spironolactone to a dual-blockade renin--angiotensin--aldosterone system (by both angiotensin-converting enzyme inhibitor \[ACE-I\] and type-2 angiotensin-receptor blockers \[ARB\]) in 11 kidney-transplant recipients with persistent proteinuria: after 6 months, proteinuria had decreased significantly with no adverse event \[[@pone.0153635.ref016]\]. Serum potassium remained stable (no severe hyperkalemia) and no serum bicarbonate was reported \[[@pone.0153635.ref016]\]. Since MRAs have never been tested in CsA-treated kidney-transplant recipients with impaired renal function, the present study was designed to test the tolerance of eplerenone in this population. Subjects and Methods {#sec007} ==================== We conducted a single-center, prospective, open-label study. The primary endpoint was the tolerance to eplerenone, assessed by the occurrence of the following expected adverse events: severe hyperkalemia (\>6 mmol/L), metabolic acidosis (serum bicarbonate \<15 mmol/L), hypotension (systolic blood pressure \<100 mmHg), acute kidney failure (increase in serum creatinine \>30% from baseline), or any adverse event that required discontinuation of eplerenone. Eplerenone was chosen due to its lower affinity for other steroid (progesterone, androgen and glucocorticoid) receptors and the absence of long-acting metabolites: this could lead to less frequent adverse events. We calculated the number of patients to include based upon the risk of severe hyperkalemia (\>6 mmol/L), which was considered as the major adverse event. Levels of kalemia were defined during the study as follow: normal (3.5 to ≤5 mmol/L), mild hyperkalemia (\>5 to 5.4 mmol/L), moderate hyperkalemia (\>5.5 to 5.9 mmol/L) and severe hyperkalemia (\>6 mmol/L). Study design {#sec008} ------------ We performed the study by using a two-steps Simon's plan (**[Fig 1](#pone.0153635.g001){ref-type="fig"}**) \[[@pone.0153635.ref017]\]. During the first step, 14 patients took eplerenone 25 mg/d for 8 weeks. This posology was chosen to be the minimum efficient. If three or more adverse events occurred, then study had to be discontinued. If not, 17 new patients were included within step 2 and also received the same treatment for 8 weeks. If four or more adverse events occurred in both steps (1 and 2), then study had to be discontinued, and the alternate hypothesis (a risk of adverse events \>20%) could not be rejected: i.e., the safety of eplerenone could not be concluded. If not, we could conclude that eplerenone at 25 mg/d could be safety used in such a population. {#pone.0153635.g001} All included patients were aged \>18 years on the date of inclusion, belonged to a healthcare system, gave their informed written consent, had a functional kidney allograft for at least 1 year from the date of inclusion, was receiving CsA, and had impaired renal function, estimated by the MDRD formula \[[@pone.0153635.ref018]\], at between 30 and 50 mL/min/1.73 m^2^. Exclusion criteria were serum potassium of ≥5 mmol/L on the date of inclusion; one or more incidences of severe hyperkalemia (≥6 mmol/L), for whatever reason; currently receiving potassium-exchange resin treatment; on-going pregnancy or lack of effective contraception during the whole study period; uncontrolled high arterial blood pressure (systolic blood pressure \>140 mmHg); orthostatic hypotension; systolic arterial blood pressure ≤110 mmHg; heart failure within the 3 months before the date of inclusion or chronic heart failure (NYHA III or IV); severe hepatic failure (Child-Pugh C score); allergy to one or more of the components of eplerenone (INSPRA^®^); on-going treatment, including spironolactone or eplerenone; on-going treatment that could not be withdrawn during the study period: e.g., potassium-sparing diuretics, potassium salts, CYP3A4 enzyme inhibitors other than CsA; malabsorption syndrome; abnormal galactose metabolism or a deficiency of galactase; on-going non-steroidal anti-inflammatory treatment, or lithium, or another nephrotoxic agent; or on-going treatment with a double-blockade of the renin--angiotensin--aldosterone system with ACE-I and ARB. The treatment could include ACE-I or ARB, but not in combination. Clinical parameters (body weight, blood pressure, and adverse events) were monitored on days (D) 0, 14, 28, and 56. Serum potassium was closely monitored on days 0, 2, 7, 14, 21, 28, 35, 42, 49, and 56. Other biological parameters (such as serum creatinine and bicarbonate) were monitored on D 0, 14, 28, and 56. At any time during the study period, adverse events that required discontinuation of eplerenone included serum potassium \>6 mmol/L, serum potassium \>5.5 mmol/L under potassium-exchange resin, metabolic acidosis assessed by a serum bicarbonate \<15 mmol/L, and any other clinical outcome that required discontinuation of eplerenone. Statistical methods and analyses {#sec009} -------------------------------- According to previously reported data, the probabilities to develop hyperkalemia (\> 6 mmol/L) during 8 weeks of eplerenone treatment are \<7% with the 25mg/d dose and \<10% with the 50mg/d dose \[[@pone.0153635.ref005], [@pone.0153635.ref006], [@pone.0153635.ref016], [@pone.0153635.ref019]\]. Included patients exerted a better renal function than the population of the present study: herein, estimated glomerular filtration rate (eGFR) range was fixed between 50 and 70 mL/min/1.73m^2^. In our population, the expected risk (null hypothesis, H~0~) was supposed to be \< 7% whereas a risk higher than 20% of developing major hyperkalemia (\>6 mmol/L) was considered unacceptable (alternative hypothesis, Ha). Using a sample proportion test, the power to detect this adverse outcome was calculated at 95% (the β risk was 5%). Otherwise, in such conditions, the risk of not identifying an unacceptable risk of major hyperkalemia (\>6 mmol/L) under eplerenone in these patients is 5%: the α risk was calculated at 22.5%. With these hypotheses, the inclusion of 31 patients was required: if 4 (/31) or more patients had to stop the treatment, the safety (\< 20%) could not be assumed. Quantitative data are described by their median and range and qualitative data as numbers and percentages. Variations of serum potassium at different times were evaluated using variance analysis for repeated measures. Comparisons between patients with mild hyperkalemia (\>5 mmol/L) during the study protocol and those with normal kalemia (\<5 mmol/L) at anytime of the study period were performed using univariate analyses (Wilcoxon\'s test or Fisher's exact test, as appropriate) and multivariate analyses (stepwise logistic regression). The multivariate stepwise logistic regression included only significant factors at *p* ≤ 0.10 with entry and removal limits set at 0.10: basal cyclosporine A posology, creatininemia, serum potassium and bicarbonate. Sensitivities and specificities of basal serum potassium and bicarbonate were calculated, and a receiver-operating characteristic (ROC) curve was calculated to determine a cut-off value with optimal sensitivity and specificity: the statistical software (SAS) calculated automatically the coordinates of the ROC curve and calculated both the sensitivity and specificity (1---specificity for more precision) for all coordinates. Then, the cut-off value obtaining the best ratio between the sensitivity and the specificity was chosen. Whatever the test used, a *p*-value \<0.05 was considered statistically significant. All statistical analyses were performed using SAS software, release 9.3 (SAS INC, Cary, California). Ethical considerations {#sec010} ---------------------- This trial (**[S1](#pone.0153635.s001){ref-type="supplementary-material"} and [S2](#pone.0153635.s002){ref-type="supplementary-material"} Figs**) received specific agreements from an appropriate independent ethics committee, was registered in the European registry (EudraCT 2011-003759-20) and in clinicaltrials.gov (NCT01834768) and has therefore been performed in accordance with the ethical standards laid down in an appropriate version of the Helsinki Declaration of 1975, as revised in 2000, as well as the Declaration of Istanbul 2008. All persons gave their informed written consent prior to their inclusion to the study. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the "Declaration of Istanbul on Organ Trafficking and Transplant Tourism". Results {#sec011} ======= A total of 31 patients were included (**[Table 1](#pone.0153635.t001){ref-type="table"}**) and all completed the study period (8 weeks), except one (last follow-up on D35 due to an unplanned move). Serum potassium increased slightly from baseline (4.2±0.4 mmol/L): on d2, serum potassium became increased and then remained in a steady state (**[Fig 2A](#pone.0153635.g002){ref-type="fig"}**). Nine patients experienced at least one episode of mild hyperkalemia (\>5 mmol/L) but there was only one episode of moderate hyperkalemia (\>5.5 mmol/L). This patient received a specific intervention (potassium-exchange resin) on D35. Half the incidences of mild hyperkalemia (\>5 mmol/L) occurred within 7 days after beginning eplerenone treatment. {#pone.0153635.g002} 10.1371/journal.pone.0153635.t001 ###### Characteristics of included patients. {#pone.0153635.t001g} ---------------------------------------------- --------------------- ***Demography*** *n* = 31  Age (years) 56 \[32--70\] Gender ratio (M/F) 18/13 Time since transplantation (months) 126 \[18--326\] Body-mass index at inclusion (kg/m^2^) 23.8 \[18.2--36.8\] Diabetes, *n* (%) 3 (10) ***Biology***   Creatininemia (μmol/L) 145 \[87--239\] eGFR (mL/min/1.73 m^2^) 41 \[26--59\] Serum potassium at inclusion (mmol/L) 4.2 \[3.4--4.9\] Serum bicarbonate at inclusion (mmol/L) 24 \[12--30\] Natriuresis at inclusion (mmol/d) 136 \[29--360\] Kaliuresis at inclusion (mmol/d) 60 \[0--176\] Proteinuria at inclusion (mg/d) 123 \[0--648\] ***Drug therapies***   Cyclosporine posology at inclusion (mg/kg/d) 2.1 \[1.4--4.0\] Cyclosporinemia at inclusion (ng/mL) 94 \[38--152\] MMF/azathioprine, *n* (%) / *n* (%) 24 (77) / 4 (1) ACE-i/ARB, *n* (%) / *n* (%) 13 (42) / 6 (2) Diuretics, *n* (%) 12 (39) β-blockers, *n* (%) 14 (45) Oral bicarbonate, *n* (%) 7 (23) Steroids, *n* (%) 5 (16) ---------------------------------------------- --------------------- M: male; F: female; eGFR: estimated glomerular-filtration rate; MMF: mycophenolate mofetil. Data are expressed by their median \[range\]. Three patients presented with other adverse events: two unspecific outcomes (diarrhea and sweats) and one acute kidney injury (\>30% increased creatininemia from baseline) on D56, secondary to acute diarrhea. None of these adverse events needed specific management. We observed no modifications to systolic blood pressure (**[Fig 2B](#pone.0153635.g002){ref-type="fig"}**), body weight (**[Fig 2C](#pone.0153635.g002){ref-type="fig"}**), or serum bicarbonate (**[Fig 2D](#pone.0153635.g002){ref-type="fig"}**). Other biological or clinical parameters remained stable. The risk of at least one episode of mild hyperkalemia (\>5 mmol/L) under eplerenone was studied using baseline data: demographic and biological parameters were analyzed as well as treatments. Two groups were individualized (patients with at least one episode of mild hyperkalemia (\>5 mmol/L) *versus* others). After stepwise multivariate analyses (including CsA posology, creatininemia, serum potassium and bicarbonate), only serum potassium and bicarbonate at baseline were independently associated with a higher risk of developing at least one episode of mild hyperkalemia (\>5 mmol/L) under eplerenone treatment (**[Table 2](#pone.0153635.t002){ref-type="table"}**). Higher serum potassium at baseline was associated with a higher risk (OR 6.5 \[1.4;30.5\]) of developing mild hyperkalemia and lower serum bicarbonate was also associated with a higher risk (OR 0.7 \[0.5;0.9\]) of developing mild hyperkalemia. 10.1371/journal.pone.0153635.t002 ###### Candidate parameters for predicting the risk of mild hyperkalemia. {#pone.0153635.t002g} Parameter Kalemia \>5 mmol/L (*n* = 9) No kalemia \> 5 mmol/L (*n* = 22) Univariate analysis[^\#^](#t002fn002){ref-type="table-fn"} Multivariate analysis[\*](#t002fn003){ref-type="table-fn"} ------------------------------------------------ ------------------------------ ----------------------------------- ------------------------------------------------------------ ------------------------------------------------------------ ----- -------------- ***Demography***             Age (years) 50.7 \[32.7--70.1\] 57.4 \[35.8--66.5\] 0.31 Gender (M/F) 7/2 11/11 0.12 Body weight (kg) 75.0 \[66--90\] 70.1 \[43.8--109\] 0.17 Body-mass index (kg/m^2^) 23.8 \[23.0--28.2\] 24.4 \[18.2--36.8\] 0.84 Diabetes at inclusion (*n*) 0 3 0.34 Time since transplantation (months) 152.0 \[24--326\] 119.5 \[18--264\] 0.29 Systolic blood pressure (mmHg) 120 \[110--140\] 130 \[110--140\] 0.42 ***Biology***             Creatininemia on day 0 (μmol/L) 170.0 \[121.0--232.0\] 138.0 \[87.0--239.0\] 0.06 eGFR (mL/min/1.73 m^2^) 36.0 \[26.0--53.0\] 44.5 \[26.0--59.0\] 0.17 Serum potassium at baseline (mmol/L) 4.7 \[4.0--4.9\] 4.1 \[3.4--4.7\] ***\<0*.*01*** ***0*.*003*** 6.5 \[1.4;30.5\] Kaliuresis (mmol/d) 62.0 \[33.0--92.0\] 57.0 \[0--176\] 0.33 Kaliuresis/creatininuria (mmol/mmol) 4.8 \[0.3--7.0\] 4.7 \[0--21.5\] 0.57 Natriuresis (mmol/d) 143 \[71--300\] 135.5 \[29--360\] 0.84 Natriuresis/creatininuria (mmol/mmol) 10 \[1.2--14.8\] 12.7 \[1.8--64.6\] 0.25 Serum bicarbonate at baseline (mmol/L) 21.0 \[12.0--25.0\] 24.0 \[19.0--30.0\] ***0*.*02*** ***0*.*007*** 0.7 \[0.5;0.9\] ***Drug therapy***             Cyclosporine A posology at inclusion (mg/d) 180 \[120--220\] 140 \[100--280\] 0.08 Cyclosporine A posology at inclusion (mg/kg/d) 2.1 \[1.6--2.8\] 2.0 \[1.4--4.0\] 0.37 Cyclosporinemia at inclusion (ng/mL) 98 \[38--145\] 92.5 \[40--152\] 0.81 ACE-I at inclusion (*n*) 3 10 0.26 ARB at inclusion (*n*) 2 4 0.36 Diuretics at inclusion (*n*) 4 8 0.29 β-blockers at inclusion (*n*) 4 10 0.31 Oral bicarbonate at inclusion (*n*) 2 4 0.36 Steroids at inclusion (*n*) 1 4 0.39 M: male; F: female; eGFR: estimated glomerular-filtration rate; OR: odds ratio; CI: confidence interval. Data are expressed as their median \[range\]. All urine tests were performed on 24-h urine collections. ^\#^ Univariate analyses using Wilcoxon tests for quantitative variables and Fisher exact test for qualitative variables. \* Multivariate analysis by stepwise logistic regression was performed including creatininemia, serum potassium, serum bicarbonate and cyclosporine A posology on day 0. ROC analyses were performed to test if a cut-off value for serum potassium and/or bicarbonate at baseline could distinguish which patients had a higher risk of developing mild hyperkalemia (\>5 mmol/L) under eplerenone treatment. Only serum potassium at baseline (**[Fig 3A](#pone.0153635.g003){ref-type="fig"}**) showed this ability (AUC = 0.846 \[0.681--1.0\]), whereas serum bicarbonate at baseline (**[Fig 3B](#pone.0153635.g003){ref-type="fig"}**) did not (AUC = 0.222 \[0.048--0.397\]). Serum potassium of \>4.35 mmol/L at baseline was a marker for a higher risk of developing mild hyperkalemia (\>5 mmol/L) during the treatment period, with a sensitivity of 78% and a specificity of 77%. {#pone.0153635.g003} Discussion {#sec012} ========== During this study, we found that eplerenone could be safely given to kidney-transplant recipients treated with CsA and impaired renal function. The only acute renal failure observed during eplerenone treatment was not considered to be associated to this treatment due to the diarrhea. This gastro-intestinal adverse outcome was no longer related to eplerenone, regarding to the context of known contact. Moreover, other ongoing drugs could have facilitated this event. After the RALES study \[[@pone.0153635.ref019]\], MRAs were considered to be at risk of major hyperkalemia \[[@pone.0153635.ref020]\], especially in patients with chronic kidney disease \[[@pone.0153635.ref021]\]. Most of the cases of severe hyperkalemia were due to the lack of serum potassium monitoring after initiating the treatment \[[@pone.0153635.ref022]\]. All CNIs increase the risk of hyperkalemia \[[@pone.0153635.ref023]\], especially after adding a renin--angiotensin--aldosterone-system blocker, such as ACE-I or ARB \[[@pone.0153635.ref024]\]: the underlying mechanisms may rely on the decreased efficacy of loop diuretics (like furosemide) \[[@pone.0153635.ref025]\], the activation of the sodium-chloride co-transporter \[[@pone.0153635.ref026], [@pone.0153635.ref027]\], and decreased ROMK channel activity \[[@pone.0153635.ref028]\]. CNIs are also associated with mild renal tubular acidosis in about one-third of patients \[[@pone.0153635.ref029]\]. The acidosis observed during CsA-treatment \[[@pone.0153635.ref030]\] can worsen potassium shift from the intracellular to the extracellular compartment: distal renal tubular acidosis \[[@pone.0153635.ref031]\] appears to be related to Na^+^/K^+^ ATPase pump impairment \[[@pone.0153635.ref032]\] under CsA-treatment. MRAs could worsen metabolic acidosis, especially when it pre-exists \[[@pone.0153635.ref033]\]. In our study, serum bicarbonate was closely monitored: if it was associated with a higher risk of developing mild hyperkalemia (\>5 mmol/L) during the treatment period, a basal cut-off value could not be proposed. The use of oral bicarbonate was allowed and was monitored due to its possible effects on preserving renal function decline \[[@pone.0153635.ref034]\] and counteracting the acidotic effect of MRAs. Even if patients that had at least one episode of mild hyperkalemia (\>5 mmol/L) had lower serum bicarbonate at baseline, they were not more frequently treated with oral bicarbonate (2/9 patients) than other patients (4/22, *p* = 0.36). Other treatments that could interact with the metabolism of potassium were screened: the frequencies of ACE-I, ARB, diuretics and/or β-blockers were not different between both groups. Renal impairment is a risk factor of hyperkalemia: during chronic kidney disease, renal potassium handling increases as glomerular-filtration rate decreases \[[@pone.0153635.ref035]\], leading to hyperkalemia because of the loss in nephron mass. In the present study (where all patients had a renal impairment), renal function assessed by the MDRD formula \[[@pone.0153635.ref018]\] was not associated with a higher risk of developing mild hyperkalemia (\>5 mmol/L) during the treatment period. Because of the creatininemia assay used in our study (modified Jaffe's method), the use of the CKD-EPI formula--which necessitates an enzymatic assay--was not appropriate \[[@pone.0153635.ref036]\]. For ranges of eGFR between 30 to 59 mL/min/1.73m^2^, the MDRD formula misclassifies 5% of patients that should be mostly in the upper eGFR group (60 to 89 mL/min/1.73m^2^) \[[@pone.0153635.ref036]\]: as the included patients in the present study had eGFR ranging from 30 to 50 mL/min/1.73m^2^, misclassifications may had been rare. Even if creatininemia at baseline tended to be different between patients that experienced at least one episode of mild hyperkalemia (\>5 mmol/L) during the study, eGFR was not different. Moreover, multivariate analysis included creatininemia at inclusion: it did not explain the higher frequency of mild hyperkalemia (\>5 mmol/L) observed in these patients. The risk of developing moderate to severe hyperkalemia during treatment with eplerenone is less than 10% in both hypertension and heart-failure indications, depending on the definition of hyperkalemia (\>5.5 mmol/L or 6 mmol/L) and drug dosage \[[@pone.0153635.ref037]\]: in our study, only one (1/31, about 3%) moderate hyperkalemia (\>5.5 mmol/L) was observed. Laboratory monitoring (serum potassium and renal function), after initiating MRA treatment, is the best way to prevent hyperkalemia and hospitalization \[[@pone.0153635.ref038]\]. Even though our cohort was relatively small (*n* = 31), higher serum potassium at baseline was associated with a higher risk of developing mild hyperkalemia (\>5 mmol/L) during the treatment period. In our study, mild hyperkalemia was not associated with a higher rate of 24-h kaliuresis, neither at baseline nor during the follow-up. At a steady state, kaliuresis reflects potassium intake and is not related to a higher risk of developing hyperkalemia: this is consistent with a previous study \[[@pone.0153635.ref039]\]. To evaluate the risk of developing mild hyperkalemia during treatment with eplerenone, a cut-off value of 4.35 mmol/L at baseline was determined to have both the best sensitivity and specificity. A previous study also reported that, during hypertension therapy, predictive factors for developing moderate hyperkalemia (\>5.5 mmol/L) under MRA treatment were eGFR \<45 mL/min/1.73 m^2^ and baseline serum potassium \>4.5 mmol/L \[[@pone.0153635.ref040]\]: this is consistent with our findings. Such data are easy to use in clinical practice, especially when hyperkalemia is feared: in our study, among patients who experienced at least one episode of hyperkalemia (\>5 mmol/L), only two had serum potassium levels at baseline that were lower than this cut-off value, defining a negative predictive value of close to 90%. The beneficial effects of MRAs have been well established during heart failure, with strong data obtained during randomized controlled trials, especially when cardiac ejection fraction is reduced \[[@pone.0153635.ref041]\]: both eplerenone and spironolactone have demonstrated improved survival benefits. During chronic kidney disease, the use of MRAs is associated with reducing proteinuria \[[@pone.0153635.ref042]\]. As proteinuria is one of the most common predictive factors for the progression of kidney disease \[[@pone.0153635.ref043]\], a beneficial effect of MRAs on kidney survival could be expected but has not been demonstrated previously, due to a lack of randomized controlled trials with kidney survival as the primary endpoint. MRAs could also be useful during CIN \[[@pone.0153635.ref044]\]: both drugs (MRAs and CsA) act on vascular function. MRAs could limit CsA-induced vascular toxicity. Several animal studies suggest a beneficial effect of MRA use under CsA treatment \[[@pone.0153635.ref009], [@pone.0153635.ref010], [@pone.0153635.ref013]\]. It could be related to vascular MR-induced remodeling \[[@pone.0153635.ref045]\]. To date, no study was published using MRAs and including tacrolimus-treated patients. The beneficial effects of MRAs could be related to their diuretic effects or their pleiotropic actions (tissue remodeling), as occurs during heart failure \[[@pone.0153635.ref041]\]. In our study, no effect was observed on systolic, diastolic or mean blood pressure nor on body weight. This is consistent with previous studies: during-end stage renal disease in anuric hemodialyzed patients, MRAs use was effective in reducing mortality without causing a diuretic effect \[[@pone.0153635.ref046]\], and post-hoc analysis of the EPHESUS trial showed that the beneficial effects of eplerenone were independent of diuretic effects \[[@pone.0153635.ref047]\]. In our study, MRA dosage was low but was efficient enough at increasing serum potassium. Dose-efficiency has been demonstrated for both spironolactone \[[@pone.0153635.ref048]\] and eplerenone \[[@pone.0153635.ref049]\] in reducing morbi-mortality. Moreover, electrolyte disturbances (hyperkalemia) appear to be also dose-dependent \[[@pone.0153635.ref041]\]. Survival benefits in heart failure have been shown with low posologies: the means were 26 mg/d for spironolactone during the RALES trial \[[@pone.0153635.ref019]\] and 42 mg/d and 39 mg/d for eplerenone during the EPHESUS \[[@pone.0153635.ref006]\] and EMPHASIS-HF \[[@pone.0153635.ref005]\] trials, respectively. Such a low dose has been shown to be efficient during end-stage renal disease in reducing mortality in hemodialyzed patients \[[@pone.0153635.ref046]\] and morbidity in peritoneal dialysis patients \[[@pone.0153635.ref050]\]. All these data are consistent with the dosage we chose in the present study: it appeared to be the best compromise between achieving higher efficacy and lower toxicity in our population. Further studies should test the safety and efficiency of higher doses (50 mg/d) that should be facilitated by the use of potassium binders \[[@pone.0153635.ref051]\]. Taken together, our data show the safe use of eplerenone in CsA-treated transplant recipients, despite renal impairment. This is consistent with a previous study in another population of chronic kidney-disease patients \[[@pone.0153635.ref015]\]. Of note, our study is the first performed on kidney-transplant recipients. Further studies are needed to analyze the potential benefits of MRAs in kidney-allograft transplantation: an adequately powered prospective randomized controlled trial should test the efficiency (and safety) of eplerenone in reducing chronic renal-allograft dysfunction, and the potential benefits to survival. Supporting Information {#sec013} ====================== ###### CONSORT/TREND statement checklist. To improve the quality of nonrandomized trials, this checklist helped verifying all items. (PDF) ###### Click here for additional data file. ###### Trial protocol (English). All the extended methods used in this trial are available here in English and was approved by legal authorities. (PDF) ###### Click here for additional data file. ###### Trial protocol (French). All the extended methods used in this trial are available here in French and was approved by legal authorities. (PDF) ###### Click here for additional data file. Reims University Hospital and the Institut National de la Santé et de la Recherche Médicale funded the present study. The authors thank Drs Aldjia Hocine and Antoine Braconnier for the follow-up of participants and Mr Eymeric Lagonotte and Dr Vincent Vuiblet for blood and urine management and storage. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: JPB CB SL OT PN PR. Performed the experiments: JPB SL OT. Analyzed the data: JPB CB PR. Contributed reagents/materials/analysis tools: JPB CB FJ PR. Wrote the paper: JPB CB.

Introduction ============ The efficacy with which epithelial cells absorb, filter, and secrete metabolites is predicated upon a polarized cell architecture and vectorial molecular trafficking ([Yeaman et al. 1999](#Yeamanetal1999){ref-type="bib"}). Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by numerous morphological and functional alterations, many of which may be explained by deficits in the cyst epithelial cell differentiation program. The aberrant expression of select basolateral proteins on the apical ADPKD cell surface ([Wilson 1997](#Wilson1997){ref-type="bib"}) is associated with the progressive accumulation and enlargement of fluid-filled cysts, which ultimately abrogates renal function ([Carone et al. 1994](#Caroneetal1994){ref-type="bib"}). Although it has long been supposed that modulations in cytoarchitecture and the fidelity of molecular targeting are central to ADPKD pathology (for review see [Wilson 1997](#Wilson1997){ref-type="bib"}), a mechanistic link between the occurrence of causal mutations and dysmorphogenesis has yet to be discovered. Recent progress concerning the genes responsible for ADPKD has been instrumental in identifying the molecular genetic basis of this disorder. Genetic lesions associated with \>85% of ADPKD cases have been mapped to the polycystic kidney disease (pkd) 1 gene ([Reeders et al. 1985](#Reedersetal1985){ref-type="bib"}), whereas the remaining cases are due to mutations in pkd2 ([Mochizuki et al. 1996](#Mochizukietal1996){ref-type="bib"}), and in rare instances a third undescribed locus ([Daoust et al. 1995](#Daoustetal1995){ref-type="bib"}). The PKD1 and PKD2 genes encode polycystin-1 and polycystin-2, respectively ([Hughes et al. 1995](#Hughesetal1995){ref-type="bib"}; [International Polycystic Kidney Disease Consortium 1995](#InternationalPolycysticKidneyDiseaseConsortium1995){ref-type="bib"}; [Mochizuki et al. 1996](#Mochizukietal1996){ref-type="bib"}). The clinically salient onset of ADPKD during mid-life is attributable to the acquisition of a second (somatic) mutation at the PKD1 or PKD2 loci ([Qian et al. 1996](#Qianetal1996){ref-type="bib"}; [Koptides et al. 1999](#Koptidesetal1999){ref-type="bib"}), which serves as the primary pathogenic stimulus that precipitates deterioration of previously normal epithelial tissue. Singular homozygous mutant cells are thought to undergo partial dedifferentiation and proliferation, leading to repopulation of the tubule wall and development of a clonal cyst ([Carone et al. 1994](#Caroneetal1994){ref-type="bib"}; [Qian et al. 1996](#Qianetal1996){ref-type="bib"}). Analyses describing protein topology and intermolecular interactions have yielded important information regarding the localization and potential roles of the polycystins. Polycystin-1 is a 480,000--mol wt putative transmembrane protein ([Hughes et al. 1995](#Hughesetal1995){ref-type="bib"}; [International Polycystic Kidney Disease Consortium 1995](#InternationalPolycysticKidneyDiseaseConsortium1995){ref-type="bib"}) localized solely along lateral contacting membranes of cultured cells ([Ibraghimov-Beskrovnaya et al. 1997](#Ibraghimov-Beskrovnayaetal1997){ref-type="bib"}). A predicted 2,500--amino acid extracellular domain is comprised of diverse motifs with putative functions in cell--cell and cell--extracellular matrix interactions ([Hughes et al. 1995](#Hughesetal1995){ref-type="bib"}; [International Polycystic Kidney Disease Consortium 1995](#InternationalPolycysticKidneyDiseaseConsortium1995){ref-type="bib"}; [Moy et al. 1996](#Moyetal1996){ref-type="bib"}), suggesting that polycystin-1 plays a role in adhesion ([Hughes et al. 1995](#Hughesetal1995){ref-type="bib"}). Polycystin-2 is an integral membrane protein consisting of domains with significant homology to polycystin-1 as well as to a family of voltage-gated ion channels ([Mochizuki et al. 1996](#Mochizukietal1996){ref-type="bib"}). Polycystin-1 and polycystin-2 interact physically via their COOH termini ([Qian et al. 1997](#Qianetal1997){ref-type="bib"}; [Tsiokas et al. 1997](#Tsiokasetal1997){ref-type="bib"}), offering an explanation for the observation that mutations in either gene results in an identical phenotype ([Tsiokas et al. 1997](#Tsiokasetal1997){ref-type="bib"}). Despite this progress, uncertainties regarding the molecular mechanism whereby alterations in the polycystins influence the phenotype of ADPKD cells have persisted. An exciting insight was provided by the recent description of intermolecular interactions between endogenous cellular polycystin-1 and E-cadherin, the integral membrane component of epithelial adherens junctions ([Huan and van Adelsberg 1999](#HuanandvanAdelsberg1999){ref-type="bib"}). E-cadherin is critical for the institution and perpetuation of epithelial cell polarity on account of its well-established role in the stabilization and elaboration of cytoskeletal--junctional complexes, and its more recently defined function in basolateral targeting patch recruitment ([Yeaman et al. 1999](#Yeamanetal1999){ref-type="bib"}). Initial E-cadherin--mediated epithelial cell adhesion is instrumental in reorganizing the cytoskeleton and exocytic transport machinery, and in establishing an apico-basolateral pole. The formation of tight junctions and the polarized delivery of vesicles and their cargo to newly defined apical and basolateral surfaces result in the genesis of two biochemically unique membrane domains. Because distinct vesicles carrying segregated apical and basolateral cargo are generated in fibroblasts as well as epithelial cells ([Yoshimori et al. 1996](#Yoshimorietal1996){ref-type="bib"}), minimal maturation of the Golgi apparatus may be required during epithelial differentiation, and the differentiating cell is likely competent to engage in vectorial trafficking as soon as vesicle targeting sites at the plasma membrane are delineated. Specification of these targeting sites occurs in response to E-cadherin--mediated adhesion. As intercellular E-cadherin interactions stabilize cytoskeletally tethered membrane proteins, a subset of cytosolic proteins are recruited to the contacting membranes. The sec proteins comprising the multimeric exocyst ([TerBush et al. 1996](#TerBushetal1996){ref-type="bib"}) are among those cytosolic proteins recruited to the basolateral membrane in response to E-cadherin ligation ([Grindstaff et al. 1998](#Grindstaffetal1998){ref-type="bib"}). These proteins demarcate a basolateral targeting patch that cooperates with the soluble *N*-ethylmaleimide--sensitive attachment protein receptor (SNARE) family of vesicular and target membrane receptors to ensure the proficiency of basolateral trafficking ([Grindstaff et al. 1998](#Grindstaffetal1998){ref-type="bib"}). The maintenance of this highly defined cellular organization is crucial in the continued performance of epithelial tissues. Because E-cadherin is critical for epithelial cell organization, it is interesting to consider the possibility that mutations in PKD1 or PKD2 disrupt normal E-cadherin--polycystin assemblies, and consequently impact ADPKD cell morphology and performance. Therefore, an assessment of ADPKD cell architecture and molecular trafficking was undertaken in order to identify specific derangements that lead to the compromised phenotypic state typical of these cells. Materials and Methods ===================== Chemical Reagents and Antibodies -------------------------------- Super Signal chemiluminescent substrate was supplied by Pierce. 4-(2-aminoethyl) benzenesulfonylfluoride, HCl (AEBSF) and Mowiol 4-88 were obtained from Calbiochem. Unless otherwise stated, all other chemical reagents were obtained from Sigma Chemical Co. Mouse mAbs against E-cadherin and rSec8 were purchased from Transduction Laboratories. A rabbit polyclonal antibody (pAb) against the COOH terminus of PR8 hemagglutinin (HA) has been described previously ([Wandinger-Ness et al. 1990](#Wandinger-Nessetal1990){ref-type="bib"}). A mouse mAb against sec6 was purchased from Stressgen Biotechnologies Corp. A rabbit pAb against occludin was supplied by Zymed Immunochemicals. Mouse mAbs against human low-density lipoprotein receptor (LDL-R) ([Van Driel et al. 1989](#VanDrieletal1989){ref-type="bib"}) and p75^NTR^ (US Patents 4,786,593 and 4,855,241) were amplified as ascites ([Harlow and Lane 1988](#HarlowandLane1988){ref-type="bib"}) using CRL-1898 and HB-8737 hybridoma cells (American Type Culture Collection), respectively. Fluorophore-conjugated secondary antibodies were purchased from Vector Laboratories, a rabbit pAb directed against mouse IgG used as a linker antibody was obtained from Jackson ImmunoResearch, and HRP-conjugated antibodies were bought from Amersham Pharmacia Biotech. Cell Culture ------------ Normal kidneys were obtained from previously healthy individuals whose acute injuries precluded organ transplantation. ADPKD kidneys were obtained after their surgical removal in preparation for transplant when patients were diagnosed with end-stage renal failure as a result of advanced ADPKD. Individual proximal tubule--derived cysts from ADPKD kidneys or proximal tubules from normal kidneys were isolated and dissected. The epithelial layer was separated from the underlying connective tissue layer and the epithelial cells isolated as described ([Carone et al. 1989](#Caroneetal1989){ref-type="bib"}). Cultures were maintained in a humidified atmosphere of 5% CO~2~ at 37°C. Cells for experiments were plated at a density of 300,000 cells per 24 mm^2^ cell culture filter insert (0.4-μm pore size) (Falcon; Becton Dickinson). Falcon tissue culture inserts were chosen based on the physical integrity of the inserts, vigorous cell growth, and morphological similarity between the two cell types. Cells were fed every other day with fresh medium until they were used for experiments on the fifth day after plating. After 5 d in culture, the integrity of all cell monolayers was assayed by light microscopy and the transepithelial resistance of select filters was measured. Once confluence was reached at culture day 3, the cells maintained a transepithelial resistance of ∼250 ohms/cm^2^, measured using a chopstick ohmmeter (World Precision Instruments). When used for immunofluorescence experiments, cells were also costained for the tight junction marker occludin to visually demonstrate cell confluence. Every experiment described in this study was performed in triplicate, using primary cells derived from normal or polycystic kidneys, in each case from three unrelated patients. The same patient samples (expanded no further than passage 4) were used throughout the study to minimize any effects of phenotypic variation. Fluorescent Lipid Transport Assay --------------------------------- 250 nmol C~6~-NBD-ceramide (Molecular Probes) in chloroform and methanol was dried to a powder under dry nitrogen and prepared as a BSA complex as described ([Pagano 1989](#Pagano1989){ref-type="bib"}). The methods used to label live cells with C~6~-NBD-ceramide and monitor lipid transport were based on published procedures ([Lipsky and Pagano 1985](#LipskyandPagano1985){ref-type="bib"}; [van Meer et al. 1987](#vanMeeretal1987){ref-type="bib"}). Immediately before the experiment, an aliquot of prepared C~6~-NBD-ceramide was thawed and diluted 1:3 in phenol red--free Hepes-buffered MEM (HMEM) containing 0.35 g/liter NaHCO~3~ and 60 mg/liter KH~2~PO~4~ (HMEM~bath~). Filter-grown cells were rinsed twice with PBS^+^ and 300 μl of the diluted fluorescent lipid was applied to the apical surface, whereas 2 ml of HMEM~bath~ equilibrated to 20°C was applied to the basolateral surface. Cells were incubated in a 20°C darkened water bath for 90 min to allow lipid accumulation in the Golgi apparatus. The cells were then rinsed twice in ice-cold PBS^+^ and cell surface lipids were removed by two incubations for 15 min each in a 10°C water bath with HMEM~bath~ containing 5 mg/ml delipidated BSA. Cell surface transport of Golgi-accumulated lipids was initiated by adding prewarmed (37°C) phenol red--free HMEM containing 3.7 g/l NaHCO~3~ and 60 mg/l KH~2~PO~4~ (1 ml apical, 2 ml basolateral). Subsequently, cells were incubated from 0--90 min at 37°C in a humidified 5% CO~2~ incubator. At the appropriate time, filter inserts were excised from the plastic holder with a scalpel. The filter was mounted cell side up on an ice-cold microscope slide with PBS^+^ and a coverslip, and confocal images were collected immediately. Immunofluorescence Microscopy ----------------------------- Filter inserts were rinsed in PBS, then fixed for 20 min at room temperature with 3% (wt/vol) paraformaldehyde, 0.1% (vol/vol) Triton X (TX)-100 (Fluka) prepared in PBS containing 1 mM CaCl~2~ and 1 mM MgCl~2~ (PBS^+^). Reactive aldehyde sites were quenched by incubation in 50 mM NH~4~Cl prepared in PBS^+^ for 20 min at room temperature. Nonspecific binding sites were blocked by incubation in PBS containing 0.2% (vol/vol) fish skin gelatin (Sigma Chemical Co.) for 30 min at room temperature. Insert supports were excised and cut into conveniently sized pieces. These pieces were incubated in primary antibody diluted in PBS containing 0.2% (vol/vol) fish skin gelatin for 1 h at 37°C in a humidified chamber. Samples were rinsed with three changes of PBS and incubated with the appropriate fluorophore-conjugated secondary antibody for 30 min at 37°C. Insert sections were washed twice for 15 min each with PBS and mounted between nail polish support posts in Mowiol 4-88. Cells were either viewed on a Zeiss Universal epifluorescence microscope or imaged using a Zeiss LSM 510 inverted laser scanning microscope equipped with He-Ne and Kr-Ar lasers. For the sec6/8 localization, several 0.4-μm-thick images taken from the cell apex to the basal substrate were assembled as an extended-focus image. For the localization of E-cadherin, epifluorescence profiles as well as images from one basolateral focal plane are shown. Cells examined during the fluorescent lipid transport assay were imaged at a single focal plane along the basolateral cell surface. Electron Microscopy ------------------- Cells grown on filters were washed in PBS and then fixed in 2.5% (wt/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 1 h. After three rinses in 0.1 M sodium cacodylate buffer, the cells were postfixed in 1% (wt/vol) aqueous OsO~4~ for 1 h at room temperature. The cells were rinsed in distilled water and stained with 1% (wt/vol) aqueous uranyl acetate in the dark for 1.5 h. The cells were dehydrated in a graded ethanol series, infiltrated, and embedded in Spurr\'s resin. Ultrathin sections were cut to 80 nm on a MT 6000-XL ultramicrotome (Ventana RMC), collected on 300 mesh nickel grids, stained with 2% (wt/vol) aqueous uranyl acetate for 4 min followed by 0.4% (wt/vol) lead citrate for 4 min. The sections were viewed on a Philips CM 120 electron microscope. Influenza Infection ------------------- Wild-type influenza (A/PR/8/34) virus stocks were grown in embryonated chicken eggs as described ([Kuchler 1977](#Kuchler1977){ref-type="bib"}). Filter-grown cells were rinsed with PBS and infected with influenza virus at 10 pfu/cell for 1 h at 37°C. The virus was diluted in DME with 0.2% BSA (DME/BSA) and 300 μl was added to the apical surface, whereas 3 ml of the same medium without virus was added basolaterally. After the 1-h virus adsorption phase, cells were washed and incubated for 4 h at 37°C in DME/BSA before either metabolic labeling or steady-state biotinylation assays. Recombinant Adenovirus Infection -------------------------------- Recombinant adenoviruses encoding either LDL-R or p75^NTR^ were kindly provided by Drs. Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX) ([Herz and Gerard 1993](#HerzandGerard1993){ref-type="bib"}) and Moses Chao (Cornell University Medical College, New York) ([Yoon et al. 1996](#Yoonetal1996){ref-type="bib"}), respectively. Large-scale virus stocks were prepared as described previously ([Spector et al. 1998](#Spectoretal1998){ref-type="bib"}). Stock virus preparations had titers of ∼10^8^ pfu/ml and infected the cells with 90% efficiency at a dilution of 1:10. Confluent filter-grown cells (72 h after initial seeding) were infected with adenoviruses (∼2 pfu/cell) for 1 h at 37°C. Viruses were diluted in serum-free DME and 300 μl was added to the apical surface, whereas 3 ml of the same medium without virus was added basolaterally. After the virus adsorption phase, media were replaced with serum-containing culture media and the cells were returned to a humidified 5% CO~2~ incubator for 36 h before metabolic labeling. Metabolic Labeling ------------------ Filter-grown cells were starved for 30 min at 37°C in DME lacking cysteine and methionine and then radiolabeled for 30 min at 37°C with 0.2 mCi/ml of ^35^S-Trans label (ICN) diluted in the same medium and added to the basolateral surface (300 μl total volume on a piece of parafilm placed in a tissue culture dish). Medium without radiolabel (1 ml) was added to the apical surface. After this radiolabeling period, cells were washed and incubated at 37°C in DME supplemented with 2 mM cysteine and methionine from 0--150 min. The chase media was collected separately from the apical and basolateral sides at each timepoint to evaluate the secreted protein profiles. Cell Surface Biotinylation -------------------------- Confluent filter-grown cells (in some cases metabolically labeled) were washed with ice-cold PBS^+^ and incubated with ice-cold E-Z Link Sulfo-NHS-LC-biotin or E-Z Link Sulfo-NHS-SS-biotin (Pierce) at 0.6 mg/ml prepared in PBS^+^, pH 8.0. Either the apical (1 ml volume) or basolateral (1.5 ml volume) surface was biotinylated twice for 15 min each. The biotinylation reaction was terminated by replacing the second biotin solution with the same volume of ice-cold 50 mM NH~4~Cl in PBS for 30 min on ice. Immunoprecipitations -------------------- ### E-Cadherin. Cells were scraped from the insert in 100 μl of 1% (vol/vol) TX-100, 0.5% (vol/vol) NP-40, 150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, CLAP (1 μM each of chymostatin, leupeptin, antipain, and pepstatin A), and 1 μM AEBSF. Detergent extracts were incubated with agitation for 1 h at 4°C, after which time insoluble material was removed by centrifugation at 15,000 *g* for 5 min at room temperature. ### LDL-R and p75NTR. Cells were scraped from the insert in 100 μl of 1% (vol/vol) TX-100, 150 mM NaCl, 15 mM Tris-Cl, pH 8.0, 4 mM EDTA, 1 μM CLAP, and 1 μM AEBSF. Detergent extracts were incubated with agitation for 1 h at 4°C, after which time insoluble material was removed by centrifugation at 15,000 *g* for 5 min at room temperature. ### Hemagglutinin. Cells were lysed by addition of 100 μl of SDS lysis buffer (1% \[wt/vol\] SDS, 15 mM Tris-Cl, pH 8.0, 4 mM EDTA, 1 μM CLAP, and 1 μM AEBSF). The extracts were boiled for 5 min to decrease viscosity of the solution. All detergent cell extracts were diluted with 900 μl of incubation buffer (0.5% \[vol/vol\] TX-100, 15 mM Tris-Cl, pH 8.0, 150 mM NaCl, 4 mM EDTA, 1 μM CLAP, 1 μM AEBSF) containing the appropriate dilution of primary antibody. Samples were incubated for 1 h at 4°C with agitation and for an additional 30 min with a rabbit pAb against mouse IgG as a linker antibody when monoclonal primary antibodies were used for immunoprecipitation. Immune complexes were recovered by incubation with 30 μl of protein A--Sepharose (100 μg total IgG binding capacity) (Amersham Pharmacia Biotech) for 1 h at 4°C with agitation. Protein A--Sepharose-bound antibody complexes were recovered after the incubation by centrifugation at 15,000 *g* for 5 min at room temperature. Immunoprecipitates were washed sequentially three times each with 1% (vol/vol) NP-40, 0.1% (wt/vol) SDS, 15 mM Tris-Cl, pH 8.0, 150 mM NaCl, 4 mM EDTA, 1 μM CLAP, 1 μM AEBSF), with the same buffer except containing 500 mM NaCl, and finally with 50 mM Tris-Cl, pH 8.0. Protein A--Sepharose beads were recovered after each wash by centrifugation at 15,000 *g* for 1 min at room temperature. The beads were resuspended in 50 μl of 10% (wt/vol) SDS and boiled for 5 min to release the antibody complexes. The supernatant fraction was collected with a narrow-bore pipette tip and 5 μl was reserved as a measure of the total immunoprecipitated protein, whereas the remainder was diluted in 900 μl of incubation buffer and reprecipitated with streptavidin-agarose to recover biotinylated proteins as described below. Streptavidin Affinity Precipitation ----------------------------------- Biotinylated samples used to analyze the steady-state distribution of cell surface proteins were solubilized in 100 μl of SDS lysis buffer. Detergent extracts were boiled for 5 min to denature nucleic acids. The lysate was subsequently diluted in 900 μl of incubation buffer containing 40 μl of streptavidin-agarose (sufficient to bind 120 μg of biotinylated protein) (Pierce), and rocked at 4°C for 1 h. Streptavidin-agarose beads were washed and recovered as described above, and boiled for 5 min in 40 μl of 2× sample buffer (100 mM Tris-Cl, pH 6.8, 4% \[wt/vol\] SDS, 0.2% \[wt/vol\] bromophenol blue, 20% \[vol/vol\] glycerol) containing 50 mM dithiothreitol. Diluted immunoprecipitates from metabolically labeled samples were incubated with 40 μl of streptavidin-agarose while rocking at 4°C for 1 h. Streptavidin-agarose beads were washed and recovered as described above, and boiled for 5 min in 40 μl of 2× sample buffer containing 50 mM dithiothreitol. SDS-PAGE and Immunoblot Analysis -------------------------------- Proteins were separated on 7 or 10% SDS polyacrylamide gels. After electrophoresis, metabolically labeled proteins were detected by drying the gels and subjecting them to phosphorimage analysis with a Fuji PhosphorImager equipped with MacBas software, or a Molecular Dynamics STORM 860 PhosphorImager equipped with ImageQuant software. For immunoblot analyses, proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). Nonspecific binding sites were blocked by a 1-h incubation at room temperature with 0.5% (wt/vol) nonfat dried milk dissolved in PBS containing 0.1% (vol/vol) Tween-20 (PBS-T). Blots were washed twice for 15 min in PBS-T and incubated with the appropriate dilution of rabbit anti-HA pAb or mouse anti--E-cadherin, anti-rsec6, or anti-rsec8 mAbs for 1 h at room temperature. After two more 15-min washes, blots were probed with the manufacturer\'s recommended dilution of HRP-conjugated secondary antibody for 30 min at room temperature, washed twice for 5 min with PBS-T, and bound antibodies were detected using chemiluminescent reagents. Results ======= ADPKD Cells Form Morphologically Typical and Functionally Intact Tight Junctions -------------------------------------------------------------------------------- Polarized epithelial cells restrict the paracellular flow of solutes (gate function) and the intermixing of apical and basolateral membrane molecules (fence function) by virtue of their apico-lateral tight junctions (for review see [Diamond 1977](#Diamond1977){ref-type="bib"}). Tight junction integrity was assayed as one measure of the ability of ADPKD cells to establish a polarized cell architecture. Immunofluorescence microscopy of the tight junction protein occludin ([Furuse et al. 1993](#Furuseetal1993){ref-type="bib"}) was used to examine tight junction morphology. The tight junctions delimited by occludin appeared identical in all patient samples ([Fig. 1](#F1){ref-type="fig"} A, upper panels). Tight junctions evident by EM consisted of closely apposed membrane contacts, which were morphologically similar in normal kidney and ADPKD cells ([Fig. 1](#F1){ref-type="fig"} A, lower panels). Although fluid accumulation within the developing cyst lumen would not be possible without an intact tight junction, paracellular gate function in explanted ADPKD cells has not been examined previously. ADPKD cells cultured on tissue culture inserts grew in tightly packed monolayers that reached a transepithelial resistance comparable to that of explanted normal kidney epithelial cells (∼250 ohms/cm^2^). This measurement confirmed that the tight junction gate in ADPKD cells was intact. The integrity of tight junction fence function was ascertained by monitoring the domain-specific localization of the apical membrane protein influenza HA ([Matlin et al. 1983](#Matlinetal1983){ref-type="bib"}). Cell surface HA was detected by membrane domain--specific biotinylation and subsequent immunoblot analysis of streptavidin-precipitated proteins. At steady state, influenza HA was correctly localized on the apical cell membrane of ADPKD cells ([Fig. 1](#F1){ref-type="fig"} B), indicating that just as in normal kidney cells, diffusion of proteins between membrane domains was prevented. This finding is in agreement with the observation that only select proteins exhibit altered membrane polarity in ADPKD cells in situ ([Wilson 1997](#Wilson1997){ref-type="bib"}). Thus, using these morphological, electrical, and biochemical criteria, ADPKD cells in culture were judged to possess tight junctions indistinguishable from those of their normal kidney counterparts. ADPKD Cell E-Cadherin Is Not Expressed at the Cell Surface and Is Sequestered in an Intracellular Compartment ------------------------------------------------------------------------------------------------------------- The adherens junctions are disposed along the lateral contacting membranes subjacent to the tight junctions, where they play a crucial role in maintaining a polarized epithelium ([Drubin and Nelson 1996](#DrubinandNelson1996){ref-type="bib"}). The immunolocalization of the integral membrane protein E-cadherin was examined to assess adherens junction architecture. Polarized monolayers were costained with antibodies against occludin and E-cadherin and imaged by epifluorescence microscopy. The contacting membranes of adjacent normal kidney and ADPKD cells were comparably demarcated by occludin ([Fig. 2](#F2){ref-type="fig"}, upper panels). Normal kidney cell E-cadherin staining ([Fig. 2](#F2){ref-type="fig"}, upper N panel, inset) coincided with that of occludin ([Fig. 2](#F2){ref-type="fig"}, upper N panel) along the lateral cell membranes. Surprisingly, the lateral membranes of ADPKD cells, defined by the disposition of occludin ([Fig. 2](#F2){ref-type="fig"}, upper P panels), were strikingly devoid of E-cadherin ([Fig. 2](#F2){ref-type="fig"}, upper P panels, insets). Because the compartment with which E-cadherin was associated in ADPKD cells was ill-defined by epifluorescence microscopy, the same samples were examined by confocal microscopy to eliminate out-of-focus information ([Fig. 2](#F2){ref-type="fig"}, lower panels). This investigation revealed that whereas E-cadherin was localized solely at the lateral membranes of normal kidney cells ([Fig. 2](#F2){ref-type="fig"}, lower N panel), E-cadherin in ADPKD cells was exclusively sequestered in perinuclear vesicular structures, completely absent from the lateral membranes ([Fig. 2](#F2){ref-type="fig"}, lower P panels). Thus, E-cadherin localization was markedly abnormal in ADPKD cells. Immunoblot analysis of biotinylated cell surface proteins corroborated the depletion of E-cadherin from the ADPKD cell membrane. E-cadherin was abundant and properly polarized at the basolateral membrane of normal kidney cells ([Fig. 3](#F3){ref-type="fig"} A, N samples). This contrasted sharply with the absence of any detectable E-cadherin on either the apical or basolateral membrane domains of ADPKD cells ([Fig. 3](#F3){ref-type="fig"} A, P samples). Since ADPKD cells appeared to contain significant amounts of E-cadherin in an intracellular E-cadherin store as judged by confocal analysis (see [Fig. 2](#F2){ref-type="fig"}), the E-cadherin content of cellular extracts was further examined by immunoblot analysis. Three different normal kidney samples expressed similar E-cadherin levels ([Fig. 3](#F3){ref-type="fig"} B, N samples). In contrast, ADPKD cell patient samples contained lower, variable levels of E-cadherin, although none of the three samples examined lacked the protein entirely ([Fig. 3](#F3){ref-type="fig"} B, P samples). These results demonstrated that ADPKD cells have reduced levels of E-cadherin, which is improperly sequestered in an intracellular pool. This could be attributed to either decreased synthesis or defective post-Golgi handling, prompting a more detailed analysis of E-cadherin biosynthesis, trafficking, and stability. Cell Surface Delivery of E-cadherin Is Impaired, while Maturation and Sorting Proceed Efficiently in ADPKD Cells ---------------------------------------------------------------------------------------------------------------- The absence of E-cadherin at the ADPKD cell membrane raised the possibility that the protein was prevented from effectively traversing the exocytic pathway. E-cadherin is initially synthesized in association with the ER as a high molecular weight precursor ([Peyrieras et al. 1983](#Peyrierasetal1983){ref-type="bib"}; [Vestweber and Kemler 1984](#VestweberandKemler1984){ref-type="bib"}). Assembly with β-catenin enables transit of E-cadherin to the Golgi apparatus ([Chen et al. 1999a](#Chenetal1999a){ref-type="bib"}), where it is subsequently processed to its mature form ([Shore and Nelson 1991](#ShoreandNelson1991){ref-type="bib"}). Information about the molecular maturation, polarized sorting, and cell surface delivery was obtained from metabolic labeling and domain-selective biotinylation experiments. The precursor form of ADPKD cell E-cadherin was synthesized in near-normal amounts and efficiently proteolytically cleaved, demonstrating that ER and Golgi apparatus functions were not generally disrupted in these cells ([Fig. 4](#F4){ref-type="fig"} A, Total N and P samples). Association of newly synthesized E-cadherin with the catenins was detected by coimmunoprecipitation under the nondenaturing conditions. Multimeric cadherin--catenin complex assembly appeared equally efficient in normal and ADPKD cells ([Fig. 4](#F4){ref-type="fig"} A, Total basolateral N and P samples). Diminished plasma membrane association of E-cadherin was therefore not due to defective catenin affiliation. However, it was noted that there was a significant decrease in E-cadherin delivery to the basolateral surface of ADPKD cells despite near-normal synthesis levels ([Fig. 4](#F4){ref-type="fig"} A, compare N and P samples in basolateral Cell Surface panel). Importantly, no missorting to the apical membrane was evident at any timepoint ([Fig. 4](#F4){ref-type="fig"} A, apical Cell Surface panel). Quantification of basolateral delivery showed that at the latest timepoint, only 20% of the protein had arrived at the ADPKD cell surface, compared with 60% in normal kidney cells ([Fig. 4](#F4){ref-type="fig"} B). Thus, inefficient transport of E-cadherin from the late Golgi cisternae to the plasma membrane is partially responsible for the diminished cell surface--associated E-cadherin observed in ADPKD cells. Cell Surface Transport of Basolateral Cargo Is Impaired in ADPKD Cells ---------------------------------------------------------------------- The polarized sorting and transport of well-characterized apical and basolateral membrane proteins was analyzed to distinguish whether decreased basolateral delivery was unique to E-cadherin or whether a more general defect in vectorial transport existed in ADPKD cells. The cell surface arrival of newly synthesized molecules was scored by metabolic labeling and cell surface biotinylation as described above. Influenza HA was used as an apical marker that is sorted in association with glycosphingolipid rafts ([Scheiffele et al. 1997](#Scheiffeleetal1997){ref-type="bib"}). Within 30 min after pulse labeling, the mature form of influenza HA appeared at the apical cell surface, where it reached maximal levels by 120 min ([Fig. 5](#F5){ref-type="fig"} A, upper panel). Both the kinetics and amounts of HA delivered to the apical membrane as a percentage of total newly synthesized HA were similar in normal kidney and ADPKD cells ([Fig. 5](#F5){ref-type="fig"} B). Delivery of HA to the basolateral membrane was undetectable in both normal and ADPKD cells ([Fig. 5](#F5){ref-type="fig"} A, lower panel). Neurotrophin receptor (p75^NTR^) served as a second apical marker that is sorted by virtue of its lumenal O-linked glycosylation ([Yeaman et al. 1997](#Yeamanetal1997){ref-type="bib"}). As was observed for influenza HA, p75^NTR^ delivery to the apical membrane of ADPKD cells was identical to that documented in normal kidney cells ([Fig. 5](#F5){ref-type="fig"} C). Approximately 30% of the total ^35^S-labeled p75^NTR^ was scored as having reached the cell surface during the chase period by biotinylation. Similar amounts of p75^NTR^ were seen delivered to the surface of MDCK cells in an analogous experiment, where it was determined that this was an under-representation of cell surface delivery most likely due to inefficient biotinylation and/or streptavidin recovery ([Grindstaff et al. 1998](#Grindstaffetal1998){ref-type="bib"}). Complementary experiments were conducted to examine the cell surface delivery of the basolaterally targeted LDL-R, the sorting signal for which resides in the cytoplasmic domain ([Matter et al. 1992](#Matteretal1992){ref-type="bib"}). In normal kidney cells, LDL-R was first detected at the cell surface within 30 min after pulse labeling and increased steadily over the course of the 120-min chase period ([Fig. 6](#F6){ref-type="fig"} A, Cell Surface samples, N lanes). The synthesis of LDL-R and processing of the precursor to the mature form proceeded with comparable kinetics in ADPKD cells and normal kidney cells ([Fig. 6](#F6){ref-type="fig"} A, Total samples, compare N and P lanes). However, the rate of LDL-R cell surface delivery was diminished twofold in ADPKD as compared with normal kidney cells ([Fig. 6](#F6){ref-type="fig"} B). Missorting of LDL-R to the apical membrane domain was not evident ([Fig. 6](#F6){ref-type="fig"} C). In two of the three ADPKD patient samples analyzed, the mobility of the mature form of LDL-R during SDS-PAGE was slightly faster than LDL-R from normal kidney cell samples ([Fig. 6](#F6){ref-type="fig"} A and data not shown), likely indicative of minor differences in posttranslational processing. Since basolateral transport efficiency was similarly diminished in all three samples and in no case was there any impact on polarized LDL-R sorting, it is felt that processing differences are inconsequential with respect to the trafficking deficit. Instead, the reduced efficiency of basolateral LDL-R delivery was reminiscent of the impaired transport of normally assembled and processed E-cadherin to the ADPKD basolateral surface. Polarized secretion of newly synthesized proteins was examined as a further measure of overall protein trafficking to the apical and basolateral plasma membrane domains. This was accomplished by collecting the media separately from the apical and basolateral chambers of metabolically labeled, filter-grown cells. Comparisons of the secreted protein profiles after SDS-PAGE and autoradiography demonstrated that most apical proteins were secreted with identical kinetics in comparable amounts in both cell types up to the 60 min timepoint ([Fig. 7](#F7){ref-type="fig"} A). It was noted that apical proteins secreted by ADPKD cells failed to increase in amounts beyond those present at the 60 min timepoint, which may represent either enhanced protein degradation on account of enhanced internalization or elevated protease activity by polycystic kidney disease cells ([Rankin et al. 1996](#Rankinetal1996){ref-type="bib"}). In marked contrast to apical protein secretion, comparisons of basolateral protein secretion revealed a disparity in the efficiency of basolateral exocytosis at all timepoints ([Fig. 7](#F7){ref-type="fig"} B). Normal kidney cells steadily secreted increasing amounts of several proteins into the basolateral medium ([Fig. 7](#F7){ref-type="fig"} B, arrowheads). Basolateral secretion from ADPKD cells was significantly reduced, with very little secreted protein detectable even at the latest timepoint ([Fig. 7](#F7){ref-type="fig"} B, P lanes), in keeping with the observations made in the analyses of individual basolateral membrane proteins. Based on this series of experiments, it was apparent that protein transport to the basolateral cell surface was impaired, whereas apical delivery proceeded relatively unimpeded. Exocytic Cargo Is Retained in the ADPKD Cell Golgi Apparatus ------------------------------------------------------------ Diminished cell surface delivery of newly synthesized basolateral cargo in the absence of any posttranslational processing deficits or apical mistargeting prompted an investigation of whether molecules might be accumulating in the ADPKD cell Golgi apparatus. To explore this possibility, the fluorescent lipid analogue C~6~-NBD-ceramide was used to assay Golgi-to-plasma membrane transport both morphologically and biochemically as described ([Lipsky and Pagano 1985](#LipskyandPagano1985){ref-type="bib"}; [van Meer et al. 1987](#vanMeeretal1987){ref-type="bib"}). Cellular membranes were labeled with C~6~-NBD-ceramide at reduced temperature (20°C) for 90 min, after which time cell surface C~6~-NBD-ceramide was removed by back-exchange at low temperature. Under these conditions, C~6~-NBD-ceramide was delivered to the Golgi apparatus, where it was similarly metabolized to C~6~-NBD-glucosylceramide and C~6~-NBD-sphingomyelin in both normal kidney and ADPKD cells (data not shown). The inhibition of vesicular transport to the cell surface caused by the 20°C incubation resulted in the accumulation of these fluorescent lipid metabolites in the TGN of both normal and ADPKD cells ([Fig. 8](#F8){ref-type="fig"}, [Fig. 010](#F10){ref-type="fig"} min). Golgi-to-cell surface transport of the C~6~-NBD-lipids was initiated by transferring the cells to 37°C and cell surface delivery was monitored by confocal microscopy ([Fig. 8](#F8){ref-type="fig"}, 30 min). These experiments revealed a dramatic defect in the ability of ADPKD cells to transport the C~6~-NBD-lipids to the cell surface ([Fig. 8](#F8){ref-type="fig"}, compare P samples with N samples). In normal kidney cells, the C~6~-NBD-lipids were first evident at the basolateral plasma membrane within 30 min after warming to 37°C, and cell surface delivery was complete by 90 min, with none remaining Golgi-associated at the latter timepoint. In contrast, basolateral delivery in ADPKD cells was not detectable after 90 min, and the C~6~-NBD-lipids remained associated with the ADPKD cell Golgi apparatus for as long as 150 min ([Fig. 8P](#F8){ref-type="fig"} 90 min sample; data not shown). Quantitative analyses of cell surface fluorescent lipids extracted from the apical or basolateral membrane domains indicated that lipid transport to the ADPKD cell basolateral membrane was reduced by 2.5--3-fold, whereas apical delivery was unimpeded (data not shown). Components of the Basolateral Targeting Patch Are Depleted from the ADPKD Cell Membrane --------------------------------------------------------------------------------------- The demonstration that C~6~-NBD-lipids failed to exit the Golgi apparatus, coupled with the observed basolateral trafficking defect, were indicative of defective vectorial trafficking from the ADPKD cell Golgi apparatus to the basolateral plasma membrane. Ineffective delivery of exocytic cargo to the ADPKD cell basolateral membrane raised the possibility that constituents of the basolateral targeting patch were improperly expressed or localized. The localization of sec6 and sec8 was therefore examined by confocal microscopy. Cells were stained to visualize sec6 or sec8 (red channel) in parallel with the tight junction protein occludin (green channel). In normal kidney cells, both sec6 and sec8 were localized in close apposition to the tight junction protein occludin ([Fig. 9](#F9){ref-type="fig"} A, left panels). In contrast, both proteins were depleted from the ADPKD cell lateral membranes and appeared diffusely dispersed throughout the cytoplasm ([Fig. 9](#F9){ref-type="fig"} A, right panels). Immunoblot analyses revealed that sec6 and sec8 protein levels were similar in all normal kidney and ADPKD samples analyzed ([Fig. 9](#F9){ref-type="fig"} B, compare N and P samples). Therefore, sec protein redistribution was not accompanied by a decline in sec protein expression, as was observed for E-cadherin. These results substantiate a marked disruption in the integrity of the basolateral cargo delivery site in ADPKD cells, likely brought about by altered E-cadherin--based adherens junction assembly. Discussion ========== This study identified the loss of cell surface E-cadherin and the exocyst components sec6 and sec8 as critical molecular deficits affecting ADPKD epithelial cells. Although ADPKD cells possessed characteristics of polarized cells, the disease cells suffered from a lack of detectable cell surface E-cadherin. Total cellular E-cadherin levels were lower than those in normal kidney cells, and existing E-cadherin was sequestered in an intracellular pool. The cell surface depletion of sec6 and sec8 in ADPKD cells devoid of plasma membrane--associated E-cadherin was correlated with significantly impaired delivery of proteins and lipids to the basolateral cell surface. Vectorial transport to the apical ADPKD cell surface, on the other hand, was functionally intact. Together, the investigations presented here serve to clarify the molecular mechanisms whereby mutations in PKD1 or PKD2 may lead to downstream alterations in cytoarchitecture and molecular trafficking in ADPKD cells. E-cadherin, the catenins, and the polycystins are all disposed within the basolateral membrane beneath the apico-lateral tight junction, where recent data suggest they are engaged in a large multimeric complex that may coordinately regulate cellular organization ([Fig. 10](#F10){ref-type="fig"}) ([Ibraghimov-Beskrovnaya et al. 1997](#Ibraghimov-Beskrovnayaetal1997){ref-type="bib"}; [Obermüller et al. 1999](#N0x274e200N0x30fa260){ref-type="bib"}; [Wu et al. 1998](#Wuetal1998){ref-type="bib"}; [Yeaman et al. 1999](#Yeamanetal1999){ref-type="bib"}). E-cadherin, as the transmembrane component of adherens junctions, constitutes the calcium-dependent molecular link between adjacent cell membranes that implements cytoskeletal organization via cytosolic catenins ([Drubin and Nelson 1996](#DrubinandNelson1996){ref-type="bib"}). Association of polycystin-1 with the E-cadherin--catenin assembly ([Huan and van Adelsberg 1999](#HuanandvanAdelsberg1999){ref-type="bib"}) is expected to tether a subset of polycystin-1 molecules in close proximity to adherens junctions. Polycystin-1, with its numerous cell--cell and cell--extracellular matrix adhesion domains, is thus poised to facilitate interactions with neighboring cells or the matrix. Demonstrated interactions between polycystin-2 and polycystin-1 ([Tsiokas et al. 1997](#Tsiokasetal1997){ref-type="bib"}) merit the inclusion of polycystin-2 within this complex. Given the likelihood of an ordered assembly between adherens junction components and the polycystins, it is interesting to consider why E-cadherin and sec6/8 might be depleted from the cell surface of ADPKD cells, and what ramifications this may have on cellular organization. Mechanisms Underlying Loss of Cell Surface E-Cadherin ----------------------------------------------------- It is plausible that the physical association of E-cadherin with mutant polycystin-1 might lead to the disruption of epithelial cell organization, particularly given the demonstrated interaction between polycystin-1 and E-cadherin ([Huan and van Adelsberg 1999](#HuanandvanAdelsberg1999){ref-type="bib"}). The developmental downregulation of polycystin-1 expression is altered in ADPKD so that mutant PKD1 products are often overexpressed ([Geng et al. 1996](#Gengetal1996){ref-type="bib"}, [Geng et al. 1997](#Gengetal1997){ref-type="bib"}). Conceivably, overexpressed mutant polycystin-1 may adversely impact E-cadherin stability. Indeed, the patient-to-patient variability in E-cadherin expression levels in ADPKD cells demonstrated here indicates that individual polycystin mutations differentially influence E-cadherin stability. Disturbances in either the stoichiometry of normal E-cadherin--polycystin-1 interactions or the structural characteristics of such complexes could account for the compromised efficiency with which E-cadherin is transported through the late exocytic pathway and stably retained at the plasma membrane in multiple ways. This report demonstrated that de novo membrane insertion, plasma membrane stabilization, and recycling or degradation of E-cadherin are all affected in ADPKD cells. Diminished E-cadherin cell surface delivery undoubtedly contributes to the reduced cell surface expression, although given the threefold decrease in delivery, this deficit cannot entirely account for the lack of ADPKD cell surface E-cadherin observed at steady state. E-cadherin was recently demonstrated to undergo dynamic recycling between the cell surface and early endosomes, traversing a circuit through which the plasma membrane disposition of E-cadherin may be regulated ([Le et al. 1999](#Leetal1999){ref-type="bib"}). In light of this finding, it is interesting to consider that modulation of a regulatory circuit may contribute to reduced cell surface expression of E-cadherin in ADPKD cells. Aberrant assembly of multimeric E-cadherin--containing complexes may cause the newly synthesized E-cadherin arriving at the basolateral cell surface to be increasingly internalized and decreasingly recycled to the plasma membrane, resulting in the observed intracellular accumulation. The existence of a pathogenic variation in this circuit is further suggested by the diminished levels of E-cadherin in ADPKD cells: once internalized, unrecycled abnormal E-cadherin complexes may be transported to late endocytic organelles and degraded. Depletion of the exocyst complex from the basolateral membrane upon the acquisition of a second, somatic genetic lesion is likely a downstream event that follows a reduction in cell surface E-cadherin. Dissociation of the basolateral cargo targeting patch from the apico-lateral apex may exacerbate the defect by precluding further delivery of newly synthesized E-cadherin, and by impacting recycling of E-cadherin to the basolateral membrane. Although future studies are needed to clarify the precise mechanisms by which mutations in PKD1 or PKD2 affect E-cadherin trafficking and stability, it is probable that deranged intermolecular interactions involving E-cadherin constitute potent stimuli culminating in cellular dysmorphogenesis in ADPKD. Cell--Cell Adhesion in the Absence of Cell Surface E-Cadherin ------------------------------------------------------------- E-cadherin has been suggested to occupy a central role in the nucleation and maintenance of epithelial cell polarity ([Drubin and Nelson 1996](#DrubinandNelson1996){ref-type="bib"}). When E-cadherin ligation was prevented, both adherens junction as well as tight junction and desmosomal junction assembly were inhibited ([Gumbiner et al. 1988](#Gumbineretal1988){ref-type="bib"}). Nevertheless, in the absence of detectable cell surface E-cadherin, explanted ADPKD cells are somehow capable of maintaining partially polarized, physically intact monolayers. The possibility that alternate cadherins are present on the ADPKD cell surface, sufficing to generate a partially polarized phenotype, was explored by immunochemical analyses of K-cadherin and the cadherins in general. K-cadherin/cadherin-6 is a cadherin family member expressed during renal development as well as in mature proximal tubule cells ([Xiang et al. 1994](#Xiangetal1994){ref-type="bib"}; [Shimoyama et al. 1995](#Shimoyamaetal1995){ref-type="bib"}; [Paul et al. 1997](#Pauletal1997){ref-type="bib"}). K-cadherin protein levels in both normal and ADPKD cells were low, and no compensatory changes were evident (data not shown). However, immunostaining using a pan-cadherin antibody directed against a highly conserved region shared by cadherin proteins demonstrated that the lateral membranes of ADPKD cells contained an alternate cadherin (data not shown). Thus, adhesion through an alternate cadherin family member, in concert with integrin- and desmosome-mediated adhesion, may compensate for the loss of cell surface E-cadherin and serve to support ADPKD cell architecture. Mechanisms Underlying Altered ADPKD Cell Polarity ------------------------------------------------- The nonpolarized distribution of certain basolateral membrane proteins in ADPKD cyst-lining cells led to the tenable hypothesis that polarized trafficking is defective ([Wilson 1997](#Wilson1997){ref-type="bib"}), which has until now remained untested. Quite unexpectedly, even in the absence of efficient basolateral delivery, E-cadherin and LDL-R were not mistargeted to the apical ADPKD cell membrane. These findings exclude promiscuous packaging of exocytic cargo within the TGN and aberrant targeting of basolateral vesicles as contributing factors in abnormal ADPKD cell polarity. Therefore, it is necessary to consider alternative explanations for immunohistochemical ([Wilson 1997](#Wilson1997){ref-type="bib"}) and pharmacological ([Du and Wilson 1995](#DuandWilson1995){ref-type="bib"}; [Wilson et al. 1991](#Wilsonetal1991){ref-type="bib"}) evidence that ADPKD cells are not fully polarized. Typically, a minor fraction of newly synthesized plasma membrane proteins escape sorting within the TGN of normal kidney epithelial cells, and as a result, are delivered to the incorrect membrane ([Simons and Fuller 1985](#SimonsandFuller1985){ref-type="bib"}). These molecules are subsequently internalized and redirected along the endocytic/transcytotic pathway to the correct plasma membrane domain. Upon arrival at the basolateral plasma membrane, proteins such as the Na^+^, K^+^ adenosine triphosphatase (Na^+^, K^+^-ATPase) become stably linked to microfilament assemblies, the organization of which depends on proper E-cadherin ligation ([Yeaman et al. 1999](#Yeamanetal1999){ref-type="bib"}). The loss of cell surface E-cadherin expression observed here is thus most likely the key to understanding deficits in ADPKD cell polarity. Given the disturbances in ADPKD cell architecture observed in situ ([Wilson et al. 1986](#Wilsonetal1986){ref-type="bib"}) coupled with the alterations in E-cadherin localization demonstrated in cultured ADPKD cells, it seems plausible that the small fraction of basolateral molecules initially mistargeted to the apical membrane (below detection levels in the cell surface delivery experiments) become stabilized within the incorrect membrane. Inappropriate apical membrane localization might occur through aberrant cytoskeletal interactions, or alternatively because endocytosis/transcytosis processes critical for retrieving mistargeted molecules to the correct membrane domain are dysfunctional. It is also possible that missorting in ADPKD is confined to select membrane proteins due to specialized, as-yet undefined transport requirements. Any of these possible mechanisms would facilitate the accumulation of basolateral cargo at the incorrect membrane domain and may account for the stable, functionally active Na^+^, K^+^-ATPase and EGF receptor at the incorrect (lumenal/apical) surface in ADPKD cysts ([Wilson et al. 1991](#Wilsonetal1991){ref-type="bib"}; [Du and Wilson 1995](#DuandWilson1995){ref-type="bib"}). Given the dramatic reorganization of ADPKD epithelial cells described in this study, it will now be of great interest to determine how the modulations in E-cadherin and sec6/8 localization contribute to the defects in ADPKD cell membrane polarity. Inefficient ADPKD Cell Trafficking: Stalling of TGN-to-Basolateral Membrane Transport ------------------------------------------------------------------------------------- Once the TGN processes of sorting and vesicle formation are complete, basolateral cargo is generally transported directly to the cell surface of kidney epithelial cells, although a subset may pass through endosomes ([Lisanti et al. 1989](#Lisantietal1989){ref-type="bib"}; [Le Bivic et al. 1990](#LeBivicetal1990){ref-type="bib"}; [Futter et al. 1995](#Futteretal1995){ref-type="bib"}; [Leitinger et al. 1995](#Leitingeretal1995){ref-type="bib"}). The experiments described in this study have spatially placed the basolateral trafficking deficit in ADPKD cells between vesicle formation at the TGN and fusion with the plasma membrane. Generalized alterations in ER-to-Golgi transport were excluded as a cause for the basolateral membrane trafficking defect in ADPKD cells by monitoring the processing of several exocytic transport markers. Posttranslational modification of apical (influenza HA) as well as basolateral (LDL-R and E-cadherin) cargo proceeded at the same rate in ADPKD and normal kidney cells. Delays in multimeric assembly of E-cadherin--catenin complexes were also not observed. Furthermore, lipid metabolism, measured as the conversion of C~6~-NBD-ceramide into C~6~-NBD-glucosylceramide and C~6~-NBD-sphingomyelin within the Golgi apparatus, occurred with similar kinetics in ADPKD and normal kidney cells (data not shown). The finding that these processes were largely unaffected pinpointed the exocytic defect to a late basolateral trafficking event. After polarized molecular sorting in the ADPKD cell Golgi apparatus, basolateral trafficking is arrested, with basolateral cargo failing to exit this organelle. Morphological evidence that export of molecules from the APDKD cell Golgi apparatus was impeded was furnished by experiments tracing the transport of fluorescent lipids. Ineffective transport was evident by the retention of C~6~-NBD lipids within the ADPKD cell Golgi apparatus long after those in normal kidney cells had traversed the Golgi apparatus and been efficiently delivered to the basolateral membrane. Impaired export from the ADPKD cell Golgi apparatus was also apparent from the delayed protein transport to the basolateral cell surface. Since ER-to-Golgi transport was unimpaired and there was no evidence of apical mistargeting, this implied that analogous to the lipid markers, basolateral proteins were detained within the Golgi apparatus. Evaluations of ADPKD cell Golgi apparatus morphology documented conspicuously dilated cisternae (Charron, A.J., R.L. Bacallao, and A. Wandinger-Ness, manuscript in preparation), which may be readily explained by the aberrant retention of basolaterally destined molecules within the Golgi apparatus. Impediment of cargo export from the Golgi apparatus may be indicative of regulatory mechanisms coordinating vesicle budding and vesicular fusion with the target (basolateral) ADPKD cell membrane. Docking and fusion of vesicles with the plasma membrane embodies the ultimate step in basolateral trafficking. This step is mediated by the concerted actions of the SNARE proteins and exocyst components ([Chen et al. 1999b](#Chenetal1999b){ref-type="bib"}; [Grindstaff et al. 1998](#Grindstaffetal1998){ref-type="bib"}). Examination of SNARE protein distribution using antiserum against the basolateral SNARE protein syntaxin 4 did not reveal noticeable differences in its distribution between the two cell types (Charron, A.J., R.L. Bacallao, and A. Wandinger-Ness, manuscript in preparation). This component of the basal docking and fusion machinery is therefore likely intact, and may partially compensate for the depletion of the basolateral targeting patch from the ADPKD cell membrane, mediating residual basolateral transport. However, the loss of sec6 and sec8 from the ADPKD cell membrane presumably impacts not only the final event in basolateral exocytosis, but also cellular organization per se. Given the central role of E-cadherin in maintaining epithelial cell organization, impaired docking and fusion of plasma membrane-bound E-cadherin-containing vesicles due to dissociation of sec6/8 from the basolateral targeting patch likely directly impacts cytoarchitecture, as discussed above. In addition, it is also conceivable that the dispersal of exocyst components affects the exocytic organelles. Basolateral cargo-bearing vesicles that leave the ADPKD cell Golgi apparatus and are transported to the membrane region normally associated with the exocyst complex may become stalled, as efficient docking and fusion is prevented. By engaging basolateral trafficking effectors in futile post-Golgi transport, this effect is expected to eventually lead to diminished basolateral vesicle budding from the ADPKD cell Golgi apparatus. If such a retrograde pathogenic mechanism issuing from the cell surface back to the Golgi exists, it is reasonable to postulate that the acquisition of mutations in the PKD1 or PKD2 loci would set into motion a cascade of events, commencing with the destabilization of cell surface E-cadherin and ultimately culminating in the ADPKD epithelial cell phenotype. We pay special tribute to the memory of Dr. Frank Carone, a friend and collaborator whose unflagging determination and pioneering spirit remain as a constant source of inspiration to all of us. We gratefully acknowledge Ms. Janice Pennington for electron microscopic imaging. Ms. Mary Slater and Ms. Elsa Romero provided valuable assistance with general laboratory management. This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases to A. Wandinger-Ness (R01 DK 50141) and R. Baccalao (R29 DK 46883). R. Baccalao is also a recipient of a Clinician Scientist Award from the National Kidney Foundation. A.J. Charron was partially supported by a National Institute of General Medical Sciences predoctoral training grant (T32 GM 08061). This study is the result of collaborative efforts between two laboratories. R. Bacallao and A. Wandinger-Ness contributed equally to the supervision of the experiments herein. *Abbreviations used in this paper:* ADPKD, autosomal dominant polycystic kidney disease; AEBSF, 4-(2-aminoethyl) benzenesulfonylfluoride, HCl; HA, hemagglutinin; HMEM, Hepes-buffered MEM; LDL-R, low density lipoprotein receptor; pAb, polyclonal antibody; PKD, polycystic kidney disease; SNARE, soluble *N*-ethylmaleimide--sensitive attachment protein receptor; TX, Triton X. {#F1} {#F4} {#F2} {#F3} {#F5} {#F6} {#F7} ![Depletion of sec8 and sec6 from the ADPKD cell membrane without loss of expression[.]{.smallcaps} (A) Confluent monolayers of normal kidney cells (N) and ADPKD cells (P) on filter inserts were processed for confocal microscopy. The association of sec8 and sec6 with the apico-lateral membrane region was assessed by coimmunostaining the cells with a rabbit pAb against occludin and mouse mAbs against either sec8 or sec6, followed by FITC-conjugated anti--rabbit and rhodamine-conjugated anti--mouse secondary antibodies. Confocal images (0.4-μm-thick focal sections) were collected along the entire height of the cells and assembled as an extended focus image of 50 such focal sections. Occludin staining appears green, whereas sec8 or sec6 staining appears red. Colocalization of occludin with either sec8 or sec6 is evident by a yellow overlap pattern. Bar, 10 μm. (B) Expression levels of sec8 and sec6 were examined by immunoblot analysis of total cellular proteins from filter-grown monolayers of two patient samples of normal kidney cells (N) and three patient samples of ADPKD cells (P). Proteins in detergent extracts (10 μg/lane) were resolved by SDS-PAGE. Gels were blotted onto nitrocellulose and blots were probed with mouse mAbs against sec8 or sec6, followed by an HRP-conjugated secondary antibody. sec8 and sec6 were detected by the addition of enhanced chemiluminescence substrate and exposure of the blot to X-ray film.](JCB9909128.f9){#F9} {#F8} {#F10}

Introduction ============ PPNAD is a rare cause of pediatric Cushing syndrome (CS). Diagnosis and management of this condition remains challenging. Although, there are diagnostic approaches and various tests to identify the cause of CS, but these tests appears to have incomplete diagnostic accuracy. Clinical case ============= A 2-year-old, previously healthy girl presented with rapid weight gain for 5 months. She had Cushingoid appearance, acne, hirsutism, hypertension, and normal pre-pubertal female genitalia. Her weight was 16.9 kg (\>97^th^ percentile) and height was 85 cm (3^rd^ percentile). Serum cortisol levels revealed loss of diurnal variation (35.0 and 30.1 μg/dl at 8.00 am and 11.00 pm, respectively). ACTH levels were unsuppressible at 22.4 (1^st^) and 20.5 pg/ml (2^nd^). Testosterone and DHEAS levels were undetectable. Abdominal USG were done twice. The first study showed enlargement of bilateral adrenals (right 1.6x1.2 cm and left 1.7x1.4 cm), but the subsequent one was unremarkable. Pituitary MRI was normal. CT chest included neck and abdomen revealed normal adrenal glands without mass. High dose dexamethasone suppression test (HDDST) revealed unsuppressible cortisol levels (baseline 51.9 and post-HDSST 47.2 μg/dl). Repeated ACTH levels were 13.6 and 15.1 pg/ml, suggested ACTH-independent CS. CT abdomen was reviewed by an adrenal radiologist expert and a small nodule (3-4 mm in size) of the right gland was identified, thus micronodular adrenocortical hyperplasia (MAD) was suspected. Laparoscopic right adrenalectomy was done. The right adrenal gland had normal gross findings, but histopathology revealed nodular thickening of the cortical tissue and internodular atrophy, which were compatible with PPNAD. After surgery, the features of CS were gradually subsided and cortisol levels were steadily declined to normal. Clinical lessons ================ Adrenal CS is more common in this age group, but initial hormonal profiles and imaging studies misled us. Non-suppressed ACTH levels could be found in adrenal CS. Other tests (CRH/HDDST) are useful to guide the diagnosis. Imaging studies should be best interpreted by experienced radiologist. Normal adrenal glands are not uncommon in MAD and its variant, PPNAD. Nodular thickening and internodular atrophy are pathognomonic of PPNAD even if no pigmentation seen in gross findings. PPNAD is caused by *PRKARIA* gene mutation which may present as isolated form or is associated with Carney's complex. Genetic testing is important because this disorder is bilateral involvement. Thus, unilateral adrenalectomy may be inadequate. Carefully follow-up is needed and contralateral adrenalectomy may be required in the future. Conclusion ========== Diagnosis and management of CS are challenges in pediatric endocrine practice, and should be best done by a team experienced in managing pediatric CS. Accurate diagnosis and localization is the key to success in managing these cases.

I am thankful to Quillien ([@B10]) for his response to my paper (Pollet, [@B7]), as it allows clarifying my position. Firstly, I would like to underline that the purpose of my paper was to flesh out the (implicit) statistical assumptions underpinning cross-cultural correlations. However, what I highlighted is but a side-issue when working with macro-level cross-cultural data (e.g., Poortinga, [@B9]; Mace and Pagel, [@B6]; Pollet et al., [@B8]). I would like to bring the discussion back to "earth" and clarify why I believe, in contrast to Quillien ([@B10]), that finite populations might be problematic in this context. I put forward that the sampling units for macro-level cross-cultural correlations are finite. In contrast, Quillien argues that this does not have to be the case. I believe for the examples I cited (Pollet, [@B7]: Table 1), the statistical data are clearly scores derived countries, states, etc. and are therefore by their (implicit) definition finite. Data at country/state/region level are very much unlike a population of people (Kuppens and Pollet, [@B5]) or an experiment where we can gather new, independent observations. Quillien presents no argument that these observed entities such as states/countries/etc. are in fact infinite but rather argues that the scores these researchers use represent something else. Let us return to a specific example we both discussed, U.S. state scores (e.g., Kanazawa, [@B4]; Eppig et al., [@B1]). I put forward that if we sampled all possible units, here: all U.S. state scores, then there is no probability for the statistical population of U.S. states (for that point in time). The sample (U.S. States) matches the population we wish to make statistical inferences about (U.S. states). If the observations we sample are from a finite population (U.S. states), this needs to be corrected for (Pollet, [@B7]). Once we have sampled the last U.S. state, the population pool is empty: we sampled and measured everything. Not explicitly defining the "population" does not alter this, nor does assuming that these state scores are part of a larger, potentially infinite, whole. Many authors, such as Quillien, might want to make statistical or logical inferences beyond these scores, for example to other macro-level units or the "human mind," but what we have in terms of data are U.S. state scores, plain and simple. These are by their very nature finite (fixed number of U.S. states). Quillien\'s argument thus seems to crucially rest on the claim that the observed unit of analysis is not a U.S. state score but rather something else. What this different unit of analysis would be is typically not clearly defined, neither by Quillien nor by the authors cited in Pollet ([@B7]: Table 1). For now, like Quillien, let us assume the unit of analysis is some (aggregate) human social unit relevant to some evolutionary process. Such a stance, i.e., the data representing something else than a U.S. state score, is in my view deeply problematic. Firstly, if one assumes the scores are something else, then one needs to explicitly define the unit of analysis *a priori*, otherwise it seems reasonable that the unit of analysis is indeed a U.S. state score. Let us tentatively define this alternative unit of analysis as "a social unit in which humans live(d) relevant to an evolutionary process." Why would a U.S. state then be representative of the pool of such social units in which humans lived? Perhaps these data can indeed tell us something about U.S. states, but it is unclear whether any documented statistical relationship would hold for other units fitting the broader definition. It is unclear whether any *statistical* inference can be done beyond U.S. states, as that is all we have. There is no logical reason to assume that any statistical relationship found for U.S. states should hold, for other "human social units," such as for example: world regions, Canadian provinces, Polynesian chiefdoms, 19th century German states, hunter gatherer populations,.... Let alone that these different "human social units" can be meaningfully lumped together and assumed to be governed by the same evolutionary process, as Quillien seems to imply. In addition, suppose that we do follow Quillien\'s logic and pretend the pool is larger, and even infinite, then a different problem still arises: we have clearly drawn a biased sample (Good and Hardin, [@B3]). "Traditional" statistical inference based on rejecting a null hypothesis in the population cannot be applied in the first place as there was no random sampling. For example, Canadian provinces are underrepresented, absent even, in our sample, while they are in our statistical population. Perhaps I am thus opposed to logical induction, and view moving from U.S. states to other not clearly defined "units" as problematic and Quillien does not. I will leave the reader to decide but it seems a much safer bet to stick to inferences about U.S. states, and not even rely on these data to make any inference on, for example, Canadian provinces. Ideally, researchers would then define and measure those, rather than assuming that one process must govern all these units. Finally, if we take these state level data to be representative of processes at an *individual* level ("the human mind"), as several authors including Quillien seem to suggest, then the ecological fallacy looms: inferences from one level of statistical analysis need not correspond to a different level of analysis (see Robinson, [@B11]; Freedman, [@B2]; Kuppens and Pollet, [@B5]; Pollet et al., [@B8]). These arguments have been made at length elsewhere and will not be reiterated here. In summary, in my view, the statistical inferences we can make based on units such as U.S. state scores can be about nothing else but U.S. states, as this is the *only* unit being sampled. One might want to strengthen the statistical and logical inference based on those data but this is likely invalid: either due to sampling bias and/or the ecological fallacy. I therefore maintain that the *p*-values commonly used for statistical inference are inappropriate for macro-level cross-cultural correlations when the sample matches the population closely. Conflict of interest statement ============================== The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. I am thankful to Gert Stulp, Toon Kuppens and Peter Dekker for helpful discussions on this topic. Any mistakes are obviously my own. [^1]: Edited by: Martin Fieder, University of Vienna, Austria [^2]: Reviewed by: Rosemary Hopcroft, University of North Carolina at Charlotte, USA; Georg Primes, University of Vienna, Austria [^3]: This article was submitted to Evolutionary Psychology and Neuroscience, a section of the journal Frontiers in Psychology

Introduction {#s1} ============ Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of dementia. AD affects 27 million people world-wide with steadily increasing numbers, thereby raising significant economic problems and tremendous personal suffer [@pone.0041457-Brookmeyer1]. The two pathological hallmarks that characterize AD are the presence of intracellular neurofibrillary tangles (NFTs) and extracellular neuritic plaques that can be found *post mortem* in the brains of patients [@pone.0041457-Braak1], [@pone.0041457-Braak2], [@pone.0041457-Selkoe1]. Neurofibrillary tangles consist of twisted filaments of hyperphosphorylated tau protein [@pone.0041457-Duff1], whereas plaques are primarily composed of amyloid-β (Aβ) [@pone.0041457-Selkoe1], [@pone.0041457-Dickson1], a 39--43 amino acid (aa) peptide derived from the amyloid precursor protein (APP) by proteolytic processing [@pone.0041457-Selkoe1], [@pone.0041457-Vetrivel1]. According to the amyloid cascade hypothesis (16), Aβ peptides and, more specifically, their aggregated forms initiate cellular events leading to the pathologic effects of AD [@pone.0041457-Haass1], [@pone.0041457-Hardy1]. *Pre mortem,* AD is usually diagnosed after the appearance of symptoms by application of tests for cognitive impairment like the mini-mental status examination (MMSE) or the Alzheimer's disease assessment scale (ADAS) [@pone.0041457-Folstein1], [@pone.0041457-Mohs1]. However, it is a great challenge to correctly diagnose AD at early presymptomatic stages [@pone.0041457-Ganguli1], [@pone.0041457-Knopman1], [@pone.0041457-Visser1]. Several publications support the finding that plaques start to accumulate 10 to 20 years before clinical symptoms appear, leading to substantial and progressive neuronal loss [@pone.0041457-Braak2], [@pone.0041457-Mintun1], [@pone.0041457-Price1]. Therefore, detection and quantitation of amyloid species in the brains of patients during the course of the disease for early diagnosis of AD and for monitoring AD-treatments is a promising and emerging field in AD research. An efficient tool for presymptomatic characterization of the brain may be imaging approaches making use of amyloid specific ligands and positron emission tomography (PET) [@pone.0041457-Silverman1] or single photon emission computed tomography (SPECT). Currently, only a few amyloid PET ligands have been applied in clinical studies (for review, see ref. [@pone.0041457-Nordberg1], [@pone.0041457-Nordberg2]). Numerous efforts are devoted to develop new, target-specific imaging agents for the detection of amyloid plaques *in vivo*. To be suitable, they should provide highly specific binding to Aβ aggregates, very selective labeling and efficient brain penetration. Moreover, imaging probes are desired with specificity for Aβ1--42 over other Aβ isoforms. The present study used a small, specific Aβ1--42 binding peptide comprising solely of D-enantiomeric amino acids, termed "D1" [@pone.0041457-vanGroen1], [@pone.0041457-Wiesehan1], [@pone.0041457-Wiesehan2], or alternatively, ACI-80 [@pone.0041457-JahanM1], [@pone.0041457-GulysB1]. ACI-80 was identified employing a mirror image phage display selection using aggregated Aβ1--42 as a target. *In vitro*, ACI-80 binds preferentially to aggregated Aβ1--42 with a K~D~ in the submicromolar range, whereas monomers are bound to a much less extent. In brain tissue sections derived from patients that suffered from AD, amyloid plaques and leptomeningeal vessels containing Aβ aggregates were stained specifically with a fluorescence-labeled derivative of ACI-80. Fibrillar deposits derived from other amyloidosis were not labeled by ACI-80 [@pone.0041457-Wiesehan1], [@pone.0041457-Wiesehan2], [@pone.0041457-Bartnik1]. We also demonstrated *in vivo* and *in vitro* that ACI-80 binds specifically to aggregated Aβ1--42 in the brains of APP/PS1 transgenic mice, where diffuse amyloid-β deposits, which do not contain Aβ1--42, were not stained [@pone.0041457-vanGroen1]. 10.1371/journal.pone.0041457.t001 ###### Pyroglutamate content of ACI-80, ACI-80-Kϕ and \[^127^I\]-ACI-80. {#pone-0041457-t001-1} ACI-80~solid~ ACI-80-Kϕ~solid~ \[^127^I\]-ACI-80~aqueous\ solution~ -------------------------------------------------- --------------- ------------------ -------------------------------------- Identified amino acid at the N-terminal position glutamine \>91.2% \>94.4% 62.6% pyroglutamate \<8.8% \<5.6% 37.4% Molecular weight 1421 Da 1907 Da 1548 Da Kϕ presents a lysine (K) linked to a fluorescein isothiocyanate (ϕ). Here, we investigate the properties of several derivatives of ACI-80. The novel compounds showed increased binding affinity and are promising candidates for further development into *in vivo* imaging compounds. Materials and Methods {#s2} ===================== Peptides {#s2a} -------- For the list of all investigated D-enantiomeric compounds see [Table 1](#pone-0041457-t001){ref-type="table"}. Aβ1--42 peptide was purchased as reversed phase high performance liquid chromatography purified product (JPT Biotech, Berlin, Germany; or Bachem AG, Bubendorf, Switzerland). Identity was confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). General Method for Synthesis of D-peptide Compounds {#s2b} --------------------------------------------------- The non-fluorinated peptides shown in [Table 1](#pone-0041457-t001){ref-type="table"} were synthesized by JPT Peptide Technologies GmbH, Berlin, Germany. 10.1371/journal.pone.0041457.t002 ###### List of investigated D-enantiomeric peptide compounds. {#pone-0041457-t002-2} Name of compound Amino acid sequence Modification --------------------- --------------------------------- ------------------------- ACI-80 QSHYRHISPAQV D1 ACI-80-Kϕ QSHYRHISPAQVKϕ D1-Kϕ ACI-87-Kϕ QSHYRHISPAQ**K**Kϕ D1-V12K-Kϕ \[^19^F\]-ACI-87-Kϕ QSHYRHISPAQ**K**K\[^19^F\]ϕ D1-V12K-K\[^19^F\]ϕ ACI-83- Kϕ **P**SHYRHISPAQVKϕ D1-Q1P-Kϕ ACI-89-Kϕ **P**SHYRHISPAQ**K-**Kϕ D1-Q1P-V12K-Kϕ \[^19^F\]-ACI-89-Kϕ **P**SHYRHISPAQ**K**K\[^19^F\]ϕ D1-Q1P-V12K-K\[^19^F\]ϕ ACI-86- Kϕ **P**S**F**YRHISPAQVKϕ D1-Q1P-H3F- Kϕ ACI-82- Kϕ SHYRHISPAQVKϕ D1-Q1X- Kϕ ACI-88-Kϕ SHYRHISPAQ**K**Kϕ D1-Q1X-V12K-Kϕ \[^19^F\]-ACI-88-Kϕ SHYRHISPAQ**K**K\[^19^F\]ϕ D1-Q1X-V12K-K\[^19^F\]ϕ ACI-85- Kϕ S**F**YRHISPAQVKϕ D1-Q1X-H3F-Kϕ ACI-81 **Z**SHYRHISPAQV D1-Q1Z ACI-81- Kϕ **Z**SHYRHISPAQVKϕ D1-Q1Z-Kϕ ACI-84- Kϕ **Z**S**F**YRHISPAQVKϕ D1-Q1Z-H3F-Kϕ Modifications in the original amino acid sequence of ACI-80 are printed in bold. Amino acid residues are given in the one-letter-code. All amino-acids are D-enantiomers. Kϕ presents a lysine (K) linked to a fluorescein isothiocyanate (ϕ). Synthesis of ^19^F-D-peptide Compounds {#s2c} -------------------------------------- \[^19^F\]-D-peptide synthesis was performed as described earlier [@pone.0041457-JahanM1]. Briefly, to an aqueous solution of peptide, borate buffer (0.5 M, pH 8.61) was added and the color of the solution changed from yellow to dark orange. Slightly excess amount of \[^19^F\]-*N*-succinimidyl−4-fluorobenzoate (SFB) in acetonitrile was added into this above solution and the reaction mixture was kept at RT for 10 min. The reaction was monitored by HPLC. The crude product was purified by an analytical HPLC column (3×300 mm, 10 µm, waters) using water with 0.1% (v/v) trifluoroacetic acid (TFA) and an acetonitrile (MeCN) gradient (20% to 50%, v/v) as eluent with a flow rate of 2 ml/min. Retention time of the three reference peptides were from 9 to 12 min at a wavelength at 234 nm. Then the product fraction was collected into a pre-filled slightly basic aqueous solution (40 ml, pH was adjusted by NaOH). This diluted fraction was passed through a C18 Sep-Pak plus cartridge (preconditioned with 10 ml ethanol +10 ml water) and the desired product was eluted with 1 ml of ethanol. The reference compounds were confirmed by LC-MS/MS. The purity and the stability of the products were checked by HPLC. 10.1371/journal.pone.0041457.t003 ###### Results of the binding assays for ACI-80-Kϕ derivatives to Aβ1--42 fibrils using surface plasmon resonance. {#pone-0041457-t003-3} Derivative/Modification Name Interaction \[%\] Dissociation \[%\] ------------------------- --------------------- ---------------------------------- -------------------- D1-Kϕ ACI-80-Kϕ used as standard and set to 100% D1-V12K-Kϕ ACI-87-Kϕ 324 443 D1-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-87-Kϕ 153 67 **D1-Q1P** **ACI-83** D1-Q1P-Kϕ ACI-83-Kϕ 600 200 D1-Q1P-V12K-Kϕ ACI-89-Kϕ 518 533 D1-Q1P-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-89-Kϕ 476 0 D1-Q1P-H3F-Kϕ ACI-86-Kϕ 21 40 **D1-Q1X** **ACI-82** D1-Q1X-Kϕ ACI-82-Kϕ 393 300 D1-Q1X-V12K-Kϕ ACI-88-Kϕ 365 667 D1-Q1X-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-88-Kϕ 294 1233 D1-Q1X-H3F-Kϕ ACI-85-Kϕ 229 40 **D1-Q1Z** **ACI-81** D1-Q1Z-Kϕ ACI-81-Kϕ 129 40 D1-Q1Z-H3F-Kϕ ACI-84-Kϕ 93 40 Interaction and dissociation was measured with respect to the maximal interaction signal during injection and the response 60 s after the end of injection, respectively. ACI-80-Kϕ binding was defined as 100%. Kϕ presents a lysine (K) linked to a fluorescein isothiocyanate (ϕ). ![Surface plasmon resonance analysis of the interaction between immobilized Aβ1--42 fibrils and ACI-80-Kϕ and various derivatives (Kϕ presents a lysine (K) covalently linked to a fluorescein isothiocyanate (ϕ).\ ACI-80 derivatives were solved in running buffer (PBS, pH 7.4). The injected volume of ACI-80 derivatives was 10 µl of a 50 µg/ml concentration using a flow rate of 5 µl/min. The response of ACI-80-Kϕ in resonance units \[RU\] was defined as 100%. Values \>100% denote increased Aβ interaction of the ACI-80 derivative in comparison to ACI-80-Kϕ. All derivatives were ϕ-labeled. Only the variations in comparison to ACI-80-Kϕ are indicated in the figure.](pone.0041457.g001){#pone-0041457-g001} Analysis of ACI-80 Stability {#s2d} ---------------------------- In solution, N-terminal glutamine peptides such as D1 are prone to convert into N-terminal pyroglutamate species: *Gln-SHYRHISPAQV → Pyr-SHYRHISPAQV*. Therefore, the stability of solid ACI-80, solid ACI-80-Kϕ (ϕ: fluorescein isothiocyanate, covalently linked to the peptide via a lysine (K)) and \[^127^I\]-ACI-80 in solution was investigated by JPT Peptide Technologies GmbH, Berlin, Germany using HPLC/ESI-MS whereby the relative content of N-terminal Gln peptide and N-terminal Pyr peptide was assessed. Surface Plasmon Resonance (SPR) {#s2e} ------------------------------- Aβ1--42 was dissolved in hexafluoroisopropanol (HFIP). After overnight incubation, HFIP was removed by evaporation. The Aβ1--42 film was dissolved in PBS buffer pH 7.4 to a concentration of 1 mg/ml and incubated for 7 days at 37°C. For the measurements, a Biacore 1000 (GE Healthcare) instrument was used. Aβ1--42 fibrils (6800 RU) were immobilized on a CM5 sensorchip (GE Healthcare) via amine coupling. The running and sample buffer was PBS, pH 7.4. To allow comparison between the ACI-80 compounds, but to avoid potential over-interpretation of the data by fitting multiple *k* ~on~ and *k* ~off~ values for a yet undefined number of different binding sites, only two values have been taken for further evaluation. Response units achieved under identical injection conditions allow comparison of binding strengths of the compounds. Dissociation rates among the compounds have been compared by measuring the remaining response units 60 s after end of injection as a measure for dissociation (*k* ~off~). All measurements have been carried out using the same flow cells with identical concentrations and injection conditions. ACI-80-Kϕ derivatives were injected as analytes in a concentration of 50 µg/ml at a flow rate of 5 µl/min for 2 minutes at ambient temperature. The data were evaluated using BiaEvaluation 4.1. The interactions between Aβ1--42 and ACI-80-Kϕ derivatives are given in resonance units (RU) and in % of ACI-80-Kϕ response units. 10.1371/journal.pone.0041457.t004 ###### ELISA: Mean binding values for compounds with concentration of 10 µg/ml. {#pone-0041457-t004-4} Modification Nomenclature Binding values and % binding relative to ACI-80-Kϕ for 10 ug/mL compoundconcentration ------------------------- --------------------- --------------------------------------------------------------------------------------- ----- -------- ----- D1-Kϕ ACI-80-Kϕ 0.49 100 0.90 100 D1-V12K-Kϕ ACI-87-Kϕ \*0.79 122 \*0.93 103 D1-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-87-Kϕ \*0.86 176 \*1.26 140 D1-Q1Z-Kϕ ACI-81-Kϕ 0.13 27 0.20 22 D1-Q1Z-H3F-Kϕ ACI-84-Kϕ 0.31 63 0.58 64 D1-Q1X-Kϕ ACI-82-Kϕ 0.82 167 1.28 142 D1-Q1X-H3F-Kϕ ACI-85-Kϕ 0.63 129 1.00 111 D1-Q1X-V12K-Kϕ ACI-88-Kϕ \*1.29 263 \*1.78 198 D1-Q1X-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-88-Kϕ \*1.05 214 \*1.76 196 D1-Q1P-Kϕ ACI-83-Kϕ 1.05 214 1.50 167 D1-Q1P-H3F-Kϕ ACI-86-Kϕ 0.80 163 1.24 138 D1-Q1P-V12K-Kϕ ACI-89-Kϕ \*1.06 216 \*1.49 165 D1-Q1P-V12K-K\[^19^F\]ϕ \[^19^F\]-ACI-89-Kϕ \*1.19 243 \*1.86 207 All values were compared to that of ACI-80-Kϕ. Compound binding to compound film, containing predominantly monomers and to fibrils was measured. Average values of two or three experiments unless marked otherwise. \*value of one single experiment only. Kϕ presents a lysine (K) linked to a fluorescein isothiocyanate (ϕ). {#pone-0041457-g002} Surface Plasmon Resonance: Single Cycle Experiments {#s2f} --------------------------------------------------- Aβ1--42 fibrils were prepared as described above. Thereafter, the sample was centrifuged for 10 min at 16000×g, the supernatant discarded and this procedure repeated for 3 times. Formation of fibrils was confirmed by a standard Thioflavin-T fluorescence assay [@pone.0041457-vanGroen2]. Aβ1--42 fibrils were covalently immobilized on a CM5 sensor chip via amine coupling. Prior to immobilization of Aβ1--42 fibrils the sample was centrifuged and fibrils resuspended in 10 mM sodium acetate buffer pH 4.0. Flow cell sensor surfaces were activated with a freshly prepared solution of 0.2 M 1-ethyl--3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.05 M N-hydroxysuccinimide (NHS) at a constant flow rate of 10 µl/min for 420 s. Aβ1--42 fibrils (∼ 110 µM monomeric Aβ1--42) were injected for 600 s with a flow rate of 10 µl/min. Deactivation of the surface was performed by injection of 1 M ethanolamine-HCl pH 8.5 at the same flow rate and duration as in the activation step. In the reference cells the deactivation step was performed directly after the activation step. {#pone-0041457-g003} 10.1371/journal.pone.0041457.t005 ###### Results for compound -- Aβ fibril interactions obtained with the heterogeneous ligand model. {#pone-0041457-t005-5} Analyte R~max~1 R~max~2 *k* ~on~1 *k* ~off~1 *k* ~on~2 *k* ~off~2 *K* ~D~1 *K* ~D~2 --------------- --------- --------- ----------- ------------ ----------- ------------ ---------- ---------- **ACI-80-Kϕ** 12.2 43.7 469 0.0252 1.37e4 1.58 5.38e-05 1.15e-04 **ACI-87-Kϕ** 43.3 36.2 206 1.84e-3 1.57e3 0.0407 8.93e-06 2.59e-05 **ACI-88-Kϕ** 9.96 63.3 1.71e4 7.22e-3 930 0.0553 4.22e-07 5.95e-05 **ACI-89-Kϕ** 21.8 19.4 2.03e4 0.162 3.34e3 0.0118 8.00e-06 3.53e-06 All SPR experiments were performed on a Biacore T100 system with series S CM5 sensor chips at 25°C. The system was run with the Biacore T100 Control Software Version 1.1.1. PBS (10 mM sodium phosphate buffer pH 7.4, 137 mM NaCl, 2.7 mM KCl) was chosen as running buffer (as previously during fibril formation) in order to minimize alterations of fibril organization. All buffers were sterile filtered (0.22 µm). After each docking of a sensor chip the detector was normalized with BIAnormalizing solution (70% glycerol, GE Healthcare) to compensate for slight differences in detector responses of individual sensor chips. For all interaction analyses the Type 1 reagent rack was used. Siliconized sample vials were used with their corresponding rubber caps (Type 2, GE Healthcare) to minimize evaporation effects. Throughout all runs the flow rate was set to 30 µl/min. All interaction studies were performed in single-cycle mode [@pone.0041457-Karlsson1]. Here, five different concentrations of analyte were passed through a reference cell and subsequently through flow cells with immobilized ligand within the same binding cycle for 60 seconds, starting with the lowest concentration. Successive injections were performed in the order of increasing concentrations. Each following concentration was a fivefold increase of the previous. The lowest analyte concentration was chosen to be 100 nM and therefore the following were 500, 2500, 12500 and 62500 nM. {#pone-0041457-g004} Biacore data were evaluated using BiaEvaluation 4.1.1 (GE Healthcare) and Biacore T100 Evaluation Software (GE Healthcare). Obtained binding data with compounds were double referenced. This was achieved by collecting the data in dual-channel mode with a reference flow cell connected upstream of the flow cell with immobilized Aβ1--42 fibrils and the subtraction of the obtained binding responses with a blank buffer injection (PBS). The double-referenced binding curves of the three lowest concentrations of each single cycle kinetics injection that showed a significant binding response were fit to a heterogeneous ligand binding model [@pone.0041457-Morton1] including a factor correcting for different refractive indices (R~I~). {#pone-0041457-g005} Enzyme-linked Immunosorbent Assays (ELISA) {#s2g} ------------------------------------------ ### Preparation of Aβ1--42 species {#s2g1} Aβ1--42 peptide film was prepared from lyophilized powder (Bachem). The powder was reconstituted in HFIP to a final concentration of 1 mM, sonicated for 15 min at room temperature (RT), agitated overnight (ON), and aliquoted into non-siliconized microcentrifuge tubes (12 µl corresponding to 55 µg). The HFIP was evaporated under a stream of argon. The resulting peptide film was vacuum dried for 10 min and stored at -80°C. For direct use as peptide film, an aliquot of Aβ1--42 peptide film was reconstituted with 0.54% dimethylsulphoxide (DMSO) in phosphate buffered saline (PBS) to obtain a final concentration of 10 µg/ml and then used for the ELISA as described below. To prepare fibrils, 55 µg aliquot of peptide film was dissolved in 55 µl of 50 mM Tris-HCl, pH 7.4 and incubated at 37°C for five days. Next, the sample was centrifuged (10'000 rpm for 5 min) and the pellet was diluted in 50 mM Tris-HCl, pH 7.4. D-peptide Compound Binding to Immobilized Aβ1--42 {#s2h} ------------------------------------------------- Aβ1--42 peptide preparations were diluted in 0.05 M bicarbonate-carbonate buffer pH 9.6, to the final concentration of 10 µg/ml and coated onto ELISA plates (MaxiSorp, Nunc). After blocking (PBS; 0.05% Tween; 1% BSA), plates were incubated with 2- or 3-fold dilutions of D-compounds (starting concentration: 10 µg/ml) and incubated for 2 h at 37°C. Plates were then washed and incubated for 2 h at 37°C with the detection antibody Rabbit-a-fluorescein isothiocyanate-AP (Sigma; 1∶10'000 dilution) followed by the incubation for 2.5 h at room temperature (RT) with 1 mg/ml of phosphatase substrate (pNPP, Sigma). The absorbance signal was read at 405 nm wavelength using a Tecan plate reader (Tecan Group Lt, Männedorf, Switzerland). D-peptide Compound Binding to Aβ1--42 in Solution {#s2i} ------------------------------------------------- ELISA plates (MaxiSorp, Nunc) were coated with anti-Aβ antibody 6E10 (Covance) at a concentration of 5 µg/ml. Either monomeric Aβ1--42 peptide film (mainly monomeric) or Aβ1--42 fibrils, which were prepared as described above, were diluted in PBS to the final concentration 10 µg/ml and mixed with 10-fold dilutions of D-peptide compounds in Eppendorf tubes (starting concentration of D-peptide compounds: 10 µg/ml). The tubes were incubated for 2 h at 37°C. Next, samples were distributed onto the ELISA plate and kept for 2 h at 37°C. Plates were washed and incubated for 2 h at 37°C with the detection antibody Rabbit-a-fluorescein isothiocyanate-AP (Sigma; 1∶10′000 dilution) following the incubation overnight (ON) at RT with 1 mg/ml of phosphatase substrate (pNPP, Sigma). The absorbance signal was read at 405 nm wavelength using the Tecan plate reader (Tecan Group Ltd, Männedorf, Switzerland). Ex vivo Staining of Mouse Brain Slices by FITC Labeled Compounds {#s2j} ---------------------------------------------------------------- *In vitro* tissue section staining was performed according to previously described protocols [@pone.0041457-vanGroen1] with slight modifications. Mouse brains were obtained from male transgenic (tg) APP (London mutation V717l) x PS1 (A246E) mice aged 13--21 months and from female wild type (wt) mice aged 9--10 months [@pone.0041457-Dewachter1]. The mice were anesthetized and transcardially perfused with saline. The brains were removed and snap frozen. 10 µm thick sagittal cryostat sections through the whole mouse brain were produced and mounted onto glass slides. ϕ-labeled compounds were applied to investigate binding. Thus, the slides were thawed, washed in PBS and fixed in 4% paraformaldehyde (20 min at RT) just before incubation with ϕ-labeled compounds. One series of sections was treated only with fluorescent compounds (0.01 mg/ml, incubation time 2 h) while another series of sections were incubated with anti-Aβ antibody 6E10 at 1∶500 dilution (SIG-39320, Covance; final antibody concentration was 2 µg/ml) in addition to ϕ-labeled compounds (0.01 mg/ml). The slices were washed in PBS. The sections incubated with ϕ-labeled compound and 6E10 antibody were further incubated for 2 h at RT with Goat-anti-Mouse IgG1-- AlexaFluor 555 (A21127, Invitrogen, 1∶1000 dilution) and washed with PBS. Finally, all sections were counterstained with 4'--6-Diamidino-2-phenylindole (DAPI, 32670, Sigma, incubation 5 min at RT) for the visualization of cell nuclei. The slides were mounted using Prolong Gold Antifade mounting medium (P36930, Invitrogen) and coverslipped. The tissues were analyzed using a fluorescent Zeiss Axioscope 2 Plus microscope using the AxioVision 4 image analysis software. A further series of slices was stained using a primary anti-fluorescein isothiocyanate antibody combined with alkaline phosphatase reaction for visualization of ϕ-labeled peptides. Briefly, the sections were treated with blocking solution (10% normal goat serum (NGS), 0.25% Triton X-100 in PBS for 1 h at RT) and incubated with primary antibody (rabbit-anti-FITC; Invitrogen) at a dilution of 1∶500. The sections were then washed and incubated for 2 h at RT with the secondary antibody goat-anti-rabbit-alkaline phosphatase (Sigma) at a dilution of 1∶100. After washing, the slides were incubated with BCIP/NBT substrate (Sigma) for 3 min, washed, dehydrated and mounted using Eukitt mounting medium. Results {#s3} ======= Analysis of ACI-80 Amino-terminal Residue Identity {#s3a} -------------------------------------------------- An important observation during initial compound stability characterization experiments was the partial conversion of the N-terminal glutamine of \[^127^I\]-ACI-80 into pyro-glutamate. It is known that peptides with an N-terminal glutamine are prone to conversion of this residue into a pyroglutamate [@pone.0041457-Abraham1]. Therefore, the composition of freshly synthesized and untreated ACI-80 and ACI-80-Kϕ as well as \[^127^I\]-ACI-80 after iodination were investigated. The results are displayed in [Table 1](#pone-0041457-t001){ref-type="table"}. It was observed that freshly synthesized ACI-80 and ACI-80-Kϕ contained only minor fractions of pyroglutamic acid while about one third of \[^127^I\]-ACI-80 in solution already converted into N-terminal pyroglutamate species. Thus, a number of novel ACI-80-derivatives were designed and synthesized with one or several amino acid deletions and/or substitutions with the aim to increase compound stability, but also Aβ binding capability. Specifically, the N-terminal glutamine was either substituted by pyroglutamate (Q1Z) or proline (Q1P) or deleted (Q1X). Furthermore, a replacement of His-3 by phenylalanine (H3F) was investigated. The Q1X deletion as well as Q1P and H3F substitutions have been proposed based on semiquantitative saturation mutagenesis peptide spot data, which predicted that the mutations would enhance Aβ binding. In the peptide spot approach, all amino-acids of ACI-80 were substituted against all other natural amino acids and the variants were tested for their ability to bind Aβ fibrils (data not shown). In addition valine was replaced by lysine at position 12 (V12K) to enable fluorination of the compound for imaging purposes. Finally, the compound was also fluorescein isothiocyanate (ϕ)-labeled via an additional C-terminal lysine residue to enable detection through fluorescence or via anti-fluorescein isothiocyanate antibodies. For a summary of all ACI-80 derivatives see [Table 2](#pone-0041457-t002){ref-type="table"}. Surface Plasmon Resonance and ELISA {#s3b} ----------------------------------- To characterize the ACI-80 derivatives in respect to their Aβ binding capabilities in comparison to ACI-80-Kϕ, a number of *in vitro* assays were performed. Surface plasmon resonance (SPR) assays were performed to analyze their interaction with immobilized Aβ1--42 fibrils ([Table 3](#pone-0041457-t003){ref-type="table"}). Aβ1--42 fibrils were immobilized on a Biacore sensor-chip as described in the methods section, and the interactions of the ϕ-labeled ACI-80 derivatives were measured and compared with original ACI-80-Kϕ. Due to the fact that Aβ1--42 fibrils represent an inhomogeneous mixture of different fibril aggregates, it is hardly possible to form a homogenous Aβ1--42 loaded SPR chip surface. Therefore we decided for semi-quantitative comparison between all compounds only. The maximal responses in resonance units (RU) of the ϕ-labeled ACI-80 derivatives during the analyte injection and the remaining response 60 s after injection end (as a semi-quantitative measure for the dissociation rate), have been related to the respective values obtained from identical concentrations of ACI-80-Kϕ in percent. The results are summarized in [Table 3](#pone-0041457-t003){ref-type="table"} and [Figure 1](#pone-0041457-g001){ref-type="fig"}. ACI-80-Kϕ derivatives ACI-82-Kϕ and ACI 83-Kϕ (aa modifications Q1X and Q1P) yielded an increased response of up to 600% as compared to ACI-80-Kϕ ([Table 3](#pone-0041457-t003){ref-type="table"}). The response for ACI-81-Kϕ (aa substitution Q1Z) was not significantly increased. The substitution at the third amino acid position (H3F) did not lead to improved binding to Aβ. While ACI-84-Kϕ behaved slightly worse than ACI-81-Kϕ, the binding efficiencies of ACI-85-Kϕ and ACI-86-Kϕ were drastically reduced as compared with those of ACI-82-Kϕ and ACI-83-Kϕ, respectively. The single substitution at aa position 12, valine to lysine (ACI-87-Kϕ), resulted in a response increase to more than 300%. Compounds ACI-88-Kϕ and ACI-89-Kϕ with two substitutions, the first one at amino acid position 1 (Q1X, Q1P) and the second one at amino acid position 12 (V12K), yielded a response increase to 350% (Q1X-V12K) and more than 500% (Q1P-V12K). The substitution V12K had an important impact with respect to dissociation. All three compounds with a V12K substitution showed decreased dissociation rates (increased remaining response 60 s after injection end) as compared to the compounds without the V12K substitution. Therefore, ACI-87-Kϕ, ACI-88-Kϕ, and ACI-89-Kϕ were the most promising compounds for further studies. In order to investigate, whether \[^18^F\]-labeling will change their binding affinities, the respective \[^19^F\]-labeled compounds have been investigated as well. Except for \[^19^F\]-ACI-87-Kϕ (Q1X-V12K), which showed a decreased response in comparison to the compound without label, the \[^19^F\]-labeled compounds showed virtually the same behavior with respect to maximum binding. The remaining response 60 s after injection end, however, was significantly changed in all three cases. Based on the remaining response 60 s after injection end, \[^19^F\]-ACI-88-Kϕ exerted the slowest off-rate of all tested compounds indicating the most suitable binding behavior for being used as a molecular probe. To verify the SPR results, ELISA was employed to assay the interaction of the ACI-80 derivatives ACI-80-Kϕ, ACI-81-Kϕ, ACI-82-Kϕ, ACI-83-Kϕ, ACI-84-Kϕ, ACI-85-Kϕ, ACI-86-Kϕ, ACI-87-Kϕ, \[^19^F\]-ACI-87-Kϕ, ACI-88-Kϕ, \[^19^F\]-ACI-88-Kϕ, ACI-89-Kϕ, \[^19^F\]-ACI-89-Kϕ) with Aβ1--42 fibrils and Aβ peptide film in solution containing mostly monomers and smaller oligomers ([Table 4](#pone-0041457-t004){ref-type="table"}). To avoid any bias by possible conformational influences the ELISA has been carried out in two versions, once with immobilized antibody and once with immobilized Aβ1--42 as described in the methods section. Both assays yielded similar results and confirmed each other. All experiments were performed twice and showed reproducible results. Panels A and B in [Figure 2](#pone-0041457-g002){ref-type="fig"} show the results for binding to Aβ1--42 fibrils and panels C and D in [Figure 2](#pone-0041457-g002){ref-type="fig"} show the results for binding to freshly prepared Aβ1--42 peptide film, which contains mostly monomeric Aβ. In [Table 4](#pone-0041457-t004){ref-type="table"}, the performance of the peptides is expressed relative to that of ACI-80-Kϕ. Briefly summarized, the binding of the ACI-80 derivatives to Aβ1--42 fibrils was generally stronger than that to monomer-enriched freshly prepared Aβ1--42. In addition, for both Aβ species a similar order of binding strengths could be established. All variants, except ACI-81-Kϕ and ACI-84-Kϕ, showed stronger Aβ binding than ACI-80-Kϕ. Substitution of glutamine to proline at position 1 or glutamine deletion had a positive effect on binding to Aβ1--42. Inversely, binding to Aβ1--42 was reduced for ACI-84-Kϕ as compared to ACI-80-Kϕ. ACI-81-Kϕ and ACI-84-Kϕ are peptides with glutamine to pyroglutamate substitution. Thus, the substitution of glutamine to pyroglutamate decreased binding to Aβ1--42. An order of binding comparing fluorinated with respect to non-fluorinated D-peptides versions was difficult to establish. The results clearly show that the fluorinated ϕ-labeled peptides \[^19^F\]-ACI-87-Kϕ, \[^19^F\]-ACI-88-Kϕ, \[^19^F\]-ACI-89-Kϕ bound well to Aβ1--42 fibrils, \[^19^F\]-ACI-88-Kϕ, \[^19^F\]-ACI-89-Kϕ being even among the very best of all variants. The order of binding strengths for the fluorinated peptides was \[^19^F\]-ACI-89-Kϕ binds stronger than \[^19^F\]-ACI-88-Kϕ much stronger than \[^19^F\]-ACI-87-Kϕ. These peptides with \[^19^F\] replaced by \[^18^F\] were used in autoradiography assays for testing the binding to human Alzheimer's brain tissue sections [@pone.0041457-GulysB1]. Briefly, the experiments, using *post mortem* human brain autoradiography in whole hemisphere human brains obtained from deceased AD patients and age matched control subjects, support the visualization capacity of the radiolabeled ACI-80 analogues of amyloid deposits in the human brain [@pone.0041457-GulysB1]. In general, the ELISA results corresponded well with the SPR results identifying the substitution of glutamine to proline or glutamine deletion at the N-terminus as useful mutation with strong benefits for the binding to Aβ fibrils and monomers. As the compounds ACI-87-Kϕ, ACI-88-Kϕ, ACI-89-Kϕ were the most promising candidates for further development, we tried to obtain more quantitative binding data and compared them to ACI-80-Kϕ. Different concentrations of the analytes were applied to an SPR chip loaded with Aβ fibrils ([Fig. 3](#pone-0041457-g003){ref-type="fig"}). A brief look at the data already revealed that about a five-fold concentrations of ACI-87-Kϕ and ACI-80-Kϕ were necessary to obtain comparable RU responses as compared to ACI-88-Kϕ and ACI-89-Kϕ. All three ACI-80 derivatives showed tighter binding to fibrils as compared to ACI-80-Kϕ. As already mentioned above, any effort to obtain an exact quantitative analysis of experimental data from SPR experiments with Aβ fibrils is prone to mis- and over-interpretation. We found, however, that the heterogeneous ligand binding model (see methods section for details) was able to yield potentially meaningful results for all four compounds without introducing too many fit parameters. With all necessary caution and taking into account only the lowest *K* ~D~ value of the two obtained from the heterogeneous binding model, the fitted *K* ~D~ values given in [Table 5](#pone-0041457-t005){ref-type="table"} confirm the order of binding: with an obtained dissociation constant in the submicromolar range ACI-88-Kϕ binds stronger than ACI-89-Kϕ and ACI-87-Kϕ, and all three of them bind stronger than ACI-80-Kϕ. Ex vivo Staining of Mouse Brain Slices by FITC Labeled Compounds {#s3c} ---------------------------------------------------------------- Brain sections from transgenic (tg) APP (London mutation V717l) x PS1 (A246E) mice were stained using ACI-80-Kϕ, ACI-87-Kϕ, ACI-88-Kϕ, ACI-89-Kϕ. Also, anti-Aβ plaque staining (using the 6E10 antibody) and DAPI nuclei counterstaining was performed on the same slides. Photomicrographs of the stained slices and triple overlay images are shown in [Figure 4](#pone-0041457-g004){ref-type="fig"}. Aβ plaques were identified in all tg animals using 6E10 anti-Aβ antibody. Moreover, plaques were stained by all tested compounds, although to different extent and with different intensity and background. Specifically, ACI-89-Kϕ and ACI-88-Kϕ stained plaques most intensively, whereas ACI-88-Kϕ additionally yielded the lowest background signal of all peptides. As shown in [Figure 4](#pone-0041457-g004){ref-type="fig"}, ACI-89-Kϕ and ACI-88-Kϕ match 6E10 anti-Aβ staining quite well giving rise to a large extent of overlay in the triple exposure. For the peptide ACI-87-Kϕ, nearly no overlay with 6E10 anti-Aβ was detected by means that only a very small fraction of the plaques identified with anti-Aβ antibody 6E10 have been stained with the D-peptide. The detection of overlay by the eye is additionally hindered by high background fluorescence. Moreover, ACI-89-Kϕ and ACI-88-Kϕ showed a slightly different staining pattern by means that ACI-88-Kϕ stained the core of plaques while ACI-89-Kϕ has a staining pattern which is more similar to the one of 6E10 which also stains diffuse Aβ plaques. Therefore, the qualitative assessment demonstrates that the peptides are able to recognize plaques in tg mouse brains with different intensities and background signals. Overall, \[^19^F\]-ACI-89-Kϕ and ACI-88-Kϕ showed a good overlap with 6E10 Aβ staining and have thus confirmed their leading roles as candidates for further evaluation. To further proof with a fluorescent independent read-out that ACI-89-Kϕ binds to plaques after peripheral injection, an antibody recognizing the FITC group in ACI-89-Kϕ was used ([Figure 5](#pone-0041457-g005){ref-type="fig"}). This antibody binds to ϕ-labeled peptides that are bound to Aβ plaques in ϕ-peptide immersed tg mouse brain slices. The alkaline phosphatase reaction showed abundant chromogenic deposits resembling the expected distribution and number of Aβ plaques in these brains identified by ACI-89-Kϕ. Thus confidence was provided that ACI-89-Kϕ binds to brain Aβ plaques following peripheral application. Discussion {#s4} ========== One of the hallmarks of AD is Aβ accumulation in plaques, probably long time before manifestation of clinical symptoms. Here, we have characterized novel ligands that we believe can have the potential to be used for diagnostic imaging in patients with AD and also in individuals that score as MCI. There is strong demand for imaging probes that allow early diagnosis of the disease, thus enabling novel therapies that allow early intervention. Also such probes will be important to monitor disease progression and therapy success in longitudinal studies. The ligands also have the potential to be used for PET imaging, for example in transgenic mouse models, that overexpress the amyloid precursor proteins and develop amyloid plaques, or in aged monkeys. Imaging of parenchymal Aβ plaques, which mainly consist of the isoform Aβ1--42 in both, transgenic animals and humans, heavily relies on molecular probes that are specifically binding to Aβ1--42 fibrils. In order to discriminate between the two most relevant Aβ depositions in AD, namely vascular Aβ, which mainly consists of Aβ1--40, and parenchymal Aβ, which mainly contains Aβ1--42, there is an urgent need for such a specific PET ligand, as it is not clear if the currently most advanced \[^11^C\]-PIB-PET compound discriminates between Aβ1--40 and 1--42 *in vivo* [@pone.0041457-Svedberg1]. The lead compounds ACI-87-Kϕ, ACI-88-Kϕ and ACI-89-Kϕ of this program were derived from ACI-80, which is a D-enantiomeric, 12 amino acid peptide that originally was selected by mirror-image phage display (24). D-peptides have several advantages over L-enantiomeric peptides. Most importantly, they are resistant to most proteases [@pone.0041457-Milton1], which can dramatically increase serum [@pone.0041457-Sadowski1] and saliva [@pone.0041457-Wei1] half-life. The need for exploring derivatives of ACI-80 was dictated by the observation that the N-terminal amino acid residue of ACI-80 converted from glutamine to pyroglutamate in aqueous solution. In addition, ACI-80 derivatives with increased binding affinity to aggregated Aβ species were desirable. The lead compounds ACI-87-Kϕ, ACI-88-Kϕ and ACI-89-Kϕ were stable in aqueous solution and showed even superior Aβ binding characteristics as compared to ACI-80-Kϕ. This was confirmed by ELISA and SPR *in vitro* binding assays. The ELISA results were fully compatible with the results from SPR. In general, a stronger binding of ACI-80-Kϕ and its derivatives to aggregated Aβ forms, in comparison to monomeric forms, could be verified by ELISA. This is in accordance to the observation previously reported for ACI-80 [@pone.0041457-Bartnik1]. Whether the ACI-80 derivatives also inherited the ACI-80 property to preferentially bind Aβ1--42 over Aβ1--40, was not investigated in the present study. All *in vitro* binding data agree that Q1X and Q1P mutations lead to an increase of binding and a decrease of dissociation rate, whereas the H3F mutation led to a decrease in binding. In line with the SPR results, the ELISA data confirmed that the substitution of glutamine to proline and the glutamine deletion increased binding to Aβ1--42 whereas the substitution of glutamine to pyroglutamate decreased binding to Aβ. Also, fluorinated, ϕ-labeled D-compounds bound well to Aβ fibrils. The SPR measurements that were carried out to compare the binding capabilities of ACI-80-Kϕ, ACI-87-Kϕ, ACI-88-Kϕ and ACI-89-Kϕ to Aβ-fibrils once more confirmed the binding order: ACI-88-Kϕ binds stronger than ACI-89-Kϕ stronger than ACI-87-Kϕ much stronger than ACI-80-Kϕ. Although not all of the fitted binding curves do perfectly fit to the experimental data, the applied evaluation procedure yielded some values for binding affinities that allowed comparison between the four compounds. *Ex vivo* staining of transgenic mouse brains showed that the FITC labeled compounds ACI-87-Kϕ, ACI-88-Kϕ and ACI-89-Kϕ and their fluorinated derivatives \[^19^F\]-ACI-87-Kϕ, \[^19^F\]-ACI-88-Kϕ, and \[^19^F\]-ACI-89-Kϕ readily recognized amyloid plaques in the mouse brain sections. This is important evidence that these compounds can be used to monitor therapy progress in AD mouse models. Interestingly, \[^19^F\]-ACI-89-Kϕ showed a different staining pattern in comparison to the other compounds, being more diffuse and comparable to the staining of 6E10 Aβ antibody. As already described in two other reports [@pone.0041457-JahanM1], [@pone.0041457-GulysB1] positive autoradiography (ARG) signals, compatible with Aβ staining, have been found in cortical gray matter using \[^18^F\]-ACI-87-Kϕ, \[^18^F\]-ACI-88-Kϕ, \[^18^F\]-ACI-89-Kϕ, \[^125^I\]-ACI-80 and \[^125^I\]-ACI-80-Kϕ ARG on human whole hemisphere brain sections of patients with AD. Brain sections from non-Alzheimer's control subjects were significantly less stained in the cortical gray matter, underpinning the specificity of the ARG signal. In conclusion, especially the ACI-80 derivatives ACI-87-Kϕ, ACI-88-Kϕ and ACI-89-Kϕ show superior binding affinities and specificities suggesting them as potential probes for specific Aβ aggregate and plaque detection in the living brain. **Competing Interests:**K.P. is employee of AC Immune. U.W. was employee of AC Immune. A.M. serves as Chief Scientific Officer, A.P. as Chief Executive Officer of AC Immune. C.S. serves as Chief Executive Officer of Prodema Management AG. This does not alter the authors\' adherence to all the PLoS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare. The other authors have declared that no competing interests exist. **Funding:**The authors have no support or funding to report. [^1]: Conceived and designed the experiments: SAF AP CS AM DW. Performed the experiments: DB JMG KP UW KW. Analyzed the data: DB JMG KP UW BG CH DW. Wrote the paper: SAF CS AP CS AM DW.

**To the Editor:** The utility of additional fixations for sliding hip screws has been well demonstrated in the study entitled "Additional fixations for sliding hip screws in treating unstable pertrochanteric femoral fractures (AO type 31-A2): short-term clinical results."[@B1] I have read with great interest the recent study, "The effect of positive medial cortical support in reduction of pertrochanteric fractures with posteromedial wall defect using a dynamic hip screw," by Cho et al.[@B2] While I appreciate citation of my paper in this article, I have some concerns as follows. First, reasoning from Table 3 of Ref. 2 and the total number of enrolled cases, I can conclude that all the cases were additionally fixed with either wire or trochanter stabilizing plate (TSP), or both: 26 cases were fixed with a wire alone; 72, with TSP alone; and 2, with both wire and TSP. Therefore, no use of wire for fragment fixation (n = 72) refers to cases with TSP alone and no use of TSP (n = 26) refers to cases with wire alone. Second, the authors[@B2] divided 100 cases into three groups by intraoperatively assessing the status of medial cortical support after reduction. In group 2, 28 of 42 cases did not undergo additional posteromedial fixation, so they could be presumed to have undergone TSP because all the cases were additionally fixed with either wire or TSP, or both. The other 14 cases underwent additional posteromedial wire fixation. The authors[@B2] stated that "Patients with additional posteromedial fixation showed significantly lower lag screw sliding (*p* = 0.02) 'than patients with TSP' might have been omitted." They consequently stated that cases with wire fixation showed significantly lower lag screw sliding than cases with TSP because there were no cases without any additional fixation (wire or TSP) and posteromedial fixation meant wiring. By the way, the authors described the effect of neck-shaft angle and lag screw sliding contingent upon the use of TSP or wire fixation of posteromedial cortex as statistically null in page 295 of Ref. 2. Therefore, they made inconsistent statements. Did they imply "no" statistical significance in the overall comparison of three groups while some statistical significance in the comparison of between two groups? This was not clearly described in the article and it does not make sense to me that there was no difference in the effect of posteromedial wiring and TSP, let alone the medial cortical variance, on the sliding distance and neck shaft angle. The effect of posteromedial wire fixation on sliding distance or neck-shaft angle should be studied between one group with it and the other group without it. It should not be studied by comparing with a group with TSP fixation because the effect of TSP should also be considered. **CONFLICT OF INTEREST:** No potential conflict of interest relevant to this article was reported.

Introduction ============ One of the fundamental questions in developmental neurobiology centers on the development of various cognitive functions and how such development derails in mental disorders. The capacity of many cognitive functions develops from childhood to adulthood.^[@bib1],\ [@bib2],\ [@bib3]^ Both cognition and working memory start to deteriorate at adolescence and predicts the onset of later schizophrenia.^[@bib4],\ [@bib5],\ [@bib6],\ [@bib7],\ [@bib8],\ [@bib9],\ [@bib10]^ The developmental maturation of working memory starts to lag during a period from adolescence to adulthood in individuals with autism spectrum disorder (ASD).^[@bib11],\ [@bib12]^ Working memory correlates to IQ^[@bib13],\ [@bib14],\ [@bib15]^ and is less developed, throughout development and in adulthood, in individuals with intellectual disabilities (ID).^[@bib16],\ [@bib17],\ [@bib18],\ [@bib19]^ Duplication, triplication and hemizygous deletion of a few hundred kb to a few Mb, collectively termed copy number variants, confer the most robust risk factors, to date, for developmental delays in cognitive function and developmental neuropsychiatric disorders.^[@bib20],\ [@bib21],\ [@bib22]^ Carriers of a 1.5 Mb to 3 Mb duplication or triplication at human chromosome 22q11.2 exhibit high rates of developmental delays in cognitive capacities^[@bib23],\ [@bib24],\ [@bib25]^ and ASD and ID.^[@bib26]^ Duplication of this chromosomal locus is also enriched in individuals with ASD and ID.^[@bib20],\ [@bib21],\ [@bib22]^ However, the precise genetic elements within 22q11.2 responsible for atypical cognitive development remain elusive due to their large duplication sizes in humans. Using mouse models with constitutive overexpression of small 22q11.2 segments,^[@bib27],\ [@bib28]^ we showed that an arrest in developmental maturation of working memory capacity is recapitulated by copy number elevation of a 190 kb region, including human catechol-*O*-methyl-transferase (*COMT*), *TXNRD2* and *ARVCF*.^[@bib29]^ Moreover, constitutive copy number elevation of another 200 kb 22q11.2 segment, containing human *SEPT5*, *GP1BB*, *TBX1* and *GNB1L*, resulted in impaired social behaviors and compulsively repetitive behaviors, the latter of which were attenuated by an antipsychotic drug, suggesting relevance of this chromosomal segment to developmental neuropsychiatric disorders.^[@bib30]^ Among these 22q11.2-encoded genes, a high activity single nucleotide polymorphism of *COMT* is associated with poor working memory after, but not before, 10 years of age, compared with a low activity *COMT* allele in humans.^[@bib31]^ Moreover, a gain-of-function mutation of *TBX1* has been identified in individuals with developmental delays in cognition.^[@bib32],\ [@bib33]^ Overexpression of no other 22q11.2 genes has been shown to impair working memory. However, mutation carriers also have other copy number variants and mutations^[@bib20],\ [@bib34]^ and the impacts of single nucleotide polymorphisms are often inconsistent, presumably due to their weak effect sizes^[@bib35]^ in humans. While *Comt* and *Tbx1* contribute to working memory and other ASD-related behavioral phenotypes in mice,^[@bib36],\ [@bib37],\ [@bib38]^ the precise neuronal and cellular mechanisms through which elevated levels of these genes contribute to developmental working memory maturation are not known in humans or mice. The targeted cell type and developmental time points are justified for four reasons. First, excitotoxic lesions of the hippocampal dentate gyrus damage adult neural stem/progenitor cells as well as mature neurons, and impair working memory in mice ([Supplementary Results 1](#sup1){ref-type="supplementary-material"} and [Supplementary Figures 1a--c](#sup1){ref-type="supplementary-material"}). Second, adult neural stem/progenitor cells have been functionally implicated in working memory in mice.^[@bib39],\ [@bib40],\ [@bib41]^ Third, adult neural stem/progenitor cells in the hippocampus express both Tbx1^[@bib36]^ and COMT ([Supplementary Results 2](#sup1){ref-type="supplementary-material"} and [Supplementary Figure 2](#sup1){ref-type="supplementary-material"}) in mice. As there is no reliable means to evaluate the proliferation and migration of adult neural progenitor cells in the brains of human carriers of 22q11.2 copy number elevation, we examined the mechanisms through which high gene doses of the two 22q11.2 genes COMT and Tbx1 affect adult neurogenesis in mouse and cell models. Fourth, constitutive overexpression of a 190 kb 22q11.2 segment, including COMT, attenuates working memory capacity at 2 months, but not at 1 month of age;^[@bib29]^ these mouse ages correspond to adulthood and periadolescence, respectively.^[@bib42]^ A similar phenomenon was observed from childhood to adulthood in humans who carry a high activity COMT allele.^[@bib31]^ Our data show that copy number elevations of the two 22q11.2 genes, *COMT* and *Tbx1*, in adult neural stem/progenitor cells of the hippocampus arrest the developmental maturation of working memory capacity and adult neurogenesis. Materials and methods ===================== We used male C57BL/6J mice. We constructed lentiviral vectors carrying enhanced green fluorescence protein (*EGFP*), *COMT*-*EGFP* or *Tbx1*-*EGFP*, all under a murine stem cell virus promoter (*MSCV*), pseudotyped with vesicular stomatitis virus (VSV-G). These vectors achieved a high degree of selectivity to infect adult neural stem/progenitor cells ([Supplementary Results 3](#sup1){ref-type="supplementary-material"} and [Supplementary Figures 3 and 4](#sup1){ref-type="supplementary-material"}). The vectors were bilaterally infused into the dorsal hippocampus and mice were evaluated 7--10 days later at 1 month or 2 months of age, in tasks designed to assess working memory, response to novelty, anxiety-like behaviors and motor behavior.^[@bib29]^ Cell types infected by the vectors were identified using flow cytometry and double fluorescence immunohistochemistry. The position of transduced cells was histochemically determined after completion of all behavioral tests. We examined the cell-autonomous effects of *COMT* or *Tbx1* overexpression on proliferation and apoptosis of adult neural stem/progenitor cells of the hippocampus in cell culture (see [Supplementary Information, Methods](#sup1){ref-type="supplementary-material"}). Results ======= Copy number elevation of *COMT* and *Tbx1* blunts the developmental expansion of working memory capacity -------------------------------------------------------------------------------------------------------- To examine the functional impact of *COMT* overexpression in adult neural stem/progenitor cells on the developmental trajectory of working memory, we infused the *COMT* vector 7--10 days before 1 or 2 months of age, corresponding with before and after adolescence, respectively,^[@bib42]^ and tested mice in a battery of behavioral assays ([Figure 1a](#fig1){ref-type="fig"}). Copy number elevation of *COMT* reduced working memory accuracy at 2 months of age, but not at age 1 month ([Figure 1b](#fig1){ref-type="fig"}). EGFP-infused mice showed 7, 10 and 13% higher rates of working memory at 0, 15 and 30 s delays, respectively, at age 2 months compared to 1 month ([Figure 1b](#fig1){ref-type="fig"} inset), thereby recapitulating the developmental maturation of human working memory capacity during this period in mice. In contrast, *COMT*-infused mice showed only 5 and 1% points higher and 13% points lower rates at 0, 15 and 30 sec delays, respectively, during the same developmental time span ([Figure 1b](#fig1){ref-type="fig"} inset); working memory with a heavy load was more severely affected. *COMT* copy number elevation reduced time spent in the open arms of the elevated plus maze ([Figure 1d](#fig1){ref-type="fig"}) and increased time spent in the margin area of the open field ([Figure 1e](#fig1){ref-type="fig"}), indicative of heightened anxiety, at age 2 months, but not 1 month. In contrast, *COMT* copy number elevation had no effect on the exploration of a novel object ([Figure 1c](#fig1){ref-type="fig"}) or motor activity in an inescapable open field([Figure 1f](#fig1){ref-type="fig"}). We next tested the effects of copy number elevation of *Tbx1* in adult neural stem/progenitor cells on working memory capacity at 1 and 2 months of age. Such treatment reduced working memory with a long delay at 2 months but not 1 month of age ([Figure 2a](#fig2){ref-type="fig"}), but had no detectable effect on responses to a novel object ([Figure 2b](#fig2){ref-type="fig"}), anxiety-like behavior in the elevated plus maze ([Figure 2c](#fig2){ref-type="fig"}), and thigmotaxis ([Figure 2d](#fig2){ref-type="fig"}) or locomotor activity ([Figure 2e](#fig2){ref-type="fig"}). Copy number elevation of *COMT* and *Tbx1* arrests migration of adult neural stem/progenitor cells -------------------------------------------------------------------------------------------------- We examined the distribution of transduced cells after completing behavioral testing, when some newly generated progenies of transduced adult neural stem/progenitor cells are expected to migrate into the granular layer from the subgranular zone. The majority of transduced cell bodies were located in and near the subgranular zone of the granular layer; their dendrites and axons reached the molecular layer of the dentate gyrus outward and extended toward the hilus and CA3 inward, respectively ([Figures 3a--d](#fig3){ref-type="fig"}, upper images), a pattern consistent with that of maturing, migrating cells derived from adult neural stem/progenitor cells. The total number of cell counts is not suitable to evaluate the impact of COMT or Tbx1 on proliferation, as the numbers are affected by the uncontrollable extent of infused viral vectors in the hippocampus. We thus used the proportion of cell numbers among the three zones for analysis. This index is independent of the extent of initial random infection. Even if proliferation is additionally affected, the proportion would not change if the same proportion of cells migrate to mid and outer zones. Thus, the ratio is a selective index for evaluation of the migration process. Transduced cells were distributed with a clear gradient from the subgranular/inner third zone to the outer third zone (see [Figures 3a--d](#fig3){ref-type="fig"}, lower images). COMT-transduced cells remained in the subgranular/inner third zone more so at age 2 months than 1 month ([Figures 3a and b](#fig3){ref-type="fig"}, bar graphs). We observed the general gradient from the subgranular/inner third zone to the outer third zone in Tbx1-transduced cells at both 1 and 2 months of age. However, *Tbx1*-transduced cells remained more in the subgranular/inner third zone than EGFP-transduced cells at 2 months of age ([Figure 3d](#fig3){ref-type="fig"}, bar graphs), but we observed a more dispersed distribution of Tbx1-transduced cells than EGFP-transduced cells at 1 month of age ([Figure 3c](#fig3){ref-type="fig"}, bar graphs). These data are consistent with our hypothesis that the migration of progenies of adult neural stem/progenitor cells is reduced by copy number elevation of *COMT* and *Tbx1* in the hippocampal granular layer at 2 months of age, when these gene over-dosages impaired working memory. Copy number elevation of *COMT* and *Tbx1* arrests proliferation of adult neural stem/progenitor cells ------------------------------------------------------------------------------------------------------ It is not possible to unequivocally evaluate how *COMT* and *Tbx1* overexpression in adult neural stem/progenitor cells alone affects the proliferation and apoptosis of transduced cells *in vivo.* To determine the cell-autonomous effects of gene overexpression on proliferation and apoptosis, we isolated adult neural stem/progenitor cells and transfected them with a plasmid containing either *EGFP*, *COMT-EGFP* or *Tbx1-EGFP* ([Figure 4a](#fig4){ref-type="fig"}). *COMT* copy number elevation significantly blunted and *Tbx1* overexpression completely blocked the proliferation of adult neural stem/progenitor cells ([Figure 4b](#fig4){ref-type="fig"}). The proportions of annexin+ cells were indistinguishable among the three groups ([Figure 4c](#fig4){ref-type="fig"}), indicating that *COMT* and *Tbx1* overexpression reduced the proliferation of adult neural stem/progenitor cells in a cell-autonomous manner without altering apoptosis rate. Discussion ========== Our data demonstrate that copy number elevations of the two 22q11.2 genes, *COMT* and *Tbx1*, in the hippocampal dentate gyrus reduced developmental maturation of working memory capacity and the proliferation---and migration of the progenies---of adult neural stem/progenitor cells. Whereas association between a *COMT* high activity allele and working memory deficits has been amply and elegantly demonstrated,^[@bib43]^ our data show that overexpression of these two genes in adult neural stem/progenitor cells does not affect working memory *per se*, but the developmental expansion of its capacity from 1 to 2 months of age in mice. COMT, but not Tbx1, overexpression in the adult, but not peri-adolescent, hippocampus exacerbated anxiety-related behaviors. This was a rather selective effect, as neither gene treatment altered novel response or motor activity. Ibotenic acid lesions severely impaired working memory, reduced response to a novel object and increased motor activity, but had no effect on anxiety-like behaviors (see [Supplementary Figures 1c--g](#sup1){ref-type="supplementary-material"}). While all the treatments equally reduced working memory capacity, these variable effects of COMT and Tbx1 overexpression and excitotoxic lesions on other behaviors indicate that working memory deficits cannot be adequately accounted for by the indirect effects of these treatments on the other behaviors. We previously demonstrated that transgenic mice constitutively overexpressing a 190 kb human chromosomal 22q11.2 segment, including *COMT*, *TXNRD2* and *ARVCF*, exhibited no maturation deficit of working memory in a no-delay version of spontaneous alternation at any age but showed deficits in working memory with delays in rewarded alternation at 2 months but not at 1 month.^[@bib29]^ Another report showed that the developmental maturation of working memory capacity is blunted from adolescence to adulthood in individuals with a high activity allele of COMT in humans.^[@bib31]^ Our current data expand these mouse and human studies in three ways. First, we identified COMT and Tbx1 in hippocampal adult stem/neural progenitor cells as a functional contributor, although we do not rule out the possibility that mature neurons in the hippocampus and elsewhere are also involved. Second, working memory with delay, as measured by spontaneous alternation, was also affected by increased *COMT* dose. Third, working memory with delay is affected by Tbx1 overexpression in the hippocampus at 2, but not 1, months of age. Our data are unique in that specific contributory genes are identified but are consistent with studies that showed the detrimental impact of inhibition of adult stem/progenitor cells on working memory in rodents.^[@bib39],\ [@bib40]^ However, some aspects of our data are seemingly inconsistent with others. One study showed inhibition of adult neurogenesis did not impair working memory tasks where no delay is imposed in rats.^[@bib44]^ However, in this study, rats did not show a robust delay-dependent performance reduction in the non-matching-to-place task in this study,^[@bib44]^ raising the possibility that the task might not have been as sensitive to working memory load as our task. Our own data are consistent with this observation in that gene elevation of COMT and Tbx1 did not affect working memory with no delay as severely as working memory with delay ([Figures 1a](#fig1){ref-type="fig"} and [2a](#fig2){ref-type="fig"}).^[@bib29]^ The interval between inhibition of adult neurogenesis and testing of working memory is another parameter that could account for diverse results. Our mice were tested 7--10 days after gene overexpression. Other studies similarly showed that while working memory is severely impaired at 1 and 3 weeks after irradiation of adult stem cells,^[@bib40]^ performance is normal at 7 weeks^[@bib40]^ and 4--8 weeks^[@bib44]^ or enhanced at 2.5--3 months^[@bib41]^ after inhibition of adult neurogenesis. Such normal performance might reflect a recovery of adult stem/progenitor cells or compensatory processes.^[@bib40]^ Moreover, as it takes approximately 2 months for adult stem/progenitor cells to differentiate into mature neurons, stem cells or their immediate progenies (for example, immature neurons) might mediate the detrimental effect of gene overdose in or functional inhibition of adult stem/progenitor cells. Taken together, these seemingly inconsistent data collectively suggest that working memory with a heavy load is more severely affected by a functional inhibition or reduction of adult stem/progenitor cells and their immediate progenies (that is, immature neurons) than of mature neurons derived from adult stem/progenitor cells. Overexpression of the protein products of these 22q11.2 genes might non-specifically reduce the functional capacity of adult stem cells in our mouse models and in human carriers of duplication and triplication. However, this possibility does not explain why the same gene overexpression did not affect working memory at 1 month of age. Certain intrinsic properties of adult neural stem/progenitor cells might underlie the age-dependent effect. There is a drastic decline in the neurogenesis rate in the hippocampal subgranular zone from age 1 month to 3 months in mice^[@bib45],\ [@bib46]^ and from infancy to adulthood in humans.^[@bib47],\ [@bib48],\ [@bib49]^ Given that *COMT* and *Tbx1* overexpressing cells tended to remain at the inner third of the granular layer more so at age 2 months than 1 month, the endogenously diminished proliferation and migration at age 2 months might make it increasingly difficult for stem cells to overcome the detrimental effects of increased gene dose. Not incompatible with this possibility, an additional potential mechanism is a developmental change in the properties of COMT and its substrates. Developmental maturation of working memory capacity from age 1 to 2 months occurred when endogenous COMT enhanced its capacity to methylate substrates in the hippocampus, presumably reflecting a higher activity ratio of membrane bound-COMT to soluble-COMT at age 2 months, compared to 1 month ([Supplementary Results 4](#sup1){ref-type="supplementary-material"}, [Supplementary Figure 5](#sup1){ref-type="supplementary-material"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"}). Catecholamine depletion reduces the proliferation rate of adult neural stem/progenitor cells in the rodent subgranular zone under certain conditions.^[@bib50],\ [@bib51]^ As COMT also methylates any protein that contains catechol, catecholamines and other catechol-carrying molecules could serve as functional substrates for this age-dependent effect of COMT. There might even be a distinct mechanism through which Tbx1 overexpression did not impair working memory at 1 month of age. Our data suggest that *Tbx1* overexpressed at 1 month facilitated migration, compared to EGFP; Tbx1 overexpression at 2 months induced the opposite pattern. It could be that newly generated cells at 1 month might have compensated for the impact of Tbx1 by facilitating migration of transduced cells due to their more active proliferation capacity, thus less detrimental effect on working memory. One unique property of our study is that we evaluated the impact of gene overexpression on working memory and other behaviors following a 7--10-day recovery period after surgical gene transduction. Some newly dividing cells can start to differentiate into neuroblasts within a few days and into neurons after a few weeks in the adult mouse brain, although it takes more than 1 week for differentiation into astrocytes.^[@bib46]^ However, this time period is not sufficient for the majority of nascent neurons to mature to extend their axons to target neurons in the hilus and CA3^[@bib52]^ and their dendrites to express spines.^[@bib53]^ Thus, the impact of cells newly generated from transduced stem cells during adulthood would be expected to remain relatively localized under our experimental protocol. As immature and maturing neurons bear a slow excitatory response to GABA inputs, COMT and Tbx1 overexpression might impair working memory by such local effects within the granular layer. A future challenge is to identify a novel substrate through which copy number variants impact working memory by altering local electrophysiologic properties of newly generated immature neurons at the time when mice exhibit blunted working memory capacity. Molecules and their associated cascades in neonatal and adult neural stem/progenitor cells can be further exploited to better understand the developmental trajectories of cognitive and affective anomalies associated with ASD, ID and schizophrenia.^[@bib37],\ [@bib54]^ The ultimate validation of our observations in mouse and cell models will be achieved only when it is shown that potential therapies developed from such hypothetical mechanisms provide beneficial effects on the atypical developmental maturation of working memory in humans. We thank Dr Arthur Nienhuis of St. Jude Children's Research Hospital for providing us with pCL20c-*MSCV*-*EGFP* plasmid. Research reported in this publication was supported by the National Institute of Mental Health of the National Institutes of Health under Award Number R01MH099660 (NH), the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R21HD053114 and U54HD090260 (NH), National Cancer Institute of the National Institutes of Health under Award Number P30CA013330 (JZ), a NARSAD Independent Investigator Award and a Maltz Foundation award (NH), the Uehara Memorial Foundation and Senshin Medical Research Foundation (SB) and Senshin Medical Research Foundation (HN).The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Dr Takeshi Hiramoto for his critical comments on an early version of the manuscript, and Drs J. Roy-Chowdhury and Xia Wang of the Gene Therapy Core, Albert Einstein College of Medicine for preparation of high titer viral particles. **Author contributions** The first three authors (SB, TI and SA) contributed equally to this work. SB, TI, SA, YN, HN, AN, TT, GK, AH, MN, SE and NH contributed to the overall design and execution of experiments and analyses. SB, TI, YN and NH wrote the manuscript. GD-T and PTM conducted Comt enzyme assays. GK, TI, TT conducted behavioral analyses. YN and KT carried out proliferation and apoptosis analyses *in vitro*. SA, HN, YN, JZ and NH conducted FACS. SK and KK provided the template plasmid of viral vectors and SB designed viral vectors. SB, TI, SA, AN, TT, AH, SE and MN carried out histochemical analyses. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies the paper on the *Molecular Psychiatry* website (http://www.nature.com/mp) The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. ###### Click here for additional data file. {#fig1} {#fig2} {#fig3} {#fig4} [^1]: These authors contributed equally to this work.

Introduction {#section1-0192513X17710773} ============ After several decades of migration research that was focused on the migration of intact family units and families being associated with the locus of residence, family migration scholars have turned their focus to transnational family arrangements resulting from international migration ([@bibr17-0192513X17710773]; [@bibr40-0192513X17710773]). Although exact numbers of transnational families worldwide are unknown, today, the number of transnational families seems to be on the rise, with both men and women migrating independently without their families ([@bibr16-0192513X17710773]; [@bibr37-0192513X17710773]; [@bibr54-0192513X17710773]; [@bibr61-0192513X17710773]). Some argue that this is the result of global capitalism, global inequalities, and the current international division of labor in which individuals are encouraged and at times forced to migrate to ensure the family's economic well-being. Especially, migrants from developing countries tend to take up low-wage service labor that is needed in industrialized countries, while leaving nonproductive family members, such as children, behind ([@bibr16-0192513X17710773]; [@bibr27-0192513X17710773]; [@bibr28-0192513X17710773]; [@bibr46-0192513X17710773]; [@bibr49-0192513X17710773]; [@bibr54-0192513X17710773]). Restrictive immigration policies, high migration costs, and difficult living and working conditions in the host country provide disincentives for families to migrate together. Yet some parents prefer to leave their children in their origin country to grow up in familiar cultural environments, supported by extended family ([@bibr10-0192513X17710773]). Transnational family scholars have argued that while transnational parenting can provide more financial security to their families, transnational parent--child separation can also have negative consequences for parents and children. It has, for example, been found that parents and children are negatively affected in their emotional well-being and health, and children in their educational performance or aspirations ([@bibr16-0192513X17710773]; [@bibr27-0192513X17710773]; [@bibr30-0192513X17710773]; [@bibr31-0192513X17710773]; [@bibr47-0192513X17710773]; [@bibr58-0192513X17710773]; [@bibr66-0192513X17710773]; [@bibr73-0192513X17710773]). Yet while family sociology and transnational migration studies have investigated the emotional consequences of transnational family life for children and their parents, other important consequences for parents' lives are less researched. As [@bibr20-0192513X17710773] indicates in her review of research on immigrant families, "there has been rather limited attention to the balance of work and family life among immigrants when compared to other domains" (p. 506). And, one aspect that has been neglected in transnational family research in particular is how being in a transnational family affects parents' labor market outcomes, especially since the economic returns of this labor are an important part of transnational parenting ([@bibr31-0192513X17710773]; [@bibr73-0192513X17710773]). The ability to send remittances and provide for the family is the main reason why some parents migrate in the first place and remittances are found to be of major importance for maintaining transnational family relations ([@bibr15-0192513X17710773]; [@bibr17-0192513X17710773]; [@bibr27-0192513X17710773]; [@bibr73-0192513X17710773]). In addition, studies from organizational psychology lead us to believe that there are various ways in which transnational parenting can be related to job outcomes. This study therefore aims to contribute to transnational family studies by exploring the association between transnational parents' work and family lives. Working-class migrants often face downward mobility and economic strain when living in the host country ([@bibr50-0192513X17710773]). On arrival, opportunities of finding work are often only available in certain low-paid sectors such as cleaning, if at all. Not speaking the new language, qualifications not being recognized, not having a work permit, and discrimination are compounding factors ([@bibr56-0192513X17710773]; [@bibr68-0192513X17710773]). The insecure but also inflexible employments migrants often find themselves in can be extra demanding and stressful when a migrant also has caregiving responsibilities, draining energy and time. A lack of kin support and the reluctance of migrants of non-Western origin to use expensive formal child care services only exacerbate the strain these migrants feel ([@bibr45-0192513X17710773]; [@bibr56-0192513X17710773]). On the other hand, child-raising arrangements in which care is arranged transnationally do not necessarily lift caregiving responsibilities and can also be a cause of stress. Transnational family studies have argued that transnational separation can lead to lower levels of well-being for parents ([@bibr3-0192513X17710773]; [@bibr26-0192513X17710773]; [@bibr28-0192513X17710773]; [@bibr30-0192513X17710773]; [@bibr46-0192513X17710773]; [@bibr54-0192513X17710773]). This in turn can affect concentration and performance at the job. Thus, the rearing of children in the country of origin could have either positive or negative effects on the job outcomes of the parents. Therefore, this study wants to explore this association and does so by comparing the job performance of Angolan parents whose children live in the home country with Angolan parents whose children live with them in the Netherlands while controlling for compounding factors. Angolans predominantly migrated as asylum seekers to the Netherlands, while most transnational parents that have been studied are labor migrants ([@bibr52-0192513X17710773]). The postconflict setting in Angola and the asylum procedure have created different transnational family arrangements in comparison with labor migrants, which can affect the effect transnational separation has on their lives in the Netherlands ([@bibr13-0192513X17710773]; [@bibr25-0192513X17710773]). Particularly now with a growing refugee population and the current "refugee crisis," we need to understand the transnational engagements of refugees and how that affects their lives better. Especially, as research has shown that, just like other migrants, transnational activities are important in refugee's lives and this can affect their life in the host country ([@bibr2-0192513X17710773]; [@bibr29-0192513X17710773]; [@bibr52-0192513X17710773]). And, as this study shows, the same holds true for transnational family life. Moreover, as a result of the fact that numerous Angolans went through the same migration trajectory many Angolans have similar socioeconomic and migration status, which makes it easier to single out the association between transnational family life and job outcomes. Work and Family Life {#section2-0192513X17710773} ==================== Significant research attention has been given to the difficulties people face balancing work and family life and on factors leading to favorable job outcomes. Yet no such research has been conducted among families in which care is arranged transnationally. Based on two bodies of literature---work--family conflict and the happy productive worker thesis---a theoretical framework is proposed to investigate the effects transnational parent--child separation has on job outcomes of migrant parents. This literature has mainly studied job outcomes by looking at job performance while the current study concentrates on job instability and absenteeism. Although the particular outcomes are thus slightly different, we can still draw important lessons from this literature. Work--Family Conflict {#section3-0192513X17710773} --------------------- Although not particularly focused on migrants, occupational stress research on work--family interactions has shown that both work and family life are negatively affected when demands of one domain affect the other ([@bibr1-0192513X17710773]; [@bibr19-0192513X17710773]; [@bibr34-0192513X17710773]). Workers with child care responsibilities, for example, experience difficulties with juggling family and work, which can result in conflict when demands of participation in the family domain are incompatible with demands in the work domain and vice versa ([@bibr1-0192513X17710773]). A longstanding and extensive literature has consistently shown that work and family life are intrinsically and reciprocally related. Higher levels of work interfering with family are related to poor family role performance, burnout, and health complaints ([@bibr4-0192513X17710773]; [@bibr7-0192513X17710773]; [@bibr59-0192513X17710773]; [@bibr65-0192513X17710773]). Conversely, family-to-work conflict is also associated with lower job satisfaction, less organizational commitment and more distress at the job ([@bibr19-0192513X17710773]; [@bibr65-0192513X17710773]; [@bibr67-0192513X17710773]). The extent to which family life creates strain on work and vice-versa is related to the nature of family, care and work arrangements, and structural and social demands of the family ([@bibr23-0192513X17710773]; [@bibr33-0192513X17710773]). A worker whose spouse stays at home to take care of the children is less likely to experience family-to-work conflict than a family of dual earners or a single parent with preschool-aged children ([@bibr1-0192513X17710773]). Especially, family support is of importance as it relieves the employee of family-related responsibilities and offers the employee more time and energy to fulfil responsibilities at work ([@bibr34-0192513X17710773]). If working parents have family to help with caregiving when children are sick, they are less likely to experience work--family conflict ([@bibr32-0192513X17710773]). Ethnicity, gender, age, marital status, social class, and occupation are also important ([@bibr64-0192513X17710773]). The presence of children, moreover, is more likely to affect job participation of mothers than that of fathers as they are often seen as the primary caregiver ([@bibr22-0192513X17710773]). Caregiving decisions are only linked to lower levels of well-being and increased conflict when they do not fit the family or work environment. For example, research has shown that the more one is devoted to caregiving the less one is able to commit to an organization, which can affect job performance ([@bibr60-0192513X17710773]). Hence, well-being and job outcomes are affected negatively when the carer has to make sacrifices in one role to fulfil another. However, what appears to be most important for the perceptions of work--family conflict are not so much the type of care arrangement used but the satisfaction the parent has with the caregiving arrangements ([@bibr33-0192513X17710773]). In transnational families, the provision of everyday practical care (containing e.g., provision of food, taking care of children when they fall ill, picking the children up from school, and attending events of the children) is not given by the migrant parent in the host country but by a caregiver in the country of origin. Based on the work--family conflict thesis, it can therefore be hypothesized as follows: 1. **Hypothesis 1:** Migrant parents whose children are in the country of origin, experience less family-to-work conflict than parents who live with their children in the host country and this leads to favorable job outcomes, as measured by job stability and attendance. Happy Productive Worker Thesis {#section4-0192513X17710773} ------------------------------ Research on well-being has consistently shown that happiness is related to various forms of success. Success leads to happiness but longitudinal research also shows that happiness can predict success ([@bibr35-0192513X17710773]). People who generally have positive emotions are more sensitive to rewards in their environment and are more likely to move toward rather than shun rewarding situations ([@bibr35-0192513X17710773]). Therefore, they are more optimistic about future events and are more proactive, resilient, and less prone to stress symptoms ([@bibr70-0192513X17710773]). People who have negative emotions, instead, become more disengaged with the world and are less optimistic that their hard work will lead to success (Wright, Cropanzano, Denney, & Moline, 2002). Positive and negative affect or happiness can be ingrained in the personality of an individual, but may also be rooted in the person's current life circumstances or a combination of these ([@bibr35-0192513X17710773]). Based on the above, we can assume that "satisfied and psychologically well employees are more likely than those less satisfied and psychologically well to have the resources necessary to foster and facilitate increased levels of job performance" ([@bibr70-0192513X17710773], p. 97). In other words, happiness stimulates activity and work involvement. This is captured in the happy productive worker thesis developed by some organizational theorists, which posits that workers who are happy perform better at their job than do employees who are unhappy ([@bibr71-0192513X17710773]; [@bibr70-0192513X17710773]). Although this thesis was received with skepticism at first, a small but growing body of empirical research has found support for the happy productive worker thesis. Evidence from cross-sectional but also longitudinal research corroborate that happy workers enjoy multiple advantages in comparison with unhappy workers, and, most important, perform better at their job. [@bibr57-0192513X17710773] found that employees who had positive emotions had greater pay 18 months later, more social support on the job and received better performance evaluations. Similarly, studies by Wright, Cropanzano, and colleagues using various methods and research designs have found that well-being leads to better job performance even when controlling for age, gender, education, and tenure at the organization (e.g., [@bibr11-0192513X17710773], [@bibr12-0192513X17710773]; [@bibr71-0192513X17710773]; [@bibr70-0192513X17710773]; [@bibr72-0192513X17710773]). People with high levels of subjective well-being are more likely to secure job interviews, appear to secure better jobs, show superior performance and productivity, are more efficient, can handle managerial jobs better, are less likely to show counterproductive workplace behavior, get more support from coworkers, and are more satisfied with their job ([@bibr35-0192513X17710773]). Happy people receive good performance evaluations and are satisfied with their jobs and consequently, they are less often absent from work, less likely to want to quit, and less likely to experience job burnout or withdrawal ([@bibr14-0192513X17710773]; [@bibr35-0192513X17710773]; [@bibr69-0192513X17710773]). Finally, gender, education, ethnicity, wealth, marriage, and family support are important resources that can act as coping mechanisms. The above body of knowledge has implications for transnational families. Various qualitative studies have indicated that when migrant parents live separated from their children they are emotionally affected. If they do not have regular physical contact with their children, parents experience difficulties in maintaining strong emotional bonds with their children. This loss of intimacy and the fact that parents long to be with their children can lead to stress, feelings of guilt, loneliness, and even to depression ([@bibr3-0192513X17710773]; [@bibr28-0192513X17710773]; [@bibr30-0192513X17710773]; [@bibr46-0192513X17710773]; [@bibr54-0192513X17710773]). These qualitative findings are partly corroborated by a larger scale study which shows that parents who live separated from their children generally experience lower subjective well-being than parents who live with their children, although contextual factors play an important role ([@bibr26-0192513X17710773]). Hence, bringing these two bodies of literature together, we can posit the opposite hypothesis than the one aforementioned: 1. **Hypothesis 2:** Migrant parents whose children live in the country of origin will have unfavorable job outcomes, as measured by job instability and absenteeism, in comparison with migrant parents who live with their children in the host country due to their lower subjective well-being. In the remainder of this article, these two contradictory hypotheses are tested. Before doing so, a short description of the group under study is given. Migration Context {#section5-0192513X17710773} ================= This article draws on data collected among Angolan migrant parents in the Netherlands, a non-Western country of origin. The majority of the evidence for the happy productive worker thesis and work--family conflict, however, comes from Westernized industrialized countries in which happiness is highly valued. Other cultures can hold different definitions of success ([@bibr35-0192513X17710773]). Also, the extent to which and the way in which individuals experience work--family conflict is dependent on the broader sociocontextual circumstances and norms and values regarding work and family that are dominant in society ([@bibr23-0192513X17710773]). For example, if hard work is valued and seen as necessary for family well-being and work--conflict is not interpreted as being stressful, it will less likely affect well-being and job or family outcomes. Therefore, these theses might not apply to the same extent to Angolans. Below the Angolan migration flow and the position this group holds in the Netherlands will be shortly described, as that might affect the outcomes of the analyses. Major migration from Angola began at the end of the 1990s as a result of a war that lasted until 2002. The Angolan migrants in the Netherlands originated predominantly from middle-class families. The main reasons for migration were fear of being forced to fight in the army, the shortage of study and work opportunities in Angola, and poverty (van Wijk, 2008). The Netherlands became a major destination country for Angolan asylum seekers because of its favorable asylum policies for this group of migrants at the time. Until 2002, it was relatively easy for Angolans to apply for asylum in the Netherlands, especially for Unaccompanied Asylum seeking Minors (UAM), that is, asylum seekers younger than age 18 migrating without parents or other family members ([@bibr63-0192513X17710773]). Consequently, about half of the Angolan migrants in the Netherlands came as an UAM resulting in a rather young Angolan population. Resulting, at its peak in 2004, in over 12,000 Angolans residing in the Netherlands ([@bibr9-0192513X17710773]). Yet as many Angolans have returned or were forced to return after the war ended in 2002, only around 8,500 Angolans remain in the Netherlands today ([@bibr9-0192513X17710773]). Because Angolans, and new immigrant groups in general, do not belong to the major migrant groups in the Netherlands, they are understudied and consequently there are no official data on their labor market participation. However, some general trends can be established based on our data and reports of others. First, the [@bibr44-0192513X17710773] reports that in 2000, 20% of the Angolans living in Organisation for Economic Co-operation and Development countries had received tertiary education, which is similar to our data in which 20% has received vocational training or higher. Second, asylum seekers generally integrate more slowly into the labor market as they are not allowed to work when awaiting asylum and traumas can stand in the way of labor market participation ([@bibr62-0192513X17710773]). Nevertheless, Angolans have been in the Netherlands for quite some time and a large proportion was able to receive residence permits and is consequently legally residing in the Netherlands. Furthermore, UAMs received a Dutch education resulting in good Dutch language proficiency and a better position in the labor market than many other new migrant groups. That is, the UAMs that came in before 2001. After 2001, less opportunities were given to UAMs. Nonetheless, our data indicate that Angolans have a relatively good position in the labor market, with only 1% being unemployed and 18% being in unskilled labor. Moreover, no differences are found in the occupational status of transnational and nontransnational Angolan parents ([@bibr24-0192513X17710773]). Thus, the fact that many Angolans followed a similar migration trajectory and hence have received similar opportunities that have resulted in a population with relatively good and comparable socioeconomic status in comparison with other new immigrant groups. Data and Analysis {#section6-0192513X17710773} ================= The present research draws on survey data collected in the TCRAf-Eu project^[1](#fn1-0192513X17710773){ref-type="fn"}^ investigating the functioning of transnational families among Angolan migrant parents in the Netherlands. The survey collected data from both transnational parents and parents who live with all their children in the Netherlands. Having both groups allows exploring specifically the effect of the separation. To be eligible for this study, respondents had to have children younger than age 21 living in the host or origin country, have lived in the Netherlands for more than 1 year consecutively, and had to be older than 16 years when coming to the Netherlands. Purposive sampling resulted in 139 transnational and 167 nontransnational Angolan parents (for more details about the data collection and characteristics of the transnational and nontransnational families in this study, see [@bibr24-0192513X17710773]). Variable Constructions {#section7-0192513X17710773} ---------------------- Two job outcomes are studied: absenteeism and job stability. These are indicators that are easy to quantify and are more objective than other measures often used such as assessment by the employer ([@bibr48-0192513X17710773]). The first is measured by days absent in the past 3 months and the second by the number of times the migrant changed jobs while being in the Netherlands. Both are treated as continuous variables. The variable days absent is positively skewed, thus, a logarithmic form is used to create a normally distributed variable. In some economic studies, job mobility is considered a positive outcome as it can reflect change to better jobs, promotion, and wage growth ([@bibr53-0192513X17710773]; [@bibr55-0192513X17710773]). However, in the Angolan case, changing jobs is a result of their insecure labor position and not because of career improvement. In fact, the data show that Angolans who were in high-skilled labor and of higher socioeconomic status have had significantly less job changes than those in unskilled and skilled labor and of low socioeconomic status. To test the first part of the hypotheses concerning the mediating role of work-to-family conflict, the following statements were used: "Worrying about my children interferes with my ability to get things done at work" and "I often miss work because I am worried about my children." Respondents could answer on a five category Likert-type scale, from *have not experienced* (0), *experienced but not at all stressful* (1) to *extremely stressful* (4). These two items were combined in one scale and averaged, with higher scores indicating increased conflict. A reliability assessment shows that the scale is reliable with a Cronbach's alpha of .74. From this measure, a binary variable was created because it was skewed. The value 0 was given to those who experienced no stress (0-1) and the value 1 was given to those who experienced various levels of stress (2-5). While we report on the binary variable, the continuous variable and binary variable resulted in similar findings. The other mediating variable pertaining to the second set of hypotheses is happiness. Happiness was assessed using a 5-point scale ranging from *not happy* to *very happy*. To account for skewness, a binary variable was created by recoding the first three answer categories in *unhappy* (0) and the last two as *happy* (1). To analyze the robustness of the results, the same models were reestimated with a measure of mental health status, namely the 12-item General Health Questionnaire developed by [@bibr21-0192513X17710773]. We also ran the analyses with happiness as a continuous variable. All these analyses produced virtually similar results. In examining (a) the association between transnational parent's work and transnational family lives and (b) the role of family-to-work conflict and subjective well-being, various commonly used controls are taken into account to avoid statistical confounds. Controls included age, sex, marital status, education, years in the Netherlands, socioeconomic status, social network, Dutch language proficiency, and whether the respondent has young children (younger than 8 years). Because only three Angolans are undocumented, legal status is not included. Educational attainment was determined using an 11-point scale, ranging from (1) *no schooling* to (11) *finished university*. To capture socioeconomic status a dummy variable is included on house ownership as earnings or other similar measures could lead to problems of endogeneity. House ownership is a more long-term and stable measure of socioeconomic status. The social network of the respondent is divided in two variables, one measuring the number of family members in the Netherlands and the other measuring the number of friends living in the Netherlands. Two respondents indicated to have over a 100 friends. We have dealt with these outliers by grouping these together with the few respondents who indicated they had 100 friends. A log transformation is applied to correct for the skewed distribution of the social network variables. Finally, to assess Dutch language proficiency respondents were asked to indicate whether they had experienced difficulties communicating in the Dutch language, from (0) *have not experienced*, (1) *not at all stressful* to (4) *extremely stressful*. Hence, higher scores indicate more problems with speaking the Dutch language and higher stress levels. Analytical Strategy {#section8-0192513X17710773} ------------------- The hypotheses developed above pertain to the mechanism explaining differences in job outcomes between transnational parents and nontransnational parents. On the one hand, we can expect transnational parents to be less happy which unfavorably affects their job outcomes. On the other hand, we can expect transnational parents to experience less family-to-work conflict leading to favorable job outcomes than nontransnational parents. These hypothesized relationships are depicted in [Figure 1](#fig1-0192513X17710773){ref-type="fig"}. ![Conceptual model of the mediation analysis.\ *Note*. Indirect effects (a1 \* b1 and a2 \* b2); direct effect (c'); total effect \[(a1 \* b1) + (a2 \* b2) + c'\]; Percentage of total effect mediated = indirect effects/total effect.](10.1177_0192513X17710773-fig1){#fig1-0192513X17710773} Binary mediation analysis is used to test these hypotheses. This analysis follows the mediation analysis as proposed by [@bibr5-0192513X17710773], but accounts for the binary nature of the mediating variables. The approach used to test mediation is to run four regression models per outcome variable. The first condition for mediation is showing that transnational parenting relates to the outcome variable. Second, transnational parenting should relate to the mediating factors happiness and family-to-work conflict, as tested by [Equations 2](#disp-formula2-0192513X17710773){ref-type="disp-formula"} and [3](#disp-formula2-0192513X17710773){ref-type="disp-formula"}. The final step establishes whether transnational parenting is associated with the job outcome variables while controlling for the mediating factors ([Equation 4](#disp-formula1-0192513X17710773){ref-type="disp-formula"}). Control variables were included in all equations to help avoid statistical confounds. $$\begin{array}{l} {{Job\ outcomes} = ß0 + \left( {ßb1\ {Happiness} + ßb2\ {Family-work\ conflict}} \right) + ßc’} \\ {{Transnational\ parenting} + ßd\ {covariates} + \varepsilon} \\ \end{array}$$ $${Mediator} = ß0 + ßa\ {Transnational\ parenting} + ßd\ {covariates} + \varepsilon$$ According to [@bibr5-0192513X17710773] if path c' in [Figure 1](#fig1-0192513X17710773){ref-type="fig"}, that is, the coefficient on transnational parenting, is close to 0 and nonsignificant when including the mediating factors and the indirect effects are significant, full mediation is found. As the two mediating variables are binary and hence part of the model is nonlinear, Stata's "binary_mediation" command is used to calculate total, indirect, and direct effects, as well as the proportion of the total effect mediated. This method standardizes the coefficients before computing the direct and indirect effects. Bootstrapping with 5,000 replications was undertaken to increase statistical power and bias-corrected nonparametric confidence intervals are utilized as the mediated effects are unlikely to be normally distributed ([@bibr36-0192513X17710773]; [@bibr51-0192513X17710773]). Variance inflation factors were below 1.7, indicating that multicollinearity among the independent variables within the models is not a serious concern. The original sample consisted of 306 Angolan respondents. For this analysis, parents who have children both in the country of origin and in the Netherlands are omitted because this would not allow exploring the different mediation paths of interest and test opposite hypotheses. Missing data and the above criterion reduced the sample to 255 Angolan respondents, including 86 transnational parents.^[2](#fn2-0192513X17710773){ref-type="fn"}^ Finally, because job absenteeism only applies to people in employment, results for the model regarding job absenteeism are based on respondents in employment at the time of interview, which resulted in 181 respondents. Results {#section9-0192513X17710773} ======= [Table 1](#table1-0192513X17710773){ref-type="table"} reports the descriptive statistics of the included variables. Looking at the mediating factors, it is found that the majority of Angolans indicate to be happy (77%) and experience limited family-to-work conflict (33%). On a scale from 0 to 5, the average score is 0.87 (*SD* = 0.89). Finally, on average Angolans report having changed jobs in the Netherlands twice and have missed on average 2.4 days of work in the 3 months prior to the interview. ###### Descriptive Statistics.  *M* *SD* Min Max -------------------------------------------------------------------------------- ------- ------- ----- ----- Transnational parenting^[a](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.34 0.47 0 1 Happiness^[b](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.77 0.42 0 1 Family-to-work conflict^[c](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.33 0.50 0 1 Age 33.60 6.28 23 59 Sex^[d](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.50 0.50 0 1 Marital status^[e](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.16 0.37 0 1 Education 7.21 2.18 0 11 Years in the Netherlands 14.31 4.46 0 25 Housing^[f](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.87 0.33 0 1 Number of friends in the Netherlands 31.47 39.21 0 294 Number of family members in the Netherlands 4.52 11.08 0 98 Dutch proficiency 1.36 1.05 0 4 Child \< 8 years^[g](#table-fn1-0192513X17710773){ref-type="table-fn"}^ 0.61 0.49 0 1 Job instability 2.00 1.97 0 9 Job absenteeism 2.41 4.01 0 24 *Note*. Superscripts indicate reference categories that include (a) nontransnational parent; (b) unhappy; (c) no work-to-family conflict (d) male; (e) married/in a relationship; (f) room, student housing, institution, other; and (g) no children \< 8 years of age. *Source*. TCRAf-Eu Angolan parent survey, The Netherlands 2010-2011. Mediation Analysis {#section10-0192513X17710773} ------------------ To test the hypotheses, we turn to regression analyses. Following the four steps as proposed by [@bibr5-0192513X17710773], it was first tested whether transnational parenting is associated with job absenteeism and job instability. These results are displayed in [Table 2](#table2-0192513X17710773){ref-type="table"}. Model 1 shows that transnational parents do not significantly differ from nontransnational parents in terms of the days they were absent from their jobs in the past 3 months. Model 2, however, indicates that, ceteris paribus, transnational parents change jobs 0.89 times more often (*p* \< .01) than nontransnational parents. Thus, the first condition for mediation is only met for job instability and not for job absenteeism. Therefore, we only continue with the next steps of the mediation analysis for job instability. ###### Results of Mediation Analyses for Job Outcomes.  Model 1 Model 2 -------------------------------------------------------------------------------- ----------------------------------------------------------------------- --------------------------------------------------------------------- Transnational parenting^[a](#table-fn3-0192513X17710773){ref-type="table-fn"}^ −0.10 (0.15) 0.89 (0.29)[\*\*](#table-fn6-0192513X17710773){ref-type="table-fn"} Age −0.00 (0.01) 0.05 (0.02)[\*](#table-fn6-0192513X17710773){ref-type="table-fn"} Sex^[b](#table-fn3-0192513X17710773){ref-type="table-fn"}^ 0.75 (0.14)[\*\*\*](#table-fn6-0192513X17710773){ref-type="table-fn"} 0.21 (0.26) Marital status^[c](#table-fn3-0192513X17710773){ref-type="table-fn"}^ 0.03 (0.22) 0.43 (0.35) Education 0.01 (0.03) 0.10 (0.06) Years in the Netherlands 0.02 (0.02) 0.06 (0.03) Housing^[d](#table-fn3-0192513X17710773){ref-type="table-fn"}^ −0.25 (0.21) 0.06 (0.41) Number of friends in the Netherlands (log) −0.06 (0.04) 0.08 (0.08) Number of family members in the Netherlands (log) 0.03 (0.06) −0.05 (0.12) Dutch proficiency 0.06 (0.07) −0.18 (0.12) Child \< 8 years of age^[e](#table-fn3-0192513X17710773){ref-type="table-fn"}^ 0.48 (0.16)[\*\*](#table-fn6-0192513X17710773){ref-type="table-fn"} 0.33 (0.31) *R* ^2^ .24 .13 *Note*. Superscripts indicate reference categories that include (a) nontransnational parent; (b) male; (c) married/in a relationship; (d) room, student housing, institution, other; (e) No children \< 8 years of age. Standard errors in parentheses *Source*. TCRAf-Eu Angolan parent survey, The Netherlands 2010-2011. *p* \< .05. \*\**p* \< .01. \*\*\**p* \< .001 (one-tailed test). [Figure 2](#fig2-0192513X17710773){ref-type="fig"} presents the results of the mediation analysis graphically and presents us with the relevant coefficients for each step of the mediation analysis. [Table 3](#table3-0192513X17710773){ref-type="table"} displays the indirect, direct, and total effects and the proportion of the total effect mediated with bias-corrected confidence intervals after bootstrapping. Although not presented, the model includes the same control variables as in [Table 2](#table2-0192513X17710773){ref-type="table"}. The second step of mediation requires the independent variable to be related to the mediating variable. Path a1 represents the association between transnational parenting and happiness and a2 between transnational parenting and family-to-work conflict. As graphically evidenced in [Figure 2](#fig2-0192513X17710773){ref-type="fig"}, both paths are significant. First, as posited in Hypothesis 2, transnational parents report to be less happy than nontransnational parents. Second, although transnational parenting is significantly associated with family-to-work conflict as postulated in Hypothesis 1, it is not in the expected direction. Instead of transnational parents reporting family-to-work conflict less often they report family-to-work conflict more often. And this is a significant difference. ![Mediation model job instability with binary mediators.\ *Note*. Indirect effects (a1 \* b1 and a2 \* b2); direct effect (c'); total effect \[(a1 \* b1) + (a2 \* b2) + c'\]; percentage of total effect mediated = indirect effects/total effect (20%); Pseudo *R*^2^ = .20; Unstandardized ordinary least squares coefficients presented, paths a1 and a2 are unstandardized logit coefficients.\ Standard errors in parentheses.\ *Source*. TCRAf-Eu Angolan parent survey, The Netherlands 2010-2011.\ \**p* \< .05. \*\**p* \< .01. \*\*\**p* \< .001 (one-tailed test).](10.1177_0192513X17710773-fig2){#fig2-0192513X17710773} ###### Test of Mediation With Bootstrapped Results for Job Instability.  Mediator Β coefficient Bias-corrected CI ------------------------------------- --------------- ------------------- ------ Indirect effect  Happiness 0.07 0.01 0.15  Conflict −0.02 −0.08 0.03  Total 0.05 −0.03 0.13 Direct effect 0.18 0.05 0.33 Total effect 0.23 0.09 0.38 Proportion of total effect mediated 0.20 *Note*. The way to interpret the confidence intervals (CIs) is as follows: If 0 lies within the interval range, no mediation is found. *Source*. TCRAf-Eu Angolan parent survey, The Netherlands 2010-2011. As step two of mediation has been found, we continue with the third and final step to investigate whether the relation between transnational parenting and job instability is eliminated or its effect diminished when including the mediating variables, hence happiness and family-to-work conflict. This is captured in path c' of the mediation model illustrated in [Figure 2](#fig2-0192513X17710773){ref-type="fig"}. If full mediation is found the effect of transnational parenting on job instability should disappear. This is not what is found. Angolan transnational parenting remains significantly related to job instability when including the mediating variables. Additionally, family-to-work conflict is not significantly associated with job instability and hence does not act as a mediator, thereby refuting Hypothesis 1. The second hypothesis is partly confirmed. Lower levels of happiness are significantly related to more job change. Ceteris paribus, respondents reporting to be unhappy change jobs 1.15 times more often than happy respondents (*p* \< .001). Plus, as displayed in [Table 3](#table3-0192513X17710773){ref-type="table"}, the indirect effect of happiness is significant, but not substantial (Β = .07) as only 20% of the effect of transnational parenting is mediated through happiness. Thus, happiness does not fully explain the association between transnational parenting and job instability. In the next section, the analysis is taken one step further by looking at possible differences between transnational parents. Analysis With Transnational Parents {#section11-0192513X17710773} ----------------------------------- The finding that transnational parents experience more not less family-to-work conflict indicates that even though transnational parents do not provide direct care, they do feel that their child caregiving responsibilities affects their work. Thus, transnational separation does not necessarily relieve migrant parents of all child caring burdens. As [@bibr33-0192513X17710773] indicate, what is more important for experiencing family-work conflict is not the type of care arrangements but the satisfaction with this arrangement. [@bibr38-0192513X17710773] has argued that a malfunctioning transnational child-raising arrangement in which parents feel their child is not being taken care of properly, can prevent the transnational parent from doing the job well because of lack of concentration or because worries can lead to sleepless nights. Yet when arrangements work well, transnational parenting is less problematic. As explored elsewhere, it seems that transnational child-raising arrangements that are poor or ill-functioning can affect the relationships parents retain with their children and also affect happiness ([@bibr25-0192513X17710773]; [@bibr26-0192513X17710773]). Therefore, one of the reasons why differences between transnational parents and nontransnational parents regarding job instability might be found could be that some of these transnational parents have ill-functioning transnational child-raising arrangements causing them to experience stress and consequently to perform less well at their jobs. To investigate the association between poor functioning transnational child-raising arrangements and job instability, a regression analysis was run including only transnational parents. A poor parent--child relationship could not be included as a measure of poor functioning arrangements because of collinearity. Therefore, the amount of contact the parent has with the child is used as a proxy for poor functioning child-raising arrangements as previous analyses indicate this to be the most important factor in explaining poor relationships with children ([@bibr25-0192513X17710773]). In addition, upholding contact is one of key ways to maintain family ties and to engage in transnational parenting ([@bibr8-0192513X17710773]; [@bibr73-0192513X17710773]). The amount of contact is a continuous measure, coded (1) not at all, (2) once or twice a month, (3) once a week, (4) a few times a week, and (5) everyday (mean is 3.43). Results of the regression analysis are presented in [Table 4](#table4-0192513X17710773){ref-type="table"}. This analysis reveals that especially poor functioning transnational child-raising arrangements are associated with job instability. That is, Angolan transnational parents who have limited contact with their children have changed their jobs in the Netherlands more often. One unit decrease in contact leads to 0.41 more job changes. Next to happiness (β = .35) the amount of contact with the child is also one of the most important predictors of job change (β = .28) in this regression model. ###### Amount of Contact With Child on Job Instability of Transnational Parents.  Job instability (*n* = 82) -------------------------------------------------------------------------------- ----------------------------------------------------------------------- ------- Happiness^[a](#table-fn9-0192513X17710773){ref-type="table-fn"}^ −1.39 (0.44)[\*\*](#table-fn11-0192513X17710773){ref-type="table-fn"} −0.35 Family-to-work conflict^[b](#table-fn9-0192513X17710773){ref-type="table-fn"}^ 0.33 (0.64) 0.07 Age −0.01 (0.05) −0.03 Sex^[c](#table-fn9-0192513X17710773){ref-type="table-fn"}^ 0.64 (0.46) 0.15 Marital status^[d](#table-fn9-0192513X17710773){ref-type="table-fn"}^ 0.35 (0.56) 0.07 Education 0.30 (0.14)[\*](#table-fn11-0192513X17710773){ref-type="table-fn"} 0.25 Years in the Netherlands 0.11 (0.05)[\*](#table-fn11-0192513X17710773){ref-type="table-fn"} 0.28 Housing^[e](#table-fn9-0192513X17710773){ref-type="table-fn"}^ 0.28 (0.45) 0.07 Number of friends in the Netherlands 0.05 (0.11) 0.04 Number of family members in the Netherlands −0.15 (0.20) −0.08 Dutch proficiency −0.07 (0.21) −0.04 Child \< 8 years of age^[f](#table-fn9-0192513X17710773){ref-type="table-fn"}^ −0.01 (0.46) −0.00 Amount of contact with child −0.41 (0.16)[\*](#table-fn11-0192513X17710773){ref-type="table-fn"} −0.28 *R* ^2^ 0.44 *Note. SE* = standard error. Superscripts indicate reference categories that include (a) unhappy; (b) no work-to-family conflict; (c) male; (d) married/in a relationship; (e) room, student housing, institution, other; and (f) no children \< 8 years of age. *Source*. TCRAf-Eu Angolan parent survey, The Netherlands 2010-2011. *p* \< .05. \*\**p* \< .01. \*\*\**p* \< .001 (one-tailed test). Discussion and Conclusions {#section12-0192513X17710773} ========================== This article aimed to complement research on transnational families by investigating absenteeism and job stability of transnational parents. While the growing body of literature on transnational families has examined how migrant parents raise their children transnationally and how they are affected in their well-being by the separation, it has not explicitly focused on labor market outcomes. This is an important gap in the literature because the fact that these parents have been found to suffer emotionally from the separation could affect their lives in other areas, such as in their professional lives. Therefore, this study set out to explore whether job outcomes are associated with transnational parenting. By combining insights from organizational psychology with transnational family studies, two opposing hypotheses have been examined. On the one hand, it was hypothesized that transnational parents would experience less family-to-work conflict because direct care of their children is provided in the country of origin. On the other hand, based on the happy productive worker thesis, it was hypothesized that as transnational parents are found to display low subjective well-being, job outcomes would be less favorable. Based on the results of the mediation analyses, the first hypothesis can be refuted. Angolan transnational parents experience more family-to-work conflict instead of less. Additionally, family-to-work conflict did not mediate the association between transnational parenting and favorable job outcomes. The second hypothesis is only partly confirmed. First, Angolan transnational parents significantly changed jobs more often than nontransnational parents (job instability), but no difference is found with regard to job absenteeism. Second, Angolan transnational parents' job changes are in part mediated by their lower happiness, but only by 20%. Hence, the direct effect of transnational parenting remains important. Further analyses indicate that especially transnational parents who are in limited contact with their children in Angola experience more job instability. Thus, it was argued that transnational parents, who engage less in transnational parenting or who are involved in poor functioning transnational child-raising arrangements, are especially affected by the parent--child separation in terms of unfavorable job outcomes. Several implications can be drawn from this exploratory research relevant for the study of transnational families. First, it is important not to overstate the extent that transnational separation affects parents' job outcomes. Only differences are found with regard to job instability, not for absenteeism. Also, the finding that especially poor functioning transnational child-raising arrangements are associated with job instability implies that when arrangements work well, transnational parenting is less problematic. Nonetheless, the fact that Angolan transnational parents report higher family-to-work conflict and more job instability suggests that transnational parent--child separation can affect migrant parents in other areas besides emotional well-being. Although care is arranged transnationally and involves limited physical contact, parents continue to be actively engaged in raising their children. Even at a distance, migrant parents keep in constant contact with their children and are affected by their children's needs and well-being ([@bibr18-0192513X17710773]; [@bibr30-0192513X17710773]; [@bibr46-0192513X17710773]). The finding that caring for a child can negatively affect transnational parents in their job outcomes, more than nontransnational parents, is perhaps because parents have less control over the care of their child, for example, in terms of parental authority, and what is happening in the country of origin ([@bibr3-0192513X17710773]; [@bibr6-0192513X17710773]; [@bibr46-0192513X17710773]). By extension, especially for Angolan transnational parents with limited contact with their children in the origin country, job instability is prevalent. It has been demonstrated empirically that limited contact is associated with poor parent--child relationships, which can cause poor parental emotional well-being ([@bibr25-0192513X17710773]; [@bibr26-0192513X17710773]). Poorly functioning arrangements can keep transnational parents from performing well at the job because worries can lead to loss of concentration or sleepless nights ([@bibr38-0192513X17710773]). Although this exploratory study has hinted at possible associations between transnational parenting and job outcomes, future research should investigate this association among different migrant groups, as the Angolan case is quite particular. First, sub-Saharan African families are fluid and the extended family plays an important role in child rearing. As many other sub-Saharan African countries, Angola has a history of child fostering in which children are relocated to other households for extended periods of time ([@bibr41-0192513X17710773]; [@bibr43-0192513X17710773]). In this context, transnational parenting is less of a break from child rearing norms than in communities where copresent parenting is the norm. The mediation between transnational parenting, happiness, and job outcomes could therefore be stronger among other migrant groups. In addition, the postconflict context of Angola is important to consider. Trauma from war can increase effects of family separation because refugees are extra sensitive to additional disruption ([@bibr58-0192513X17710773]). Furthermore, leaving a child in a postconflict/conflict and thus unsafe setting can lead to worry and anxiety ([@bibr58-0192513X17710773]). Also, the war has led to family dislocation, breakup of affective bonds and, hence, fragmented care networks because almost all families have lost a member through war violence ([@bibr42-0192513X17710773]). These fragmented care networks limit the pool of good careers and could lead to worries regarding child care, which can result in increased work--family conflict and loss of concentration at work. In fact, a study indicates that Angolan children in transnational families show lower emotional well-being than children in transnational families in Nigeria and Ghana; countries that have comparable child-raising norms ([@bibr39-0192513X17710773]). This suggests that transnational family separation may have a larger impact on children living in a postconflict setting, which in turn can affect transnational parents' well-being. Finally, war and postconflict settings can keep family members from being in regular contact and lead to long and unplanned separations. This can lead to strained relationships, which are also found to affect parental well-being ([@bibr26-0192513X17710773]). There are potential limitations of this study that have to be taken into consideration. First, measures of job outcomes in this study are objective measures that can be asked to respondents themselves. Some studies investigating job outcomes or performance use supervisory ratings. Although this latter measure is less objective, it can capture the phenomenon of people feeling stressed and performing less well at the job, while still keeping their job. The measures used in the current study are more conservative as they measure days absent from a job and whether someone keeps a job. Consequently, the effects of transnational parenting on job outcomes may be underestimated, which might explain why no difference is found with regard to job absenteeism. At the same time, the fact that some results are significant is all the more telling given the conservative measures used. Second, the sampling strategy might have led to some bias. Random sampling is not possible in this study that requires specific inclusion criteria and as a result the data are not nationally representative. Third, the analysis is based on a relatively small number of observations and hence has limited statistical power, especially with regard to the last analysis including only transnational parents. Fourth, the data are cross-sectional and we can therefore not be conclusive about the causality of the effects. In particular, the association between well-being and job outcomes may reflect reverse causation and should therefore be interpreted as associations. Longitudinal organizational behavioral research has proved job performance and well-being are bidirectional and hints at the reciprocal nature of the relation. Thus, the mediation analysis should be treated as descriptive. Future qualitative and longitudinal studies are required to validate the meditational relations and explore the different mechanisms that are at play. Despite these limitations, this research is a first attempt to study the association between transnational parenting and job outcomes. It does so by bringing together previously separate bodies of literature: organizational psychology and transnational family studies. This study indicates that transnational parenting can affect job outcomes of parents and thus have direct consequences for participation in the host society. This is especially true when parents and children are in limited contact leading parents to worry more about their children. Matters that happen thousands of miles away can negatively affect migrants' lives in the receiving country significantly and in various ways. However, when arrangements work well, transnational parenting is less problematic. **Author's Note:** The TCRAf-Eu project is coordinated by Maastricht University (V. Mazzucato) and is formed, additionally by University College Cork (A. Veale), University of Lisbon (M. Grassi) and FAFO Institute for Applied International Studies (C. Øien). **Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. **Funding:** The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Results presented in this article have been obtained within the Transnational Child-Raising Arrangements between Africa and Europe project (TCRAf-Eu). The research leading to these results has received funding from NORFACE Research Programme "Migration in Europe---Social, Economic, Cultural and Policy Dynamics" (Grant number 315). For more information on the project, refer to [www.tcra.nl](http://www.tcra.nl). The questionnaire and the codebook can also be obtained here. Two respondents indicated to have changed jobs 20 times while being in the Netherlands; these outliers have been omitted from the analyses.

Background ========== It has been more than 30 years since human T-cell leukemia virus type-I (HTLV-1) was shown to be the causative agent of adult T-cell leukemia (ATL) \[[@B1],[@B2]\]. However, understanding the true nature of the multiple leukemogenic events \[[@B3]\] that are essential for this aggressive transformation remains elusive \[[@B4]-[@B9]\]. Although approximately 5% of HTLV-1-infected individuals develop ATL after a long latency period, the majority remain asymptomatic carriers (ACs) throughout their lifetimes. However, there are not enough clear determinants to distinguish between individuals who eventually develop ATL and those who remain as ACs \[[@B10],[@B11]\]. To discover the factors associated with disease development, long-term prospective studies have assessed the correlation between disease outcome and proviral load (PVL), that is, the percentage of infected cells among the total peripheral blood mononuclear cells (PBMCs) \[[@B10]-[@B12]\]. The 'Joint Study on Predisposing Factors of ATL Development' (JSPFAD) \[[@B13]\] showed that a PVL higher than 4% is one of the indications of risk for progression to ATL \[[@B10]\]. Although an elevated PVL is currently the best characterized factor associated with a high risk of ATL development, a high PVL alone is not sufficient for disease prediction, suggesting the need to discover additional predictive factors \[[@B10],[@B11]\]. Because ATL is a malignancy caused by HTLV-1 infection, both the integration of provirus into the host genome and the clonal expansion of infected cells are highly critical leukemogenic events \[[@B6],[@B7],[@B14],[@B15]\]. Although many studies have addressed these aspects, the mechanism of HTLV-1 clonal expansion has not been elucidated \[[@B15]-[@B35]\]. Accurate monitoring for changes in clonality occurring before, during, and after ATL development is of great interest and of major clinical significance not only to clarify the underlying mechanisms but also to discover reliable predictive biomarkers for disease progression. A broad range of evidence strongly supports that most neoplasms are composed of clonally expanded cell populations \[[@B36]-[@B38]\]. Owing to its biological significance, the concept of clonal expansion in cancer biology has been investigated using a variety of approaches in many tumor types \[[@B36]-[@B39]\], including ATL \[[@B6],[@B15],[@B16],[@B18]-[@B20],[@B22],[@B24],[@B29]-[@B32]\]. Clonal proliferation of HTLV-1-infected cells was first detected as monoclonal-derived bands by southern blotting \[[@B33]\]. Early studies found that monoclonal integration of HTLV-1 is a hallmark of ATL cells \[[@B16]\]. Furthermore, it was suggested that detecting a monoclonal band is useful for diagnosis and is associated with a high risk of ATL development \[[@B29],[@B30]\]. Subsequent PCR-based methods included inverse PCR, linker-mediated PCR, and inverse long PCR, which enabled analysis of samples with clonality below the detection threshold of southern blotting \[[@B17],[@B25],[@B31],[@B34]\]. Based on the observed banding patterns, the clonality of the samples was described as having undergone monoclonal, oligoclonal, or polyclonal expansion. Such PCR-based analyses revealed that, in addition to a monoclonal proliferation of infected cells, a monoclonal or polyclonal proliferation occurs even in non-malignant HTLV-1 carriers \[[@B31],[@B35]\]. Moreover, considering the stability of the HTLV-1 proviral sequence, it was hypothesized that maintaining a high PVL is achieved by persistent clonal proliferation of infected cells *in vivo*\[[@B25]\]. This hypothesis was further supported by the detection of a particular HTLV-1 clone in the same carrier over the course of several years \[[@B18]\]. Two Miyazaki cohort studies focused on the maintenance and establishment of clonal expansion: Okayama *et al.* analyzed the maintenance of a pre-leukemic clone in an AC state several years prior to ATL onset \[[@B19]\], and Tanaka *et al.* assessed the establishment of clonal expansion by comparing the clonality status of long-term carriers with that of seroconverters. They showed that some of the clones from long-term carriers were stable and large enough to be consistently detectable by inverse long PCR; however, those from seroconverters were unstable and rarely detectable over time \[[@B20]\]. Knowledge provided by conventional studies has shed light on the next challenges worthy of further investigation. Owing to technical hurdles, however, previous studies isolated small numbers of integration sites from highly abundant clones and detected low abundant clones in a non-reproducible manner \[[@B22],[@B34]\]. Furthermore, conventional techniques could not provide adequate information regarding the number of infected cells in each clone (clone size) \[[@B22]\]. To effectively track and monitor HTLV-1 clonal composition and dynamics, we considered devising a new method that would not only enable the high-throughput isolation of integration sites but also provide an accurate measurement of clone size. PCR is a necessary step for the integration site isolation and clonality analysis. However, bias in the amplification of DNA fragments (owing to issues such as extreme fragment length and high GC content) is intrinsic to any PCR-based method \[[@B40]-[@B45]\]. Different fragment amplification efficiencies make it difficult to calculate the amount of starting DNA (the original distribution of template DNA) from PCR products. Hence, estimating HTLV-1 clonal abundance, which requires calculating the number of starting DNA fragments, is only achievable by avoiding the PCR bias. Recently, Bangham's research group analyzed HTLV-1 clonality and integration site preference by a high-throughput method \[[@B22]\]. In the method developed by Gillet *et al.*, clone sizes were estimated using length of DNA fragments (shear sites generated by sonication) as a strategy for removing PCR bias \[[@B22]\]. Owing to the limited variation in DNA fragment size observed with shearing, the probability of generating starting fragments of the same lengths is high, leading to a nonlinear relationship between fragment length and clone size \[[@B22],[@B46]\]. Therefore, Gillet *et al.* used a calibration curve to statistically correct the shear site data \[[@B22]\]. Later, Berry *et al.* introduced a statistical approach, and further addressed the difficulties of estimating clone size from shear site data \[[@B46]\]. Their approach estimates the size of small clones with little error, but estimates for larger clones have greater error \[[@B46]\]. A parameter adopted from the Gini coefficient \[[@B47],[@B48]\] and termed the oligoclonality index was used to describe the size and distribution of HTLV-1 clones \[[@B22]\]. It has been demonstrated that the oligoclonality index differs between malignant and non-malignant HTLV-1 infections, and also a high PVL of HTLV-1-associated myelopathy is due to cells harboring large numbers of unique integration sites \[[@B22]\]. Furthermore, genome-wide integration site profiling of clinical samples revealed that the abundance of a given clone *in vivo* correlates with the features of the flanking host genome \[[@B22],[@B24]\]; although there was not a specific hotspot, HTLV-1 more frequently integrated in transcriptionally active regions of the host genome \[[@B22],[@B24]\]. These findings further clarified the characteristics of HTLV-1 integration sites, and strongly suggested the importance of HTLV-1 clonal expansion *in vivo*. Here we introduce a method that overcomes many of the limitations of currently available methods. Taking advantage of next-generation sequencing (NGS) technology, nested-splinkerette PCR, and a tag system, we designed a new high-throughput method that enables specific isolation of HTLV-1 integration sites and, most importantly, allows for the quantification of clonality not only from the major clones and high-PVL samples but also from low-abundance clones (minor clones) and samples with low PVLs. Moreover, we conducted comprehensive internal validation experiments to assess the effectiveness and accuracy of our new methodology. A preliminary validation was conducted by analyzing DNA samples from HTLV-1-infected individuals with different PVLs and disease status. Subsequently, an internal validation was performed that included an appropriate control with known integration sites and clonality patterns. We present our methodology, which illustrates that employing the tag system is effective for improving quantification of clonal abundance. Methods ======= Our clonality analysis method included two main aspects: (1) wet experiments, and (2) *in silico* analysis (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1). A general explanation of materials and methods is provided here, and detailed protocols of the wet experiments are included in Additional file [1](#S1){ref-type="supplementary-material"}: Notes. The *in silico* analysis is further described in Results and discussion. NGS data have been deposited in the Sequence Read Archive of NCBI with access number of (SRP038906). Wet experiments --------------- ### ***Biological samples: specimens and cell lines*** Specimens: In total five clinical samples were provided by a biomaterial bank of HTLV-1 carriers, JSPFAD \[[@B13],[@B49]\]. The clinical samples were a part of those collected with an informed consent as a collaborative project of JSPFAD. The project was approved by the Institute of Medical Sciences, the University of Tokyo (IMSUT) Human Genome Research Ethics Committee. Information about the disease status of samples was obtained from JSPFAD database in which HTLV-1-infected individuals were diagnosed based on the Shimoyama criteria \[[@B50]\]. In brief, genomic DNA from PBMCs was isolated using a QIAGEN Blood kit. PVLs were measured by real-time PCR using the ABI PRISM 7000 Sequence Detection System as described in \[[@B10]\]. Cell lines: An IL2-dependent TL-Om1 cell line \[[@B51]\] was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 1% penicillin-streptomycin (GIBCO), and 10 ng/mL IL2 (R&D systems). The same conditions as those of patient samples were used to extract DNA and measure PVL. ### ***Illumina-specific library construction*** We employed a library preparation protocol specifically designed to isolate HTLV-1 integration sites. The final products in the library that we generated contained all the specific sequences necessary for the Illumina HiSeq 2000 platform (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S2). These products included a 5′-flow cell binding sequence, a region compatible with read-1 sequencing primer, 5-bp random nucleotides, 5-bp known barcodes for multiplexing samples, HTLV-1 long terminal repeat (LTR), human or HTLV-1 genomic DNA, a region compatible with read-2 and read-3 sequencing primers, 8-bp random tags, and a 3′-flow cell binding sequence from 5′ to 3′, respectively (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S2B). Incorporating the 5-bp random nucleotides downstream of the region compatible with the read-1 sequencing primer was critical and resulted in high-quality sequence data. We used a library designed without the first 5-bp of random nucleotides as input for the HiSeq 2000 sequencer in our first samples (S-1, S-2, S-3, and S-4). Because all fragments began with the same LTR sequence, clusters generated in the flow cells could not be differentiated appropriately. These samples resulted in low-quality sequence data (see Additional file [1](#S1){ref-type="supplementary-material"}: Notes). Designing the first 5-bp randomly resulted in high-quality sequence data for the remaining samples because clusters were differentiated with no problem during the first five cycles of sequencing (data not shown). Our library construction pipeline comprised the following four steps (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S2) (Additional file [1](#S1){ref-type="supplementary-material"}: Notes): {ref-type="supplementary-material"}: Figure S2 for a simple image from an integration site and its shear sites. **(A)** Depicted is the complex population of uninfected cells (grey circles) together with infected clones (circles of different colors). A clone is shown as a group of sister cells (circles of the same color) having the same integration site (IS). Different clones are distinguishable based on differing integration sites, and thus the number of integration sites represents the number of infected clones. For example, the six different unique integration sites refer to six unique clones. **(B)** Genomic DNA fragmented by sonication generates random shear sites (fragments of different length). Fragment size, measured by an Agilent Bioanalyzer, ranged from 300 to 700 bp. This size range can theoretically provide approximately 400 variations. **(C)** The size distribution of fragments decreased following amplification by integration-site-specific PCR. From the deep sequencing data, the original number of starting fragments could be estimated by removing PCR duplicates and counting fragments with different lengths. For example, five different lengths of PCR amplicons represent five infected sister cells. **(D)** We analyzed four samples, including (S-1: asymptomatic carrier (AC), (8% PVL)), (S-2: smoldering (SM), (9% PVL)), (S-3: smoldering, (31% PVL)), and (S-4: acute, (33% PVL)). Using our method, the clone sizes were quantified by considering only shear sites. The first major clone (the largest clone) of each sample was mapped to (chr 11-41829319 (+)), (chr 15: 59364370 (+)), (chr 4-563543 (-)), and (chr X - 83705328 (-)), respectively. The shear site variations of each major clone were 209, 119, 242, and 222, respectively. Different colors on the pie graphs indicate different integration sites, and the size of each piece represents the clone size.](gm568-1){#F1} \(1\) DNA isolation: DNA was extracted as described above, and the concentration of extracted DNA was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific). We recommend using 10 μg of DNA as the starting material. However, in practice there are some rare clinical samples with limited DNA available. In order to be able to handle those samples, the method was also optimized for 5 μg and 2 μg of starting DNA.(2) Fragmentation: According to the protocol provided in Supplementary Notes, the starting template DNA was sheared by sonication. The resulting fragments represented a size range of 300 to 700 bp as checked by an Agilent 2100 Bioanalyzer and DNA 7500 kit (Figure [1](#F1){ref-type="fig"}B). \(3\) Pre-PCR manipulations: Four steps of end repair, A-tailing, adaptor ligation, and size selection were performed as described in Additional file [1](#S1){ref-type="supplementary-material"}: Notes. \(4\) PCR: To amplify the junction between the genome and the viral insert, we used nested-splinkerette PCR (a variant of ligation-mediated PCR \[[@B52],[@B53]\]) (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S2). We confirmed that the technique specifically amplifies HTLV-1 integration sites; since there was no non-specific amplification neither from human endogenous retroviruses nor from an exogenous retrovirus such as HIV (see Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Figure S1). Information on oligonucleotides, including adaptors and primers, and the LTR and HTLV-1 reference sequences \[[@B54]\] are provided in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1. The final PCR products were sequenced using the HiSeq 2000 platform. *In silico* analysis -------------------- Raw sequencing data were processed according to the workflow described in the Results and discussion section. The initial forward read (100-bp) was termed Read-1 and the reverse read (100-bp) was termed Read-3 and an index read (8-bp) was termed Read-2. In brief, analysis programs were written in Perl language and run on a supercomputer system provided by The University of Tokyo's Human Genome Center at The Institute of Medical Science \[[@B55]\]. The sequencing output was check for quality using the FastQC tool \[[@B56]\]. The regions corresponding to the LTR and HTLV-1 genome were subjected to a blast search against the reference sequences described in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1. Following isolation of the integration sites, the flanking human sequences were mapped to the human genome (hg19) (the UCSC genome browser \[[@B57]\]) by Bowtie 1.0.0 \[[@B58]\]. The final processed data included information about shear sites (R1R3), tags (R1R2), and a combination of tags and shear sites (R1R2R3). Fitting the data to the zero truncated Poisson distribution for retrieving correlation coefficients were done by the R-package 'gamlss.tr' \[[@B59]\]. The Gini coefficient was calculated by StatsDirect medical statistics software \[[@B60]\]. Results and discussion ====================== General concepts ---------------- We originally designed our method to overcome the limitations of conventional techniques \[[@B31],[@B34]\] and to make improvements in the only existing high-throughput method \[[@B22]\]. In general, our method includes two main sets of wet experiments and an *in-silico* analysis. We used genomic DNA as the starting material to prepare an appropriate library for Illumina sequencing. Subsequently, deep-sequencing data were analyzed by a supercomputer. The resulting information represents the clonality status of each sample (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S1). There are complex populations of infected clones and uninfected cells in a given HTLV-1 infected individual. High-throughput clonality analysis requires monitoring two main characteristics of clones: HTLV-1 integration sites and the number of infected cells in each clone (clone size). Each HTLV-1-infected cell naturally harbors only a single integration site \[[@B23]\]. Therefore, the number of detected unique integration sites corresponds to the number of infected clones. Based on our analysis, which is consistent with the data of Gillet *et al*. \[[@B22]\], employing high-sensitivity deep sequencing allowed for the isolation of a large number of unique integration sites (UISs), including samples with low PVLs (Figure [1](#F1){ref-type="fig"}). We analyzed four samples from HTLV-1-infected individuals with different PVLs, disease status, and expected clonality patterns. The samples include S-1: AC (8% PVL); S-2: smoldering ATL (SM) (9% PVL); S-3: SM (31% PVL); and S-4: acute ATL (33% PVL). Based on the final optimized conditions, 1030, 39, 265, and 384 UISs were isolated from each sample, respectively (Figure [1](#F1){ref-type="fig"}). The most challenging aspect of our clonality analysis was estimating the number of infected cells in each clone. Although a necessary step in the analysis, PCR introduces a bias in the frequency of starting DNA material \[[@B40]-[@B45]\]. Because amplification causes significant changes in the initial frequency of starting materials, PCR products cannot be used directly to estimate the amount of the starting DNA material. To overcome this problem, we needed to manipulate DNA fragments to make them unique prior to PCR amplification. Thus, if each DNA fragment could be marked with a unique feature, it would then be possible to calculate its frequency based on the frequency of that unique feature. When a single unique stretch of DNA is amplified by PCR, the resulting product is a cluster of identical fragments termed PCR duplicates. Therefore, to estimate the frequency of starting DNA fragments, one should count the number of clusters with unique features. The remaining technical question then becomes how to mark the starting DNA prior to PCR amplification. In the following section, we compare and discuss two main strategies, namely (1) shear sites and (2) a tag system, which enable DNA fragments to be uniquely marked. Estimating the size of clones by shear sites -------------------------------------------- The first strategy, described by Gillet *et al.*, relies on shearing DNA by sonication, resulting in fragments of random length \[[@B22]\]. Sonication-derived shear sites were thus used as a distinguishing feature to make fragments unique prior to PCR. Clone sizes were then estimated by statistical approaches \[[@B22],[@B46]\]. To directly assess the effectiveness of the shear site strategy, we analyzed the clonality of the aforementioned clinical samples (S-1, S-2, S-3, and S-4). Genomic DNA was cleaved by sonication with fragments in the 300- to 700-bp range, theoretically providing approximately 400 possible variations in fragment size (Figure [1](#F1){ref-type="fig"}A and B). Following library construction, however, the final product represented smaller size ranges, implying a relatively limited number of variations (Figure [1](#F1){ref-type="fig"}C). Finally, the number of PCR amplicons with unique shear sites was retrieved from deep-sequencing data. See Additional file [2](#S2){ref-type="supplementary-material"}: Figure S2 for a simple image from an integration site and its shear sites. The data obtained from the shear site experiments were not fitted to calibration curves or statistical treatments, which were used by Gillet *et al.* and Berry *et al.*, respectively (See Additional file [1](#S1){ref-type="supplementary-material"}: Notes) \[[@B22],[@B46]\]. For clarity, only the information relating to the major clone of each sample is provided in Figure [1](#F1){ref-type="fig"}D. The shear-site variations of the major clone were 209, 119, 242, and 222 for samples S-1 through S-4, respectively. Even in the case of control samples with 100% PVLs, the shear sites did not provide more than 225 variations (see Validation of the methodology). However, it was expected that samples with differing PVLs and disease status would harbor varying numbers of sister cells, at least in their major clones. Similar variations of shear sites were observed in major clones of AC, SM, and acute samples. These data suggest that, because the number of sister cells in each clone exceeded the shear site variations, the size of the clones was underestimated (Figure [1](#F1){ref-type="fig"}). This is most problematic in the case of large clones and leads to an underestimation of the clone size. Measuring the size of clones by the tag system ---------------------------------------------- We developed an alternate strategy to remove PCR bias and to estimate starting DNA. We designed a tag system in which 8-bp random nucleotides are incorporated at the end of DNA fragments during adaptor ligation step. Each tag acts as a molecular barcode, which gives each DNA fragment a unique signature prior to PCR. Information on the frequency of observed tags from the deep-sequencing data can be used to remove the PCR duplicates and thereby estimate the original clonal abundance in the starting sample. Owing to their random design, the tags could theoretically provide approximately 65,536 variations. This degree of potential variation is expected to provide a unique tag for a large number of sister cells in each clone (Figure [2](#F2){ref-type="fig"}). {#F2} We analyzed samples S-1, S-2, S-3, and S-4 to assess the effectiveness of our tag system for estimating clone size. The major clone of each sample showed tag variations of 393, 142, 1751, and 2675, respectively (Figure [2](#F2){ref-type="fig"}D). Similar variations of tags and shear sites were observed in the largest clones of S-1 and S-2 ((shear sites *vs.* tags): (209 *vs.* 393) and (119 *vs.* 142)) (Figure [1](#F1){ref-type="fig"}D and Figure [2](#F2){ref-type="fig"}D). In all four samples, those variations were also similar in the minor clones of which the clone sizes did not exceed shear sites variations (approximately \<200 variations) (See Additional file [1](#S1){ref-type="supplementary-material"}: Table S3 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S1 for information on the ten largest clones). However, the variations covered by tags were significantly greater than those of shear sites, especially for large clones like those observed in the major clones of S-3 and S-4 ((shear sites *vs.* tags): (242 *vs.* 1751) and (222 *vs.* 2675)). The variations covered by tags and combinations were almost the same for all four samples ((tags *vs.* combinations): (393 *vs.* 296), (142 *vs.* 119), (1751 *vs.* 1192), and (2675 *vs.* 2038)). Upon comparison of the tag system data with the shear site data, it was clear that both strategies yield essentially the same results when the size of clones is small enough to be covered by the number of shear site variations generated. However, the tag system provides a much better estimation of clonality when the number of sister cells in each clone exceeds shear site variations. Therefore, clone size was underestimated when considering only shear sites in expanded clones like samples S-3 and S-4. Given this, our tag system should be used for samples with different clonality status to avoid underestimation of the size of clones. See Additional file [2](#S2){ref-type="supplementary-material"}: Figure S3 for a simple comparison of shear site and tag variations. Validation of the methodology ----------------------------- Our newly developed method - the tag system and the related data analysis - were successfully validated, internally. As mentioned above, the initial validation was done by analyzing samples from different HTLV-1-infected individuals (Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Finally, we conducted a comprehensive internal validation by using an appropriate control with known integration sites and clonality patterns to provide direct evidence for the effectiveness of our system in the clonality analysis. We designed a suitable control because there was not an appropriate control available. Using our system, we could evaluate the method and confirm its accuracy, sensitivity, and reproducibility. We selected two samples with the following special conditions as starting materials for preparing the control system.Sample one (M): DNA from an acute ATL patient with 100% PVLs and a single integration site in the major clone (Figure [3](#F3){ref-type="fig"}A). The integration site of this sample was first checked with conventional splinkerette PCR, which detected a single major integration site. Subsequently, deep-sequencing data (tags only and combinations) showed that approximately 99% of the PVL accounted for the major clone with an integration site at chromosome 12:94976747(-). A small numbers of clones occupied approximately 1% of the PVL of this sample. Those clones were only detected in the second trial samples for which the external PCR products were not diluted. Therefore, to simplify the overall analysis, we removed those low-abundance clones (data not shown).Sample two (T): DNA was isolated from a fresh culture of TL-Om1, which is a registered monoclonal ATL cell line with 100% PVL and a single integration site at chromosome 1:121251270(-) in each cell (Figure [3](#F3){ref-type="fig"}A).Having prepared these two samples, they were sonicated and mixed in proportions of 50:50 and 90:10 (Figure [3](#F3){ref-type="fig"}B). These known proportions were thus expected to generate specific patterns that could be verified with our subsequent analysis. We conducted two independent sets of trials.In the first trial, samples were named as 'first trial control 1 \~ 4' and abbreviated as 1st T-cnt-1 \~ 4. Various amounts of DNA (μg) from samples M and T were mixed to prepare the final expected clone sizes as shown in Figure [3](#F3){ref-type="fig"}C. A 1-μL sample of a 10-fold dilution of external PCR product was used as the starting material for nested PCR for this trial. The samples were run in separate lanes of HiSeq 2000.We named the samples of the second trial as second trial control-1 \~ 4 and abbreviated them as 2nd T-cnt-1 \~ 4. DNA samples were mixed similarly to that for the first trial except for sample four (Figure [3](#F3){ref-type="fig"}D). In contrast to the first trial, we used 1 μL of the external PCR product without any dilution as a starting material for the nested PCR. These samples were multiplexed and run in the same lane of HiSeq 2000. The purpose of the second trial was to test both method reproducibility and the effect that the dilutions had on the results. {#F3} The samples of both the first and second trials were analyzed under the same conditions, except where noted above. For each control sample, expected patterns and experimentally observed patterns were calculated for (a) raw sequence reads, (b) shear sites, (c) only tags, and (d) the combination of tags and shear sites (Figure [4](#F4){ref-type="fig"}). Figure [4](#F4){ref-type="fig"} shows the data when the optimal conditions were considered. Additional file [1](#S1){ref-type="supplementary-material"}: Figure S3 includes most of the data accumulated during optimization of the method. {ref-type="supplementary-material"}: Figure S4 for information on merging approach.](gm568-4){#F4} Evaluating the accuracy of the clonality analyzed based on shear sites *vs.* tags system ---------------------------------------------------------------------------------------- The 'absolute error', a technique used to evaluate system accuracy \[[@B61]\], was used to assess our method. The experimental values were subtracted from expected values (Figure [5](#F5){ref-type="fig"}A). Taking advantage of our control system (the first and second trial samples), the clone size was calculated by considering (a) sequencing reads without removing PCR duplicates, (b) only shear sites, (c) only tags, and (d) the combination of tags and shear sites (Figure [5](#F5){ref-type="fig"}B and C). The absolute errors of raw sequence reads for the first trial samples were 23.58, 6.26, 4.57, and 5.72, whereas those of the second trial samples were 44.66, 9.50, 6.88, and 60.24. The magnitude of errors in the first trial was lower than that of the second trial probably due to the dilution of the external PCR products in the first trial. However because dilution reduced the number of covered integration sites, it should be done sparingly and with the purpose of the experiments in mind. The errors when considering only shear sites were 1.72, 34.33, 21.76, and 18.73 for the first trial and 0.47, 38.29, 36.72, and 40.47 for the second trial. Underestimations caused by low shear site variation did not affect the relative size of clones when the expected size of the clones was 50% *vs.* 50%. In this situation, shear sites had the smallest error: 1.72 for 1^st^ T-cnt-1 and 0.47 for 2^nd^ T-cnt-1. {ref-type="fig"}). The *y* axis represents the percentage of absolute errors in different conditions including: (1) raw sequencing reads without removing duplicated PCR, (2) only shear sites, (3) only tags, and (4) the combination of tags and shear sites. The absolute errors of the final optimal condition: the first trial: (Bowtie parameters: -v 3 - - best, and filtering condition: (merging approach) JT-10), and the second trial: (Bowtie parameters: -v 3 - - best, and filtering condition: (merging approach) JT-10-1%) are presented in this figure. Please refer to Additional file [1](#S1){ref-type="supplementary-material"}: Figure S6 for the absolute errors in all examined conditions. **(B)** The absolute errors of the first trial. **(C)** The absolute errors of the second trial. See Additional file [1](#S1){ref-type="supplementary-material"}: Figure S4 for information on merging approach.](gm568-5){#F5} The errors were reduced in the data using the tag system: 7.27, 5.23, 14.49, and 6.50 for the first trial, and 6.67, 7.07, 10.07, and 13.16 for the second trial. In the case of the combination of tags and shear sites, errors were: 6.98, 4.06, 0.21, and 1.31 for the first trial and 3.42, 10.51, 12.26, and 5.83 for the second trial. Interestingly, the samples 'tags only' and 'combinations' showed similar error levels. Based on these data, our system showed lower absolute errors than when considering only shear sites (Figure [5](#F5){ref-type="fig"}) (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S4). Owing to differences in analyzed samples and system setups, we could not directly compare our data with published data \[[@B22],[@B46]\]. Indirect evidence, however, provided by shear site analysis of our own data illustrated that our system has lower absolute errors than using the shear site-based methodology. *In-silico* analysis -------------------- Processing, management, and analysis of the large amount of data generated by deep sequencing require special infrastructures and bioinformatics skills. We designed a data analysis and interpretation pipeline specific for HTLV-1 integration sites and clonality studies. The workflow is provided in Figure [6](#F6){ref-type="fig"}. First, the raw data for high-throughput sequencing were checked for quality by the FastQC tool. We then removed the first 5-bp random nucleotides from read-1 and de-multiplexed those samples that were run in the same lane of the HiSeq 2000 based on 5-bp of the known sequence (Figure [6](#F6){ref-type="fig"} and Additional file [1](#S1){ref-type="supplementary-material"}: Figure S2). The downstream 23 nucleotides, which represented LTR-specific primers, were also trimmed before further analysis. We then separated the remaining sequence of read one into two different datasets: (1) LTR sequence and (2) HTLV-1 or human sequence. The former comprises the 27-bp sequence remaining from the LTR, whereas the latter is composed of the 41-bp or 45-bp HTLV-1 or human sequence. In the case of multiplexed and non-multiplexed samples, different lengths (that is, 41-bp and 45-bp) were available for analysis. Both sets were subjected to blast analysis against LTR and HTLV-1 reference sequences with one or two mismatches permitted, respectively. Reads for which the sequence did not match HTLV-1 were presumed to be human as long as their 27-bp LTR sequences matched the LTR reference sequence. The resulting human reads were mapped to the human genome (hg19) using Bowtie 1.0.0 \[[@B58]\]. We employed various parameters of Bowtie and different lengths of read three to obtain the optimal mapping yield (Additional file [1](#S1){ref-type="supplementary-material"}: Table S2). These conditions were achieved when a maximum of three mismatches were permitted (-v parameter) and when the best alignment regarding the number of mismatches was reported (\--best parameter). In addition, use of the same length of read-1 as in read-3 allowed for better mapping results. Mapping results are further discussed in Additional file [1](#S1){ref-type="supplementary-material"}: Notes.The 5′-mapped regions were considered to be the positions of integration sites and reported as (chromosome: position: (strand)) for example, (chr1:121251270: (-)). In addition, 3′-mapped regions from read-3 were reported as shear sites for each corresponding position. Information on the tags, obtained from read-2, was used to determine the size of clones as described in subsection: Measuring the size of clones by the tag system. Final outputs of our analysis - the three main reports: R1R3, R1R2, and R1R2R3 - include information on shear sites, tags, and a combination of tags and shear sites, respectively (Figure [6](#F6){ref-type="fig"}). {#F6} Removing background noise ------------------------- Data obtained from next-generation sequencers are not error free \[[@B40],[@B62]-[@B65]\]. There are many reports on the error rate of Illumina sequencers \[[@B66],[@B67]\]. Teemu Kivioja *et al.* recently developed a system named unique molecular identifiers (UMIs) for quantifying mRNAs and employed filtering criteria to remove false UMIs generated by sequencing errors \[[@B68]\]. In our study, consistent with the data of Kivioja *et al*. \[[@B68]\], the sequencing errors produced false tags with low frequencies. A filtering system was required to remove those tags, which could affect interpretation of our clonality data and reduce the accuracy of the clone size measurement. To minimize the effect of sequencing errors on data interpretation, we tested different filtering conditions to remove background noise. Here, we report our proven filtering approach (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S4). Considering that tags are designed randomly, each tag has an equal probability of being observed. Hence, the distribution of tags should be fitted to the zero truncated Poisson distribution \[[@B59],[@B68]\]. Therefore, we test data fit to the Poisson distribution to determine the efficacy of each filtering condition. The distribution of tags for each sample was measured by the R-package 'gamlss.tr' \[[@B59]\], and the correlation coefficient was compared before and after filtering (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S6). We used a filtering system, which we named the merging approach. The merging approach was conducted by clustering the tags and allowing only one mismatch so that unique tags, differing only in one nucleotide (one-mismatch permission), were merged. Subsequently, if the frequency of observed tag reads (PCR duplicates) was greater than 10, those unique tags were employed in further analysis. Otherwise, they were considered as artifacts. We referred to this filtering approach as 'Join Tag- remove10' (JT-10) in the Figure legends. To facilitate understanding, these filtering conditions are illustrated in Additional file [1](#S1){ref-type="supplementary-material"}: Figure S4. Final discussion ---------------- The advent of NGS technologies holds promise to reveal the complex nature of neoplasms and to move past the limitations of previous methods. Using different approaches starting from early cytogenetic analysis to later, more elaborate studies with NGS technologies, the clonal composition of different tumors has been analyzed \[[@B36]-[@B39]\]. Robust monitoring and tracking of clonal dynamics using provirus integration sites allow for the assessment of clonal composition of HTLV-1-infected individuals from early infection to the final stage of ATL development. To meet the technical requirements for such type of analysis, we combined our expertise in the field of HTLV-1 research and NGS analysis and developed the high-throughput methodology described herein. Gillet *et al.* also recently introduced a high-throughput method to extensively characterize HTLV-1 integration site preferences and quantify clonality (further discussed in Additional file [1](#S1){ref-type="supplementary-material"}: Notes) \[[@B22]\]. They statistically analyzed shear site data to estimate clone size. According to their published data \[[@B22],[@B46]\] and as well as our current data, the limited variation in shear sites leads to an underestimation of the size of large clones. Considering that the incidence of large clones increases with disease progression from the healthy AC state to the malignant states of smoldering, chronic, or acute \[[@B22],[@B46]\], an accurate measurement of clone size - particularly large clones - is of great clinical significance. Our study is the first in which the size of large clones was experimentally measured without using statistical estimation. We have provided details of the method design, optimized experiment protocols, and *in-silico* data processing workflow. To validate our methodology and assess its accuracy, we analyzed eight control samples with known integration sites and clone sizes, and four clinical samples. We subjected the samples to deep sequencing so that they had enough read coverage for each integration site and to ensure accurate measurement of clone size (See Additional file [1](#S1){ref-type="supplementary-material"}: Notes). We proved our methodology to be reliable for isolating large numbers of integration sites and to be accurate for quantifying clone size. Because the tag system could provide a sufficient number of variations regardless of clone size, we were able to demonstrate that the measurements are accurate. Preliminary experiments on the clinical samples with differing PVLs and disease status showed different clonality patterns specific to AC and different ATL disease subtypes. S-1 was selected to represent still-healthy but infected individuals (ACs), S-2 and S-3 to represent indolent types of ATL, and S-4 to represent a typical aggressive type of ATL. Despite similar PVLs, S-1 and S-2 could be distinguished based on clonality patterns (polyclonal *vs.* a shift towards oligoclonal): S-1: AC, 8% PVL, and S-2: SM, 9% PVL. The clones of AC showed a uniform distribution pattern with no large difference in clone size; clones of S-2, however, had non-uniform size. S-2 and S-3 (S-3: SM, 31% PVL) are both smoldering subtypes of ATL progression with differing PVLs (9% *vs.* 31%) and showed similar clonality patterns but a different number of infected cells in each clone. S-3 and S-4 had similar PVL (S-4: acute, 33% PVL) but exhibited different clonality patterns: oligoclonal for S-3 (three or four relatively large clones at the top surrounded with other clones) *vs.* monoclonal for S-4 (a large major clone surrounded with some small clones in the background). After ranking the clones in order of descending size, we noted that the size of the largest clone in the acute sample was 10 times that of the next clone (tags: (chr X: 83705328 (-)) = 2675 *vs.* (chr 14: 30655896 (+)) = 209). Relative size of the major clone (chr X: 83705328 (-)) was also estimated by another method (PCR-southern) (detailed information is provided in Additional file [2](#S2){ref-type="supplementary-material"}: Figure S3 and Additional file [2](#S2){ref-type="supplementary-material"}: Supporting experiments). Samples with distinct disease status (AC, SM, and acute) manifested different clone sizes (Additional file [1](#S1){ref-type="supplementary-material"}: Table S3 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S1 include the number of infected cells in the top 10 clones), but S-1 *vs.* S-2 (0.60 *vs.* 0.67) and S-3 vs. S-4 (0.84 *vs.* 0.80) could not be discriminated based on their oligoclonality index (Additional file [1](#S1){ref-type="supplementary-material"}: Figure S7) (See Additional file [1](#S1){ref-type="supplementary-material"}: Notes for further discussion). Therefore, it can be inferred that, with an accurate measurement of clone size, the application of this method will aid in the discrimination of ATL subtypes. These results suggest a possible association between disease status, PVLs, and clonality patterns. Hence, HTLV-1-infected individuals could be classified in different groups based on their clonality patterns, which could ultimately affect their choice of therapy and estimation of prognosis. Moreover, by interpreting information from previous studies on HTLV-1 clonality \[[@B15],[@B18]-[@B20],[@B22],[@B27],[@B31],[@B32],[@B35]\] and considering the data provided in our present paper, it appears that ACs harbor a polyclonal population of HTLV-1-infected cells, whereas ATL patients show monoclonal patterns. Thus, changes in the clonality pattern and onset of a clonal expansion of HTLV-1-infected cells seem to be potentially applicable as a prognostic indicator of ATL onset. For these purposes, it is necessary to analyze appropriate pools of samples from ACs and different subtypes of ATL and to conduct a cohort study on the clonality patterns of the sequential samples available over time. Conclusions =========== We took advantage of next-generation sequencing technology, a tag system, and an *in-silico* analysis pipeline to develop and internally validate a new high-throughput methodology. The method was proved to accurately measure the size of clones by analyzing control samples with already known clone sizes and clinical samples. We also discussed the novelty, significance, and applications of our method, and compared it with the only existing high-throughput method devised by Gillet *et al.*\[[@B22]\]. Employing our new methodology and the analysis of an appropriate pool of samples provided by JSPFAD \[[@B13]\] will be helpful not only for diagnosis and prediction but also for elaborated understanding of the underlying mechanism of ATL development. The methodology described here could be adapted to investigate and quantify other genome-integrating elements (such as proviruses, transposons, and vectors in gene therapy). In addition, the tag system can be used for quantifying DNA/RNA fragments in RNA expression \[[@B68]\] or in metagenomics for determining the size of bacterial populations. Competing interests =================== The authors declare that they have no competing interests. Authors' contributions ====================== TW, TY, YS, SS, and SF conceived the project. SF designed and carried out the experiments and wrote the manuscript. YL prepared the Perl scripts. YL and SF performed *in-silico* data analysis. SF and TY analyzed and interpret the data. YS, SS and SF contributed in sequencing the samples. YS and KN contributed to *in-silico* data analysis. TY, YL, TW and YS assisted in drafting the manuscript. TY and YS advised the direction of study. TW supervised the study. All authors read and approved the final manuscript. Supplementary Material ====================== ###### Additional file 1 **Supplementary data include (1) Supplementary Notes: '*Supplementary materials and method*' and '*Supplementary results and discussion*' (2) Supplementary figures and tables: seven figures, and three tables provided in a PDF file.** ###### Click here for file ###### Additional file 2 Additional supporting data include (1) Additional supporting protocols and (2) Additional supporting experiments: four figures and one table provided in a PDF file. ###### Click here for file Acknowledgements ================ We gratefully appreciate: JSPFAD for providing clinical samples; M. Nakashima and T. Akashi for maintenance of JSPFAD; Sung-Joon Park, Riu Yamashita, and Kuo-ching Liang for their invaluable advice on *in-silico* analysis; K. Abe, K. Imamura, T. Horiuchi, and M. Tosaka for sequencing technical support; Sara Firouzi and Unes Firouzi for comments on the design of figures. SF expresses deep respect and gratitude to the NITORI scholarship foundation for supporting her during undergraduate studies. Computational analyses were provided by the Super Computer System, Human Genome Center, Institute of Medical Science, at The University of Tokyo. Funding ------- This work was supported by the Japanese Society for the Promotion of Science (JSPS) - DC1 (24.6916 to SF); Third Term Comprehensive Control Research for Cancer, Ministry of Health, Labour and Welfare (H24-G-004 to TW); JSPS KAKENHI (23390250 to TW, 24591383 to TY); and MEXT KAKENHI (221S0001to TW, 221S0002 to YS).

This classic review article published in Survey of Ophthalmology is reprinted with permission from Elsevier. Copyright (2004). Shields CL, Shields JA. Tumors of the conjunctiva and cornea. Surv Ophthalmol 2004;49:3-24. General Considerations {#sec1-1} ====================== Tumors of the conjunctiva and cornea occupy a large spectrum of conditions ranging from benign lesions such as limbal dermoid or myxoma to aggressive, life-threatening malignancies such as melanoma or Kaposi\'s sarcoma.\[[@ref23][@ref88]\] The clinical differentiation of these tumors is based on the patient\'s medical background as well as certain typical clinical features of the tumor. The recognition and proper management of such tumors requires an understanding of the anatomy of the conjunctiva and cornea and knowledge of general principles of tumor management, both of which are described below. The specific clinical and histopathologic features as well as the management of each tumor is discussed, based on the authors\' personal experience with over 1,600 patients with conjunctival tumors during a 30-year period (Shields CL, submitted for publication). In this report, we review and illustrate the features of conjunctival and corneal tumors for the general ophthalmologist as well as the specialist who might occasionally examine an affected patient and want a quick reference for recognition and therapy. Anatomy {#sec2-1} ------- The conjunctiva is a continuous mucous membrane that covers the anterior portion of the globe. It extends from the eyelid margin onto the back surface of the eyelid (palpebral portion), into the fornix (forniceal portion), onto the surface of the globe (bulbar portion), and up to the corneoscleral limbus (limbal portion). The conjunctiva is composed of epithelium and stroma. The epithelium consist of both stratified squamous and columnar epithelium.\[[@ref89]\] The squamous pattern is found near the limbus and the columnar pattern is found near the fornix. The stroma is composed of fibrovascular connective tissue that thickens in the fornix and thins at the limbus. Special regions of the conjunctiva include the plica semilunaris and caruncle. The plica semilunaris is a vertically oriented fold of conjunctiva, located in the medial portion of the bulbar conjunctiva. It is speculated that the plica semilunaris represents a remnant of the nictitating membrane found in certain animals. The caruncle is located in the medial canthus between the upper and lower punctum. It contains both conjunctival and cutaneous structures such as nonkeratinized stratified squamous epithelium overlying the stroma of fibroblasta, melanocytes, sebaceous glands, hair follicles, and striated muscle fibers. Neoplasms can arise in the conjunctiva from both its epithelial and stromal structures. These are similar clinically and histopathologically to tumors that arise from other mucous membranes in the body. However, unlike other mucous membranes in the body, the conjunctiva is partially exposed to sunlight, which may be a factor in the development of some tumors. Similarly, the cornea can develop epithelial tumors, but corneal stromal tumors are uncommon. The caruncle, with its unique composition of both mucous membrane and cutaneous structures, can generate tumors found both in mucosa and skin. Diagnostic approaches {#sec2-2} --------------------- Unlike many other mucous membranes in the body, the conjunctiva is readily visible. Thus, tumors and related lesions that occur in the conjunctiva are generally recognized at a relatively early stage. Because many of these tumors have typical clinical features, an accurate diagnosis can often be made by external ocular examination and slit-lamp biomicroscopy, provided that the clinician is familiar with their clinical characteristics. A diagnostic biopsy is not usually necessary in cases of smaller tumors (≤4 clock hours limbal tumor or ≤15 mm basal dimension) that appear benign. If a smaller tumor does require a biopsy, it is often better to completely remove the lesion in one operation (excisional biopsy). In cases of larger lesions (\>4 clock hour limbal tumor or \>15 mm basal dimension), however, it may be appropriate to remove a portion of the tumor (incisional biopsy) to obtain a histopathologic diagnosis prior to embarking upon more extensive therapy, as conjunctival tumors are readily accessible to incisional biopsy. Occasionally, exfoliative cytology\[[@ref90]\] and fine-needle aspiration biopsy can provide useful information on the basis of a few cells. In addition to evaluation of the conjunctival lesion, meticulous slit-lamp examination of the cornea is essential in patients with suspected conjunctival tumors. Invasion of squamous cell carcinoma and melanoma into the peripheral cornea may appear as a subtle, gray surface opacity. It is important to completely outline such corneal involvement prior to surgery, because it is often less visible through the operating microscope than it is with slit-lamp biomicroscopy in the office. Management {#sec2-3} ---------- Depending on the presumptive diagnosis and the size and extent of the lesion, management of a conjunctival tumor can consist of serial observation, incisional biopsy, excisional biopsy, cryotherapy, chemotherapy, radiotherapy, modified enucleation, orbital exenteration or various combinations of these methods.\[[@ref1][@ref78][@ref79][@ref80][@ref86]\] If large areas of conjunctiva are removed, mucous membrane grafts from the conjunctiva of the opposite eye, buccal mucosa, or amniotic membrane may be necessary.\[[@ref42][@ref60]\] ### Observation {#sec3-1} Observation is generally the management of choice for most benign, asymptomatic tumors of the conjunctiva. Selected examples of lesions that can be observed without interventional treatment include pingueculum, dermolipoma, and nevus. External or slit-lamp photographs are advisable to document all lesions and are critical to follow-up of the more suspicious lesions. Most patients are examined every 6 to 12 months looking for evidence of growth, malignant change, or secondary effects on normal surrounding tissues. ### Incisional biopsy {#sec3-2} Incisional biopsy is reserved for extensive suspicious tumors that are symptomatic or suspected to be malignant. Examples include large squamous cell carcinoma, primary acquired melanosis, melanoma, and conjunctival invasion by sebaceous gland carcinoma. It should be understood that if tumors occupy 4 clock hours or less on the bulbar conjunctiva, excisional biopsy is generally preferable to incisional biopsy. However, larger lesions can be approached by incisional wedge biopsy or punch biopsy. Definitive therapy would then be planned based on the results of biopsy. Incisional biopsy is also appropriate for conditions that are ideally treated with radiotherapy, chemotherapy, or other topical medications. These lesions include lymphoid tumors, metastatic tumors, extensive papillomatosis, and some cases of squamous cell carcinoma and primary acquired melanosis. Incisional biopsy should generally be avoided for melanocytic tumors, especially melanoma, as this can increase the risk for numerous tumor recurrences.\[[@ref64]\] ### Excisional biopsy {#sec3-3} Primary excisional biopsy is appropriate for relatively smaller tumors (≤4 clock hours limbal tumor or ≤15 mm basal dimension) that are symptomatic or suspected to be malignant. In these situations, excisional biopsy is preferred over incisional biopsy to avoid inadvertent tumor seeding. Examples of benign and malignant lesions that are ideally managed by excisional biopsy include symptomatic limbal dermoid, epibulbar osseous choristoma, steroid-resistant pyogenic granuloma, squamous cell carcinoma, and melanoma. When such lesions are located in the conjunctival fornix they can be completely excised and the conjunctiva reconstructed primarily with absorbable sutures, sometimes with fornix deepening sutures or symblepharon ring to prevent adhesions. If the defect cannot be closed primarily, then a mucous membrane graft can be inserted. Most primary malignant tumors of the conjunctiva, like squamous cell carcinoma and melanoma, arise in the interpalpebral area near the limbus and the surgical technique for limbal tumors is different than that for forniceal tumors.\[[@ref72][@ref78][@ref79]\] Limbal neoplasms possibly can invade through the corneal epithelium and sclera into the anterior chamber and also through the soft tissues into the orbit. Thus, it is often necessary to remove a thin lamella of sclera to achieve tumor-free margins and to decrease the chance for tumor recurrence. In this regard, we employ a partial lamellar sclerokeratoconjunctivectomy with primary closure in for such tumors ([Fig. 1](#F1){ref-type="fig"}). Because cells from these friable tumors can seed into adjacent tissues, a gentle technique without touching the tumor (*no touch technique*) is advised. Additionally, the surgery should be performed using microscopic technique and the operative field should be left dry so that cells adhere to the resected tissue. It is wise to avoid wetting the field with balanced salt solution until after the tumor is completely removed to minimize seeding of cells. There are no published comparative reports of the various surgical techniques for tumor excision, but discussions at the 1997 International Congress of Ocular Oncology in Jerusalem supported the above surgical principles. {#F1} The technique for resection of limbal tumors is shown in [Fig. 1](#F1){ref-type="fig"}. Using retrobulbar anesthesia and the operating microscope, the corneal epithelial component is approached first and the conjunctival component is dissected second, with the goal of excising the entire specimen completely in one piece. Absolute alcohol soaked on an applicator is gently applied to the entire corneal component. This causes epithelial cellular devitalization and allows easier release of the tumor cells from Bowman\'s layer. A beaver blade is used to microscopically outline the malignancy within the corneal epithelium using a delicate epithelial incision or epitheliorhexis technique 2 mm outside the corneal component. The beaver blade is then used to sweep gently the affected corneal epithelium from the direction of the central cornea to limbus, into a scroll that rests at the limbus. Next, a pentagonal or circular conjunctival incision based at the limbus is made 4--6 mm outside the tumor margin. The incision is carried through the underlying Tenon\'s fascia until the sclera is exposed so that full thickness conjuctiva and Tenon\'s fascia is incorporated into the excisional biopsy. Cautery is applied to control bleeding. A second incision is then outlined by a superficial scleral groove approximately 0.2 mm in depth and 2.0 mm outside the base of the overlying adherent conjunctival mass. This groove is continued anteriorly to the limbus. The area outlined by the scleral groove is removed by flat dissection of 0.2-mm thickness within the sclera in an attempt to remove a superficial lamella of sclera, overlying Tenon\'s fascia and conjunctiva with tumor, and the scrolled corneal epithelium. In this way, the entire tumor with tumor-free margins is removed in one piece without touching the tumor itself (no touch technique). The removed specimen is then placed flatly on a piece of thin cardboard from the surgical tray and then placed in fixative and submitted for histopathologic studies. This step prevents the specimen from folding and allows better assessment of the tumor margins histopathologically. The used instruments are then replaced with fresh instruments for subsequent steps, to avoid contamination of healthy tissue with possible tumor cells. After excision of the specimen, cryotherapy is applied to the margins of the remaining bulbar conjunctiva. This is performed by freezing the surrounding bulbar conjunctiva as it is lifted away from the sclera using the cryoprobe. When the ice ball reaches a size of 4--5 mm, it is allowed to thaw and the cycle repeated once more. The cryoprobe is then moved to an adjacent area of the conjunctiva and the cycle is repeated until all of the margins have been treated by this method. It is not necessary to treat the corneal margins with cryoapplication. The tumor bed is treated with absolute alchohol wash on cotton tip applicator and bipolar cautery, avoiding cryotherapy directly to the sclera. Using clean instruments, the conjunctiva is mobilized for closure of the defect by loosening the intermuscular septum with Steven\'s scissor spreading and creation of transpositional conjunctival flaps. Closure is completed with interrupted absorbable 6-0 or 7-0 sutures. If the surgeon prefers, an area of bare sclera can be left near the limbus, but we prefer complete closure as this promotes better healing and allows for facility of further surgery if the patient should develop recurrence. The patient is treated with topical antibiotics and corticosteroids for two weeks and then followed at 3- to 6-month intervals. ### Cryotherapy {#sec3-4} In the management of conjunctival tumors, cryotherapy can be used as a supplemental treatment to excisional biopsy as described above. The advantages of cryotherapy include elimination of subclinical, microscopic tumor cells and prevention of recurrence of malignant tumors, including squamous cell carcinoma and melanoma.\[[@ref14][@ref64]\] It can also be used as a principal treatment for primary acquired melanosis and pagetoid invasion of sebaceous gland carcinoma. If cryotherapy can devitalize the malignant or potentially malignant cells in these instances, radical surgery like orbital exenteration can often be delayed or avoided. The disadvantages of cryotherapy include conjunctival chemosis that may last over one week and if the technique is misused and the globe is accidentally frozen, cataract, uveitis, scleral, and corneal thinning, and phthisis bulbi can occur. ### Chemotherapy {#sec3-5} Recent evidence has revealed that topical eyedrops comprised of mitomycin C, 5-fluorouracil, or interferon are effective in treating epithelial malignancies such as squamous cell carcinoma, primary acquired melanosis, and pagetoid invasion of sebaceous gland carcinoma.\[[@ref19][@ref20][@ref32][@ref38][@ref53][@ref56][@ref57][@ref96][@ref97]\] Mitomycin C or 5-fluorouracil are employed most successfully for squamous cell carcinoma, especially after tumor recurrence following previous surgery. This medication is prescribed topically 4 times daily for a 1-week period followed by a 1-week hiatus to allow the ocular surface to recover \[[Table 1](#T1){ref-type="table"}\]. This cycle is repeated once again so that most patients receive a total of two weeks of the chemotherapy topically. Both mitomycin C and 5-fluorouracil are most effective for squamous cell carcinoma and less effective for primary acquired melanosis and pagetoid invasion of sebaceous gland carcinoma. Caution should be used with this medication as it is most effective for intraepithelial disease and much less effective or ineffective for deeper disease. Toxicities include most commonly dry eye findings, superficial punctate epitheliopathy, and punctal stenosis. Corneal melt, scleral melt, and cataract can develop if these agents are used with open conjunctival wounds or used excessively. Topical interferon can be effective for squamous epithelial malignancies and is less toxic to the surface epithelium, but this medication may require many months of use to effect a result.\[[@ref32]\] ###### Protocol for Use of Mitomycin C for Conjunctival Squamous Cell Neoplasia and Primary Acquired Melanosis Time Medication and Frequency -------- ---------------------------------------------------- Week 1 Slit-lamp biomicroscopy Place upper and lower punctal plugs Cycle 1: mitomycin C 0.04% qid to the affected eye Week 2 No medication Week 3 Cycle 2: mitomycin C 0.04% qid to the affected eye Week 4 No medication Slit-lamp biomicroscopy Prescribe more cycles if residual tumor exists Remove punctal plugs after all medication complete ### Radiotherapy {#sec3-6} Two forms of radiotherapy are employed for conjunctival tumors, namely external beam radiotherapy and custom-designed plaque radiotherapy. External beam radiotherapy to a total dose of 3,000--4,000 cGy is used to treat conjunctival lymphoma and metastatic carcinoma when they are too large or diffuse to excise locally. Side effects of dry eye, punctate epithelial abnormalities, and cataract should be anticipated. Custom-designed plaque radiotherapy\[[@ref65]\] to a dose of 3,000--4,000 cGy can be used to treat conjunctival lymphoma or metastasis. A higher dose of 6,000--8,000 cGy can be employed to treat the more radiation resistant melanoma and squamous cell carcinoma. In general, plaque radiotherapy is reserved for those patients who have diffuse tumors that are incompletely resected and for those who display multiple recurrences. The two designs for conjunctival custom plaque radiotherapy include a conformer plaque technique with six fractionated treatment sessions as an outpatient or a reverse plaque technique with the device sutured onto the episcleral as an inpatient. In unique instances, plaque radiotherapy to a low dose of 2,000 cGy is employed for benign conditions, including steroid resistant pyogenic granuloma that show recurrence after surgical resection.\[[@ref26]\] This treatment should be performed by experienced radiation oncologists and ocular oncologists. There is no published report on a comparison of these radiotherapy techniques. ### Modified enucleation {#sec3-7} Modified enucleation is a treatment option for primary malignant tumors of the conjunctiva that have invaded through the limbal tissues into the globe, producing secondary glaucoma. This occurrence is quite rare but can occasionally be found with squamous cell carcinoma and melanoma. The uncommon mucoepidermoid variant and spindle cell variant of squamous cell carcinoma of the conjunctiva has a greater tendency for intraocular invasion.\[[@ref2][@ref7][@ref25]\] At the time of enucleation, it is necessary to remove the involved conjunctiva intact with the globe so as to avoid spreading tumor cells. Thus, the initial peritomy should begin at the limbus, but when the tumor is approached, the incision should proceed posteriorly from the limbus to surround the tumor-affected tissue by at least 3--4 mm. The tumor will remain adherent to the globe at the limbus. Occasionally, a suture is employed through the surrounding conjunctiva into the episclera to secure the tumor to the globe so that it will not be displaced during subsequent manipulation. The remaining steps of enucleation are gently performed and the globe is removed with tumor adherent after cutting the optic nerve from the nasal side. The margins of the remaining, presumed unaffected conjunctiva are treated with double freeze-thaw cryotherapy. Often this surgical technique leaves the patient with a limited amount of residual unaffected conjunctiva for closure. In these instances, a mucous membrane graft or amniotic membrane graft may be necessary for adequate closure and to provide fornices for a prosthesis. In some instances, a simple horizontal inferior forniceal conjunctival incision from canthus to canthus may suffice, as long as the conformer is constantly worn as a template so the new conjunctival fornix grows deep and around this structure. ### Orbital exenteration {#sec3-8} Orbital exenteration is probably the treatment of choice for primary malignant conjunctival tumors that have invaded the orbit or that exhibit complete involvement of the conjunctiva.\[[@ref64][@ref84][@ref86]\] Either an eyelid-removing or eyelid-sparing exenteration is employed, depending on the extent of eyelid involvement. The eyelid-sparing technique is preferred in that the patients have better cosmetic appearance and heal within 2 or 3 weeks. Specifically, if the anterior lamella of the eyelid is uninvolved with tumor, an eyelid-sparing (eyelid-splitting) exenteration may be accomplished.\[[@ref78][@ref84][@ref86]\] Other options to exenteration are radiotherapy using the external beam approach or the brachytherapy approach. There are too few cases in the literature to do a scientific comparison. ### Mucous membrane graft {#sec3-9} Mucous membrane grafts are occasionally necessary to replace vital conjunctival tissue after removal of extensive conjunctival tumors. The best donor sites include the forniceal conjunctiva of the ipsilateral or contralateral eye and buccal mucosa from the posterior aspect of the lower lip or lateral aspect of the mouth. Such grafts are usually removed by a freehand technique, fashioned to fit the defect, and secured into place with cardinal and running absorbable 6-0 or 7-0 sutures. Currently, in most instances, we employ a donor amniotic membrane graft to replace lost conjunctiva.\[[@ref42][@ref60]\] The tissue is delivered frozen and must be defrosted for 20 minutes. The fine, transparent material is carefully peeled off its cardboard surface, laid basement membrane side up, and sutured into place with absorbable sutures. Topical antibiotic and steroid ointments are applied following all conjunctival grafting procedures. It is important that the surgeon use a minimal manipulation technique for tumor resection. For graft harvest and placement, we prefer to use clean, sterile instruments at both the donor and the recipient sites to avoid transfer and implantation of tumor cells into previously uninvolved areas. Congenital Lesions {#sec1-2} ================== A variety of tumors and related conditions may be present at birth or become clinically apparent shortly after birth.\[[@ref10][@ref74]\] Most of the lesions to be considered here are choristomas, consisting of displaced tissue elements normally not found in these areas. A simple choristoma is comprised of one tissue element such as epithelium whereas a complex choristoma represents variable combinations of ectopic tissues like bone, cartilage, and lacrimal gland. Despite their presence at a young age, all of the conjunctival choristomas discussed herein are sporadic, without hereditary tendency. Dermoid {#sec2-4} ------- Conjunctival dermoid is a congenital well-circumscribed yellow-white solid mass that involves the bulbar conjunctiva or at the corneoscleral limbus.\[[@ref10][@ref11][@ref49][@ref72][@ref74]\] It characteristically occurs near the limbus inferotemporally and often this tumor has fine white hairs, best seen with slit lamp biomicroscopy ([Fig. 2](#F2){ref-type="fig"}). In rare cases, it can extend to the central cornea or be located in other quadrants on the bulbar surface. There are three types of dermoids, classified on the extent of involvement. The first type includes the small limbal dermoid, straddling the limbus and approximately 5 mm in diameter. The second type is larger, often involving the entire surface of the cornea, but not deeper than Descemet\'s membrane. The third type is most extensive and the dermoid involves the cornea, anterior chamber, and iris stroma and its posterior aspect is lined by the iris pigment epithelium. The various types are related to the time during fetal development in which the dermoid develops, with more severe types occurring earlier. {#F2} Conjunctival dermoid may occur as an isolated lesion or it can be associated with Goldenhar\'s syndrome. Hence, the patient should be evaluated for ipsilateral or bilateral preauricular skin appendages, hearing loss, eyelid coloboma, orbitoconjunctival dermolipoma, and cervical vertebral anomalies that comprise this nonheritable syndrome. Histopathologically, the conjunctival dermoid is a simple choristomatous malformation that consists of dense fibrous tissue lined by conjunctival epithelium with deeper dermal elements including hair follicles and sebaceous glands. The management of an epibulbar dermoid includes simple observation if the lesion is small and visually asymptomatic. It is possible to excise the lesion for cosmetic reasons, but the remaining corneal scar is sometimes cosmetically unacceptable. Larger or symptomatic dermoids can produce visual loss from astigmatism. These can be approached by lamellar keratosclerectomy with primary closure of overlying tissue if the defect is superficial or closure using corneal graft if the defect is deep or full thickness. It has been reported that the cosmetic appearance may improve, but the refractive and astigmatic error and visual acuity may not change.\[[@ref49]\] When the lesion involves the central cornea, a lamellar or penetrating keratoplasty may be necessary and long-term amblyopia can be a problem.\[[@ref72]\] Occasionally, extensive dermoids involve the lateral canthus and carefully planned excision with lateral canthal repair is necessary. Dermolipoma {#sec2-5} ----------- Dermolipoma is believed to be congenital and present at birth, but it typically remains asymptomatic for years and may not be detected until adulthood when it protrudes from the orbit through the conjunctival fornix superotemporally ([Fig. 3](#F3){ref-type="fig"}). It appears as a pale-yellow, soft, fluctuant, fusiform mass below the palpebral lobe of the lacrimal gland, best visualized with the eye in inferonasal gaze. It usually extends for a variable distance into the orbital fat and onto the bulbar conjunctiva, and occasionally it can extend anteriorly to reach the limbus. Unlike herniated orbital fat, dermolipoma can contain fine white hairs on its surface and it cannot be reduced with digital pressure into the orbit. {#F3} With computed tomography (CT) or magnetic resonance imaging (MRI), dermolipoma has features similar to orbital fat from which it may be indistinguishable. Histopathologically, it is lined by conjunctival epithelium on its surface and the subepithelial tissue has variable quantities of collagenous connective tissue and adipose tissue. Pilosebaceous units and lacrimal gland tissue may occasionally be present. The majority of dermolipomas require no treatment, but larger symptomatic ones or those that are cosmetically unappealing can be managed by excision of the entire orbitoconjunctival lesion through a conjunctival forniceal approach or by simply removing the anterior portion of the lesion in a manner similar to that used to remove prolapsed orbital fat. Epibulbar osseous choristoma {#sec2-6} ---------------------------- Epibulbar osseous choristoma is a rigid deposit of bone generally located in the bulbar conjunctiva superotemporally ([Fig. 4](#F4){ref-type="fig"}).\[[@ref70]\] It is believed to be congenital and typically remains undetected until personally palpated by the patient in the preteen years. It is clinically suspected due to its rock-hard consistency on palpation, although fibrous tissue tumors can feel similar. The diagnosis can be confirmed with ultrasonography or computed tomography to illustrate the calcium component. This tumor is generally best managed by periodic observation. Occasionally patients report a foreign body sensation and symptomatic lesions can be excised with a circumtumoral conjunctival incision followed by dissection to bare sclera for full thickness conjunctival resection. For those tumors that might be adherent to the sclera, a superficial sclerectomy might be warranted.\[[@ref70]\] {#F4} Lacrimal gland choristoma {#sec2-7} ------------------------- Lacrimal gland choristoma is a congenital lesion, discovered in young children as an asymptomatic pink stromal mass, typically in the superotemporal or temporal portion of the conjunctiva.\[[@ref45]\] It is speculated that this lesion represents small sequestrations of the embryonic evagination of the lacrimal gland from the conjunctiva. The lacrimal gland choristoma can masquerade as a focus of inflammation due to its pink color. Rarely, a cystic appearance ensues from this secretory mass if there is no connection to the conjunctival surface. Excisional biopsy is usually performed to confirm the diagnosis. Respiratory choristoma {#sec2-8} ---------------------- In unique instances, a cystic choristoma, appearing as congenital sclerocorneal ectasia, is found. In one report, this was found to manifest respiratory mucosa.\[[@ref98]\] Complex choristoma {#sec2-9} ------------------ The conjunctival dermoid and epibulbar osseous choristoma are termed simple choristomas as they contain one tissue type such as skin or bone. A complex choristoma contains a greater variety of tissue like dermal appendages, lacrimal gland tissue, cartilage, bone, and occasionally other elements. Complex choristoma contains tissue derived from two germ layers. It is quite variable in its clinical appearance and may cover much of the epibulbar surface or it may form a circumferential growth pattern around the limbus ([Fig. 5](#F5){ref-type="fig"}). For example, a tumor with extensive lacrimal tissue appears as a lobular pink mass whereas one with dermal tissue appears yellow and thick and one with cartilage displays a smooth blue-gray hue. The complex choristoma has a peculiar association with the linear nevus sebaceous of Jadassohn.\[[@ref58][@ref75][@ref82]\] The nevus sebaceous of Jadassohn includes cutaneous features with sebaceous nevus in the facial region and neurologic features including seizures, mental retardation, arachnoid cyst, and cerebral atrophy. The ophthalmic features of this syndrome include epibulbar complex choristoma and posterior scleral cartilage.\[[@ref82]\] {#F5} The management of the complex choristoma depends upon the extent of the lesion. Observation or wide local excision with mucous membrane graft reconstruction are options. In the rare case of a very extensive lesion, where the lesion causes dense amblyopia with no hope for visual acuity, modified enucleation with ocular surface reconstruction may be necessary. Benign Tumors of Surface Epithelium {#sec1-3} =================================== Several benign tumors and related conditions can arise from the squamous epithelium of the conjunctiva. Papilloma {#sec2-10} --------- Squamous papilloma is a benign tumor, documented to be associated with human papillomavirus (subtypes 6, 11, 16, and 18) infection of the conjunctiva.\[[@ref55][@ref88]\] This tumor can occur in both children and adults. It is speculated that the virus is acquired through transfer from the mother\'s vagina to the newborn\'s conjunctiva as the child passes through the mother\'s birth canal. Papilloma appears as a pink fibrovascular frond of tissue arranged in a sessile or pedunculated configuration. The numerous fine vascular channels ramify through the stroma beneath the epithelial surface of the lesion. In children, the lesion is usually small, multiple, and located in the inferior fornix ([Fig. 6](#F6){ref-type="fig"}). In adults, it is usually solitary, more extensive, and can often extend to cover the entire corneal surface simulating malignant squamous cell carcinoma. Histopathologically, the lesion shows numerous vascularized papillary fronds lined by acanthotic epithelium. {#F6} In the case of a small sessile papilloma in a child, there are several treatment options. Sometimes, periodic observation allows for slow spontaneous resolution of the viral-produced tumor. Larger or more pedunculated lesions are generally symptomatic with foreign body sensation, chronic mucous production, hemorrhagic tears, incomplete eyelid closure, and poor cosmetic appearance. These lesions are unlikely to show a favorable response to observation or steroids and are best managed by surgical excision. Complete removal of the mass without direction manipulation of the tumor (no touch technique) is generally advisable to avoid spreading of the tumor-related virus. Double freeze-thaw cryotherapy is applied to the remaining conjunctiva around the excised lesion in order to help prevent tumor recurrence. In some instances, the pedunculated tumor is frozen alone and allowed to slough off the conjunctival surface later. For some large unwieldy pedunculated tumors, complete cryotherapy of the mass down its stalk to its base is performed and excision while the mass is in the frozen state is achieved. This is especially important for large lesions to allow for traction on the tumor without forcep manipulation. Closure is completed with absorbable sutures. Topical interferon and mitomycin C have been employed for conjunctival papillomas.\[[@ref27][@ref47]\] For those lesions that show recurrence, oral cimetidine for several months can resolve the papilloma virus-related tumor by boosting the patient\'s immune system and stimulating regression of the mass ([Fig. 6](#F6){ref-type="fig"}).\[[@ref55]\] Keratoacanthoma {#sec2-11} --------------- The conjunctiva can give rise to benign reactive inflammatory lesions that simulate carcinoma including pseudocarcinomatous hyperplasia and its variant, keratoacanthoma.\[[@ref39]\] In some instances a distinct nodule is found. This lesion appears gelatinous or leukoplakic, similar to squamous cell carcinoma of the conjunctiva, but its onset may be more rapid. Massive acanthosis, hyperkeratosis and parakeratosis is found histopathologically.\[[@ref39]\] Treatment is complete resection as this may be difficult to differentiate from carcinoma both clinically and histopathologically. Hereditary benign intraepithelial dyskeratosis {#sec2-12} ---------------------------------------------- Hereditary benign intraepithelial dyskeratosis (HBID) is a peculiar condition seen in an inbred isolate of white, African-American, and Native American (Haliwa Indians). This group resided initially in North Carolina. Hereditary benign intraepithelial dyskeratosis has subsequently been detected in several other parts of the United States. It is an autosomal dominant disorder characterized by bilateral elevated fleshy plaques on the nasal or temporal perilimbal conjunctiva ([Fig. 7](#F7){ref-type="fig"}).\[[@ref63]\] Similar plaques can occur on the buccal mucosa. It can remain relative asymptomatic or it can cause severe redness and foreign body sensation. In some instances it can extend onto the cornea. It has no known malignant potential. It is characterized histopathologically by acanthosis, dyskeratosis on the epithelial surface and deep within the epithelium, and prominent chronic inflammatory cells. {#F7} Hereditary benign intraepithelial dyskeratosis is a benign condition that does not usually require aggressive treatment. Smaller, less symptomatic lesions can be treated with ocular lubricants and judicious used of topical corticosteroids. Larger symptomatic lesions can be managed by local resection with mucous membrane grafting if necessary. Epithelial inclusion cyst {#sec2-13} ------------------------- Conjunctival cysts can occur spontaneously or following inflammation, surgery, or nonsurgical trauma. Histopathologically, they are lined by conjunctival epithelium and are filled with clear fluid that often contains desquamated cellular debris ([Fig. 8](#F8){ref-type="fig"}). These cysts can be simply observed or they can be excised completely with primary closure of the conjunctiva. {#F8} Dacryoadenoma {#sec2-14} ------------- Dacryoadenoma is a rare conjunctival tumor, noted in patients as a pink mass. In one report, this tumor was found in the inferior bulbar region of a 48-year-old woman.\[[@ref30]\] It is uncertain if the lesion is congenital or acquired. This benign tumor appears to originate from the surface epithelium and proliferate into the stroma, forming glandular lobules similar to the lacrimal gland. Keratotic plaque {#sec2-15} ---------------- Keratotic plaque is a white limbal or bulbar conjunctival mass, usually in the interpalpebral region.\[[@ref76]\] It is composed of acanthosis and parakaratosis with keratinization of the epithelium. It appears similar to squamous cell carcinoma with leukoplakia. Actinic keratosis {#sec2-16} ----------------- Actinic keratosis is a frothy, white lesion usually located over a chronically inflamed pingueculum or pterygium.\[[@ref76]\] It is also referred to as dysplasia, actinic keratosis variety. Histopathologically, it is composed of a proliferation of surface epithelium with keratosis. Clinically, it resembles squamous cell carcinoma of the conjunctiva. Malignant Lesions of Surface Epithelium {#sec1-4} ======================================= Squamous cell neoplasia can occur as a localized lesion confined to the surface epithelium (conjunctival intraepithelial neoplasia or dysplasia) or as a more invasive squamous cell carcinoma that has broken through the basement membrane and invaded the underlying stroma.\[[@ref2][@ref4][@ref25][@ref29][@ref36][@ref85][@ref92]\] The former has no potential to metastasize but the latter can gain access to the conjunctival lymphatics and occasionally metastasize to regional lymph nodes. It has been found that most squamous cell neoplasia is related to human papillomavirus infection of the conjunctival epithelium and this is most certain in those patients with bilateral squamous cell neoplasia and those immunosuppressed patients who develop this disease.\[[@ref48]\] The currently accepted term for the localized variety is conjunctival intraepithelial neoplasia (CIN), but others prefer the terms dysplasia (mild, moderate, or severe) and carcinoma-in-situ. When the abnormal cellular proliferation involves only partial thickness of the epithelium it is classified as mild CIN, a condition also called mild or moderate dysplasia. When it affects full thickness epithelium it is called severe CIN, a condition also called severe dysplasia. In these cases, there may be an intact surface layer of cells. Where there are no longer normal surface cells then the process is termed carcinoma-in-situ. It is stressed that these are histopathologic terms and the differential between CIN mild and CIN severe cannot be made clinically. Conjunctival intraepithelial neoplasia (CIN) {#sec2-17} -------------------------------------------- Clinically, CIN appears as a fleshy, sessile or minimally elevated lesion usually at limbus in the interpalprebal fissure and less commonly in the forniceal or palpebral conjunctiva ([Fig. 9](#F9){ref-type="fig"}). The limbal lesion may extend for a variable distance into the epithelium of the adjacent cornea. A white plaque (leukoplakia) may occur on the surface of the lesion due to secondary hyperkeratosis. {#F9} Histopathologically, mild CIN (dysplasia) is characterized by a partial thickness replacement of the surface epithelium by abnormal epithelial cells that lack normal maturation. Severe CIN (severe dysplasia) is characterized by a nearly full-thickness replacement of the epithelium by similar cells. Carcinoma-in-situ represents full thickness replacement by abnormal epithelial cells. Squamous cell carcinoma {#sec2-18} ----------------------- Squamous cell carcinoma an extension of abnormal epithelial cells through the basement membrane to gain access to the conjunctival stroma. Clinically, invasive squamous cell carcinoma is generally larger and more elevated than CIN ([Fig. 10](#F10){ref-type="fig"}). Leukoplakia may be variable. Uncommonly, lesions that are untreated or incompletely excised can invade through the corneoscleral lamella into the anterior chamber of the eye or they can transgress the orbital septum and invade the soft tissues of the orbit adjacent to the globe.\[[@ref29][@ref85]\] A rare variant of squamous cell carcinoma of the conjunctiva is the mucoepidermoid carcinoma. Clinically, this variant occurs in older patients and has a yellow globular cystic component due to the presence of abundant mucous-secreting cells within cysts. It tends to be more aggressive than the standard squamous cell carcinoma and, therefore, deserves wider excision and closer follow-up.\[[@ref2][@ref25]\] The spindle cell variant of squamous cell carcinoma is likewise aggressive.\[[@ref7]\] {#F10} Histopathologically, invasive squamous cell carcinoma is characterized by malignant squamous cells that have violated the basement membrane and have grown in sheets or cords into the stromal tissue. As mentioned above, the mucoepidermoid variant contains mucous-secreting cells that often produce mucous-containing cysts within the lesion. Even though the cells of invasive squamous cell carcinoma gain access to the blood vessels and lymphatic channels, regional and distant metastases are both rather uncommon. Patients who are medically immunosuppressed for organ transplantation or those with human immunodeficiency virus are at particular risk to develop conjunctival squamous cell carcinoma. In these cases, the risk for life- threatening metastatic disease is greater.\[[@ref51]\] The management of squamous cell carcinoma of the conjunctiva varies with the extent of the lesion. In general, the management of lesions in the limbal area involves alcohol epitheliectomy for the corneal component and partial lamellar scleroconjunctivectomy with wide margins for the conjunctival component followed by freeze-thaw cryotherapy to the remaining adjacent bulbar conjunctiva, similar to the method used for limbal conjunctival melanoma.\[[@ref78][@ref79][@ref80]\] In some cases, microscopically controlled excision (Mohs surgery) is performed at the time of surgery to ensure tumor-free margins.\[[@ref3]\] Those tumors in the forniceal region can be managed by wide local resection and cryotherapy. In cases where excessive conjunctiva is sacrificed, a mucous membrane graft or amniotic membrane graft may be employed for reconstruction. In all cases, the full conjunctival component along with the underlying Tenon\'s fascia should be excised using the no touch technique as mentioned previously. A thin lamella of underlying sclera should be removed with the tumor for those in the limbal region where the tumor is adherent to the globe. The surgical management of conjunctival squamous cell carcinoma is similar to the management of conjunctival melanoma and is discussed further in the subsequent section on melanoma. For those patients with extensive tumors or those tumors that are recurrent, especially those with extensive corneal component, treatment with topical mitomycin C, 5-fluorouracil, or interferon is advised.\[[@ref19][@ref20][@ref32][@ref38][@ref56][@ref96][@ref97]\] We generally use mitomycin C for two cycles with close monitoring of the patient \[[Table 1](#T1){ref-type="table"}\].\[[@ref56]\] Melanocytic Tumors {#sec1-5} ================== There are several lesions that arise from the melanocytes of the conjunctiva and episclera \[[Table 2](#T2){ref-type="table"}\]. The most important ones include nevus, racial melanosis, primary acquired melanosis, and malignant melanoma. Ocular melanocytosis should be included in this discussion as its scleral pigmentation can masquerade as conjunctival pigmentation. ###### Differential Diagnosis of Pigmented Epibulbar Lesions Condition Anatomical Location Color Depth Margins Laterality Other Features Progression ---------------------------------- ------------------------------------------ ----------------- ------------ -------------- ---------------------------------- ------------------------------------------------------------------------------- ----------------------------------------------------------------------------------- Nevus Interpalpebral limbus usually Brown or yellow Stroma Well defined Unilateral Cysts \<1% progress to conjunctival melanoma Racial melanosis Limbus bulbar palpebral conjunctiva Brown Epithelium Ill defined Bilateral Flat, no cysts Very rare progression to conjunctival melanoma Ocular melanocytosis Bulbar conjunctiva Gray Episclera Ill defined Unilateral moreso than bilateral Congenital, usually 2 mm from limbus, often with periocular skin pigmentation \<1% progress to uveal melanoma Primary acquired melanosis (PAM) Anywhere, but usually bulbar conjunctiva Brown Epithelium Ill defined Unilateral Flat, no cysts Progresses to conjunctival melanoma in nearly 50% cases that show cellular atypia Malignant melanoma Anywhere Brown or pink Stroma Well defined Unilateral Vascular nodule, dilated feeder vessels, may be non-pigmented 32% develop metastasis by 15 years See [Figure 11](#F11){ref-type="fig"}, [Figure 12](#F12){ref-type="fig"}, [Figure 13](#F13){ref-type="fig"}, [Figure 14](#F14){ref-type="fig"}, [Figure 15](#F15){ref-type="fig"} for clinical illustrations Nevus {#sec2-19} ----- The circumscribed nevus is the most common melanocytic tumor of the conjunctiva. It generally becomes clinically apparent in the first or second decade of life as a discrete variably pigmented, slightly elevated, sessile lesion that usually contains fine clear cysts on slit-lamp biomicroscopy ([Fig. 11](#F11){ref-type="fig"}).\[[@ref21][@ref54]\] It is typically located in the interpalpebral bulbar conjunctiva near the limbus and remains relatively stationary throughout life with less than 1% risk for transformation into malignant melanoma.\[[@ref21][@ref54]\] The interpalpebral location is so classic that one should doubt the diagnosis of nevus if a patient presents with a forniceal or palpebral pigment mass and suspect primary acquired melanosis, racial melanosis, or malignant melanoma. Over time, a nevus can become more pigmented and the previously inapparent nonpigmented portions can acquire pigment, simulating growth. {#F11} Histopathologically, a conjunctival nevus is composed of nests of benign melanocytes in the stroma near the basal layers of the epithelium.\[[@ref9]\] Like cutaneous nevus, it can be junctional, compound, or deep. The best management is usually periodic observation with photographic comparison and if growth is documented then local excision of the lesion should be considered. In some cases, excision for cosmetic reasons is desired. At the time of excision, the entire mass is removed using the no touch technique and, if it is adherent to the globe, then a thin lamella of underlying sclera is remove intact with the tumor.\[[@ref78]\] Standard double freeze-thaw cryotherapy is applied to the remaining conjunctival margins. These precautions are employed to prevent recurrence of the nevus and also to prevent recurrence should the lesion prove to be a melanoma. Racial melanosis {#sec2-20} ---------------- Racial melanosis is a relatively common, bilateral condition of flat conjunctival pigmentation found in darkly pigmented individuals. This pigment is generally present at the limbus, often for 360°, and a variable amount of this pigment can be noted on the limbal cornea and bulbar conjunctiva ([Fig. 12](#F12){ref-type="fig"}). Uncommonly, this pigment involves the fornix and rarely the palpebral conjunctiva. This pigmentation can occasionally be mottled with a patchy appearance. It is extremely rare for conjunctival melanoma to arise from racial melanosis. Histopathologically, the pigmented cells are benign melanocytes located in the basal layer of the epithelium. The recommended management is periodic observation. {#F12} Ocular melanocytosis {#sec2-21} -------------------- Ocular melanocytosis is a congenital pigmentary condition of the periocular skin, sclera, orbit, meninges, and soft palate. Typically, there is no conjunctival pigment. However, this condition is commononly confused with primary acquired melanosis because of their similar appearance. In ocular melanocytosis, flat, gray-brown pigment scattered posterior to the limbus on the sclera is visualized through the thin overlying conjunctival tissue ([Fig. 13](#F13){ref-type="fig"}). The entire uvea is also generally affected by similar increased pigment. This condition imparts a 1 in 400 risk for the development of uveal melanoma and not conjunctival melanoma.\[[@ref87]\] Affected patients should be followed once or twice yearly for the development of uveal, orbital, or meningeal melanoma. {#F13} Primary acquired melanosis (PAM) {#sec2-22} -------------------------------- Primary acquired melanosis is an important benign conjunctival pigmented condition that can give rise to conjunctival melanoma. In contrast to conjunctival nevus, it is acquired in middle age and appears diffuse, patchy, flat, and noncystic \[[Fig. 14](#F14){ref-type="fig"}\]. In contrast to ocular melanocytosis, the pigment is acquired, located within the conjunctiva, and appears brown, not gray, in color. The pigmentation can wax and wane over time.\[[@ref17][@ref18][@ref53]\] In contrast to racial melanosis, PAM generally is found in fair-skinned individuals as a unilateral patchy condition.\[[@ref22]\] {#F14} Histopathologically, PAM is characterized by the presence of abnormal melanocytes near the basal layer of the epithelium. Pathologists should attempt to classify the melanocytes as having atypia or no atypia based on nuclear features and growth pattern.\[[@ref17][@ref18]\] PAM with atypia carries nearly 50% risk for ultimate evolution into malignant melanoma whereas PAM without atypia carries nearly 0% risk for melanoma development \[[Table 3](#T3){ref-type="table"}\].\[[@ref17][@ref18]\] ###### Histopathologic Classification of Primary Acquired Melanosis of the Conjunctiva and Risks for Evolution into Conjunctival Melanoma General Classification Risk for Development of Conjunctival Melanoma --------------------------------------------------------------------------------------------------- ----------------------------------------------- Primary acquired melanosis without atypia 0% Primary acquired melanosis with atypia 46% If atypical melanocytes in the epithelium located in other than the basal layer of the epithelium 90% If atypical melanocytes showing epithelioid cellular features (abundant cytoplasm) 75% From Folberg *et al*.^17^ x17. Folberg, R., McLean, I.W., and Zimmerman, L.E. Primary acquired melanosis of the conjunctiva. Hum Pathol. 1985; 16: 136-143 The management of PAM depends on the extent of involvement and the association with melanoma. If there is only a small region of PAM, occupying less than three clock hours of the conjunctiva, then periodic observation or complete excisional biopsy and cryotherapy are options.\[[@ref78]\] If the PAM occupies more than three clock hours, then incisional map biopsy of all four quadrants is warranted, followed by double freeze-thaw cryotherapy to all affected pigmented sites. If the patient has a history of previous or current conjunctival or cutaneous melanoma or if there are areas of nodularity or vascularity within the presumed PAM that are suspicious for melanoma, then a more aggressive approach is warranted with complete excisional biopsy of the suspicious areas using the no touch technique as described previously. Additional small incisional map biopsies should be performed in the regions of flat PAM and even in the apparently uninvolved quadrants of the bulbar conjunctiva to determine if there are melanocytes with atypia. Cryotherapy should be applied to all remaining pigmented areas. We manage patients who have PAM associated with melanoma more aggressively than those with PAM alone. If there is recurrent PAM on follow-up, prompt excisional biopsy and cryotherapy in the operating room or in the outpatient clinic setting is provided. Topical mitomycin C can also be beneficial, especially if there is recurrent corneal PAM; however, mitomycin C is not as effective for PAM as it is for squamous epithelial neoplasia. Malignant melanoma {#sec2-23} ------------------ Malignant melanoma of the conjunctiva most commonly arises from PAM, but it can also arise from a pre-existing nevus or de novo.\[[@ref50][@ref64]\] It typically arises in adults at a median age of 62 years, but rare cases of conjunctival melanoma in children have been recognized.\[[@ref64][@ref91]\] Conjunctival melanoma shows considerable clinical variability. It is generally a pigmented or tan, elevated conjunctival lesion that can be located on the limbal, bulbar, forniceal, or palpebral conjunctiva ([Fig. 15](#F15){ref-type="fig"}). Occasionally, the tumor shows predominance on the cornea, despite origin from the conjunctiva.\[[@ref93]\] Often prominent feeder vessels and surrounding flat PAM are present. Conjunctival melanoma can show both local tumor recurrence and distant metastasis (Tables [4](#T4){ref-type="table"}--[6](#T6){ref-type="table"}).\[[@ref40][@ref41][@ref50][@ref64]\] Multiple recurrences, especially those that occur within the orbit, frequently necessitate orbital exenteration.\[[@ref40][@ref64][@ref84]\] Metastases to ipsilateral facial lymph nodes, brain, lung, and liver are the most common sites.\[[@ref15][@ref64]\] Histopathologically, conjunctival melanoma is composed of variably pigmented malignant melanocytes within the conjunctival stroma. There may be microscopic evidence of PAM or a nevus. {#F15} ###### Risks for Local Tumor Recurrence, Exenteration, Metastasis, and Death in Patients with Conjunctival Melanoma Outcome Length of follow-up^a^ ------------------ ------------------------ ---- ---- Recurrence (%) 26 51 65 Exenteration (%) 8 16 32 Metastasis (%) 16 26 32 Death (%) 7 13 na ^a^Kaplan Meier life table analysis. na=not available. From Shields *et al*.^64^ x 64. Shields, C.L., Shields, J.A., Gunduz, K. *et al*. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Arch Ophthalmol. 2000; 118: 1497-1507 ###### Clinical Factors Predictive of Local Tumor Recurrence Following Resection of Conjunctival Melanoma Factor *P* Relative Risk ---------------------------------------------------------- ------ --------------- Tumor location extralimbal 0.01 2.3 Tumor extending to surgical margin (histopathologically) 0.02 2.9 From Shields *et al*.^64^ x 64. Shields, C.L., Shields, J.A., Gunduz, K. *et al*. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Arch Ophthalmol. 2000; 118: 1497-1507 ###### Clinical Factors Predictive of Tumor Metastasis from Conjunctival Melanoma Factor *P* Relative Risk ---------------------------------------------------------- ------- --------------- Tumor extending to surgical margin (histopathologically) 0.005 5.7 Tumor location extralimbal 0.03 3.1 From Shields *et al*.^64^ x 64. Shields, C.L., Shields, J.A., Gunduz, K. *et al*. Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Arch Ophthalmol. 2000; 118: 1497-1507 The management of conjunctival melanoma varies with the extent of the lesion.\[[@ref52]\] This malignancy is particularly difficult to treat. Despite excellent microscopic excision of the mass, further disease can develop from associated PAM in 26% of patients by 5 years and 65% of patients by 15 years follow-up \[[Table 4](#T4){ref-type="table"}\].\[[@ref64]\] Classic limbal tumors are removed by absolute alcohol epitheliectomy for the flat corneal component and wide no touch technique, partial lamellar scleroconjunctivectomy with 4 mm margins followed by double freeze-thaw cryotherapy for the conjunctival portion. Larger lesions that extend into the forniceal region or orbit may require more extensive excision, always with tumor free margins encapsulating the tumor and with no touch, dry technique ([Fig. 1](#F1){ref-type="fig"}). Closure is achieved by primary apposition of conjunctiva or with conjunctival rotational flaps, mucous membrane graft from the opposite eye or buccal mucosa, or amniotic membrane transplantation.\[[@ref60]\] Often, fornix deepening sutures or a symblepharon ring is required to reform the fornix. Lesions that extend into the globe may require a modified enucleation and those that extend into the orbit may require orbital exenteration as described above.\[[@ref18][@ref64][@ref81][@ref84]\] Paridaens and associates found that early exenteration did not improve life prognosis.\[[@ref40]\] Shields and associates found tumor related death occurred in 7% of patients at 5 years and 13% at 8 years.\[[@ref64]\] The risk factors for death using multivariate analysis included initial symptoms (lump) (*p* = 0.004) and pathology findings (de novo melanoma without primary acquired melanosis) (*p* = 0.05). The technique of initial surgery (using complete excisional biopsy with the no touch technique combined with cryotherapy to remaining tumor free margins) was shown to be an important factor in preventing eventual tumor recurrence (*p* = 0.07), metastasis (*p* = 0.03), and death (*p* = 0.006) in the univariate analysis, but did not reach significance in the multivariate analysis.\[[@ref64]\] Conditions that can simulate conjunctival melanocytic tumors {#sec2-24} ------------------------------------------------------------ There are several benign, non-neoplastic conditions that can resemble conjunctival PAM or melanoma and these include pingueculum, pterygium, Axenfeld\'s nerve loops at the site of a scleral emissarial canal, mascara deposition in the inferior fornix, silver deposition on the entire conjunctival surface in patients who have used argyrol eyedrops, gunpowder deposition in patients exposed to gunpowder explosions, adrenochrome pigment in the inferior fornix in patients using epinephrine eyedrops, hemorrhagic conjunctival cyst following previous surgery, pigmented cells trapped within a non-melanocytic tumor (fellow travelers),\[[@ref83]\] ochronosis pigmentation at the site of muscle insertion and in pingueculum in patients with alkaptonuria, and calcified Cogan\'s scleral plaque at the horizontal rectus muscle insertions in older adults.\[[@ref76]\] Understanding and recognition of these pseudomelanomas should be achieved by clinicians managing patients with conjunctival malignancies. Vascular Tumors {#sec1-6} =============== Pyogenic granuloma {#sec2-25} ------------------ Pyogenic granuloma is a proliferative fibrovascular response to prior tissue insult by inflammation, surgery, or nonsurgical trauma. It is sometimes classified as a polypoid form of acquired capillary hemangioma.\[[@ref16]\] It appears clinically as an elevated red mass, often with a florid blood supply. Microscopically, it is composed of granulation tissue with chronic inflammatory cells and numerous small caliber blood vessels ([Fig. 16](#F16){ref-type="fig"}). Because the lesion is rarely pyogenic nor granulomatous, the term "pyogenic granuloma" may be a misnomer. Pyogenic granuloma will sometimes respond to topical corticosteroids but many cases ultimately require surgical excision. In bothersome recurrent cases, low-dose plaque radiotherapy can be applied.\[[@ref26]\] {#F16} Capillary hemangioma {#sec2-26} -------------------- Capillary hemangioma of the conjunctiva generally presents in infancy, several weeks following birth, as a red stromal mass, sometimes associated with cutaneous or orbital capillary hemangioma ([Fig. 17](#F17){ref-type="fig"}). Similar to its cutaneous counterpart, the conjunctival mass might enlarge over several months and then spontaneously involute. Management includes observation most commonly, but surgical resection or local or systemic prednisone can be employed. {#F17} Cavernous hemangioma {#sec2-27} -------------------- Cavernous hemangioma of the conjunctiva is rare.\[[@ref94]\] This benign tumor appears as a red or blue lesion usually in the deep stroma in young children ([Fig. 18](#F18){ref-type="fig"}). It may be similar to the orbital cavernous hemangioma that is generally diagnosed in young adults. It can be managed by local resection. {#F18} Racemose hemangioma {#sec2-28} ------------------- Occasionally, dilated arteriovenous communication without intervening capillary bed (racemose hemangioma) is found in the conjunctiva. This appears as a loop or neatly wound monolayer of a dilated, noncrossing vessel in the stroma with no evident stimulus or planned direction. It can remain stable for years and is generally monitored conservatively. It is important to rule out Wyburn--Mason syndrome in these cases. Lymphangioma {#sec2-29} ------------ Conjunctival lymphangioma can occur as an isolated conjunctival lesion or, more often, it represents a superficial component of a deeper diffuse orbital lymphangioma.\[[@ref95]\] It usually becomes clinically apparent in the first decade of life and appears as a multliloculated mass containing variable-sized clear dilated cystic channels ([Fig. 19](#F19){ref-type="fig"}). In most instances, one sees blood in many of the cystic spaces. These have been called "chocolate cysts." The treatment of conjunctival lymphangioma is often extremely difficult because surgical resection or radiotherapy cannot completely eradicate the mass. {#F19} Varix {#sec2-30} ----- Varix is a venous malformation that can be found in the orbit and rarely the conjunctiva. It is a mass of dilated venous channels that can enlarge with Valsalva maneuver. Some authorities believe that this condition is in the spectrum of lymphangioma. Treatment involves cautious observation. If clotted and painful, cold compresses and aspirin may be useful. Surgical resection should be cautiously employed due to the risk for prolonged bleeding at surgery.\[[@ref71]\] Hemangiopericytoma {#sec2-31} ------------------ Hemangiopericytoma is a tumor composed of the pericytes that surround blood vessels.\[[@ref24]\] It can show both benign and malignant cytological features. It appears as a red conjunctival mass originating from the stroma. Wide surgical resection with tumor-free margins is advised. Kaposi\'s sarcoma {#sec2-32} ----------------- Kaposi\'s sarcoma is best known as a cutaneous malignancy that occurs in elderly immunosuppressed patients. With the advent of acquired immune deficiency syndrome (AIDS), this tumor has become more common and often affects mucous membranes, including conjunctiva. Clinically, it appears as one or more reddish vascular masses that may resemble a hemorrhagic conjunctivitis ([Fig. 20](#F20){ref-type="fig"}). It is moderately responsive to chemotherapy and markedly responsive to low dose radiotherapy.\[[@ref69]\] {#F20} Fibrous Tumors {#sec1-7} ============== Fibroma {#sec2-33} ------- Fibroma is a rare conjunctival tumor that appears as a white stromal mass, either unifocal or multifocal.\[[@ref31]\] Surgical resection is advised. Fibrous histiocytoma {#sec2-34} -------------------- Fibrous histiocytoma is a rare mass of the conjunctiva and is comprised of fibroblasts and histiocytes. Clinically and histopathologically it resembles many other amelanotic stromal tumors. In the conjunctiva it can be benign, locally invasive, or malignant. Wide excision with tumor-free margins is advised. Nodular fasciitis {#sec2-35} ----------------- Nodular fasciitis is a benign proliferation of connective tissue that most commonly occurs in the skin and less commonly in the eyelid, orbit, and conjunctiva. Clinically and histopathologically it can resemble fibrosarcoma. The lesion appears as a solitary white mass in Tenon\'s fascia. Complete excision is advised as the lesion can recur. Neural Tumors {#sec1-8} ============= Neural tumors of the conjunctiva are rare. They tend to manifest a more yellow appearance than the fibrous tumors. Neurofibroma {#sec2-36} ------------ Neurofibroma can occur in the conjunctiva as a solitary mass or as a diffuse or plexiform variety. The former is not usually associated with systemic conditions and the latter is generally a part of von Recklinghausen\'s neurofibromatosis.\[[@ref58][@ref75]\] The solitary tumor is a slowly enlarging elevated stromal mass that is best managed by complete surgical resection. The plexiform type is more difficult to surgically excise and debulking procedures are often necessary. Neurilemoma {#sec2-37} ----------- Neurilemoma, also known as schwannoma, is a benign proliferation of Schwann cells that surround the peripheral nerves. This tumor more commonly arises in the orbit, but there are reports of similar rare tumor in the conjunctiva.\[[@ref44]\] Clinically, this lesion is a yellowish-pink, nodular mass in the stroma. Complete excision is warranted to minimize recurrence. Granular cell tumor {#sec2-38} ------------------- Granular cell tumor is a rare tumor and of disputed origin, but currently, most authorities speculate that it is of Schwann cell origin.\[[@ref76]\] This benign tumor clinically appears smooth, vascular, and pink, and is located in the stroma or within Tenon\'s fascia. Histopathologically, it is comprised of large round cells with pronounced granularity to the cytoplasm. Complete excision is advised. Histiocytic Tumors {#sec1-9} ================== Xanthoma {#sec2-39} -------- Xanthoma most often occurs within the cutaneous dermis, near extensor surfaces and its location on the conjunctiva is exceptionally rare. Conjunctival xanthoma appears as a yellow subepithelial smooth mass affecting one or both epibulbar surfaces. Bilateral conjunctival involvement has been found in a condition termed xanthoma disseminatum. Histopathologically, subepithelial infiltrate of lipidized histiocytes, eosinophils, and Touton giant cells are seen. Juvenile xanthogranuloma {#sec2-40} ------------------------ Juvenile xanthogranuloma is a relatively common cutaneous condition that presents as painless, pink skin papules with spontanteous resolution, generally in children under the age of 2 years. Rarely, conjunctival, orbital, and intraocular involvement is noted. In the conjunctiva, the mass appears as an orange-pink stromal mass, typically in young adults ([Fig. 21](#F21){ref-type="fig"}). If the classic skin lesions are noted, the diagnosis is established clinically and treatment with observation or topical steroid ointment is provided. Otherwise, biopsy is suggested and recognition of the typical histopathologic features of histiocytes admixed with Touton\'s giant cells confirms the diagnosis. {#F21} Reticulohistiocytoma {#sec2-41} -------------------- Reticulohistiocytoma is a rare tumor, often found as part of a systemic multicentric reticulohistiocytosis. Clinically, the tumor appears as a pink, vascular limbal mass in an adult. Histopathologically, it is comprised of large histiocytes with granular cytoplasm.\[[@ref13]\] Myxoid Tumors {#sec1-10} ============= Myxoma {#sec2-42} ------ Myxoma is a rare conjunctival tumor that appears as an orange-pink mass within the stroma. The tumors are slow growing, freely movable solitary lesions located usually in the temporal bulbar conjunctiva. Histologically, they are hypocellular and were composed of stellate and spindle-shaped cells interspersed in a loose stroma.\[[@ref43][@ref68][@ref77]\] Myogenic Tumors {#sec1-11} =============== Rhabdomyosarcoma {#sec2-43} ---------------- Ophthalmic rhabdomyosarcoma is generally regarded as a primary orbital tumor; however, it can occur primarily in the conjunctiva and even within the globe.\[[@ref66]\] Conjunctival rhabdomyosarcoma appears as a pink, vascular mass with rapid growth, usually over 1 to 2 months. Complete excisional biopsy is advised and adjunctive therapy with chemotherapy and possibly radiotherapy is warranted depending on many factors.\[[@ref66]\] Lipomatous Tumors {#sec1-12} ================= Lipoma {#sec2-44} ------ Conjunctival lipoma is quite rare and generally is found in adults as a yellowish-pink stromal mass.\[[@ref68][@ref77]\] They are generally of pleomorphic type with large lipid vacuoles surrounded by stellate cells. Herniated orbital fat {#sec2-45} --------------------- Occasionally, orbital fat presents in the conjunctiva as a herniation from the superotemporal orbit. The condition is often bilateral and represents deficiency in the orbital connective tissue to maintain the proper location of the normal orbital fat. Clinically, the mass is deep to Tenon\'s fascia and is most prominent on inferonasal gaze ([Fig. 22](#F22){ref-type="fig"}). Digital reposition of the fat into the orbit can be performed, but is only temporary. Management is observation, unless the condition causes symptoms of dry eye from eyelid malposition. In these cases, resection of the herniated fat and resuspension of the orbit position of the fat is advised. Histopathologically, the tissue comprises large lipid cells. Daniel and coauthors recently described six patients with typical herniated orbital fat that proved on histopathology to have pleomorphic lipoma, with large pleomorphic cells within the adipose tissue arranged in a floret-like pattern.\[[@ref12]\] They noted the clinical overlap between these two conditions. {#F22} Liposarcoma {#sec2-46} ----------- Liposarcoma of the conjunctiva has been rarely recognized and shows clinical features similar to lipoma. Histopathologically, neoplastic stellate lipid cells and signet-ring type cells have been observed.\[[@ref77]\] Lymphoid Tumors {#sec1-13} =============== Lymphoid tumors can occur in the conjunctiva as isolated lesions or they can be a manifestation of systemic lymphoma.\[[@ref6][@ref8][@ref34][@ref37][@ref61]\] Clinically, the lesion appears as a diffuse, slightly elevated pink mass located in the stroma or deep to Tenon\'s fascia, most commonly in the forniceal region ([Fig. 23](#F23){ref-type="fig"}). This appearance is similar to that of smoked salmon; hence it is termed the "salmon patch."\[[@ref61]\] It is not usually possible to differentiate clinically between a benign and malignant lymphoid tumor. Therefore, biopsy is necessary to establish the diagnosis and a systemic evaluation should be done in all affected patients to exclude the presence of systemic lymphoma \[[Table 7](#T7){ref-type="table"}\]. Histopathologically, sheets of lymphocytes are found and classified as reactive lymphoid hyperplasia or malignant lymphoma. Most are B cell lymphoma (non-Hodgkin\'s type). Rarely, T cell lymphoma is noted.\[[@ref62]\] Treatment of the conjunctival lesion should include chemotherapy if the patient has systemic lymphoma or external beam irradiation (2,000--4,000 cGy) if the lesion is localized to the conjunctiva. Other options include excisional biopsy and cryotherapy,\[[@ref14]\] local interferon injections, or observation. {#F23} ###### Risks for the Development of Systemic Lymphoma in Patients who Present with Conjunctival Lymphoid Infiltrate and No Sign of Systemic Lymphoma Development of Systemic Lymphoma ----------------------------------------------- ---------------------------------- ---- ---- Generally, if conjunctival lymphoid tumor (%) 7 15 28 Specifically, if conjunctival lymphoma (%) 12 38 79 From Shields *et al*.^61^ x 61. Shields, C.L., Shields, J.A., Carvalho, C. *et al*. Conjunctival lymphoid tumors: clinical analysis of 117 cases and relationship to systemic lymphoma. Ophthalmology. 2001; 108: 979-984 Leukemia {#sec1-14} ======== Leukemia generally manifests in the ocular region as hemorrhages from associated anemia and thrombocytopenia rather than leukemic infiltration.\[[@ref46]\] However, leukemic infiltration can be found with chronic lymphocytic leukemia. In these cases, the tumor appears as a pink smooth mass within the conjunctival stroma either at the limbus or the fornix, similar to a lymphoid tumor. Biopsy reveals sheets of large leukemic cells. Treatment of the systemic condition is advised with secondary resolution of the conjunctival infiltration. Metastatic Tumors {#sec1-15} ================= Metastatic tumors rarely occur in the conjunctiva but conjunctival metastasis can occur from breast carcinoma, cutaneous melanoma, and other primary tumors.\[[@ref33]\] Metastatic carcinoma appears as one or more fleshy pink vascularized conjunctival stromal tumors ([Fig. 24](#F24){ref-type="fig"}). Metastatic melanoma to the conjunctiva usually is pigmented.\[[@ref33]\] {#F24} Secondary Conjunctival Involvement from Adjacent Tumors {#sec1-16} ======================================================= The conjunctiva can be secondarily involved by tumors of adjacent structures, particularly by direct extension from tumors of the eyelids. The most important tumor to exhibit this behavior is sebaceous gland carcinoma of the eyelid.\[[@ref5][@ref28]\] This tumor can exhibit pagetoid invasion and extend directly into the conjunctival epithelium. This can result in a clinical picture compatible with chronic unilateral blepharoconjunctivitis. Uveal melanoma in the ciliary body region can extend extrasclerally into the subconjunctival tissues, simulating a primary conjunctival tumor. Rhabdomyosarcoma of the orbit, a tumor typically found in children, occasionally presents first with its conjunctival component before the mass is discovered in the orbit.\[[@ref66][@ref73]\] Caruncular Tumors and Cysts {#sec1-17} =========================== The caruncle is a unique anatomic structure that contains elements of both conjunctiva and skin. The tumors and related lesions that develop in the caruncle are similar to those that occur in mucous membranes and cutaneous structures. By histopathologic analysis, 95% of caruncular tumors are benign and 5% are malignant.\[[@ref35]\] The most common lesions include papilloma and nevus \[[Table 8](#T8){ref-type="table"}\] ([Fig. 25](#F25){ref-type="fig"}).\[[@ref35][@ref67]\] Other caruncular lesions include pyogenic granuloma, inclusion cyst, sebaceous hyperplasia, and sebaceous adenoma, and oncocytoma.\[[@ref59]\] Malignant tumors such as squamous cell carcinoma, melanoma, lymphoma, and sebaceous carcinoma are relatively rare in the caruncle. The oncocytoma is a benign tumor that occurs more commonly in the lacrimal or salivary glands. In the caruncle it probably arises from accessory lacrimal gland tissue and often has a blue cystic appearance ([Fig. 25](#F25){ref-type="fig"}). The treatment of most caruncular masses is either observation or local resection, depending on the final diagnosis. ###### Types and Frequency of Tumors of the Caruncle: Comparison of Two Major Survey Lesions (%) Luthra *et al*.^35^ (*n*=112) Shields *et al*.^67^ (*n*=57) ------------------------------- ------------------------------- ------------------------------- Papilloma 13 32 Nevus 43 24 Pyogenic granuloma 3 9 Epithelial inclusion cyst 4 7 Chronic inflammation 4 7 Oncocytoma 4 4 Normal caruncle 0 4 Sebaceous gland hyperplasia 8 2 Sebaceous gland adenoma 0 2 Lipogranuloma 0 2 Seborrheic keratosis 1 2 Lymphangiectasia 0 2 Histiocytic lymphoma 0 2 Squamous cell carcinoma 0 2 Basal cell carcinoma 0 2 Reactive lymphoid hyperplasia 4 0 Foreign body granuloma 3 0 Malignant melanoma 2 0 Capillary hemangioma 2 0 Senile keratosis 1 0 Freckle 1 0 Adrenochrome pigment 1 0 Cavernous hemangioma 1 0 Dermoid 1 0 Granular-cell myeloblastoma 1 0 Plasmacytoma 1 0 Apocrine hydrocystoma 1 0 Pilar cyst 1 0 Sebaceous gland carcinoma 1 0 Ectopic lacrimal gland 1 0 From Luthra, C.L., Doxanas, M.T., and Green, W.R. Lesions of the caruncle. A clinicopathologic study. Surv Ophthalmol. 1978; 23: 183-195 and Shields, C.L., Shields, J.A., White, D., and Augsburger, J.J. Types and frequency of lesions of the caruncle. Am J Ophthalmol. 1986; 102: 771-778 {#F25} Miscellaneous Lesions that can Simulate Conjunctival Neoplasms {#sec1-18} ============================================================== A number of non-neoplastic conditions can simulate neoplasms. These include pingueculum, pterygium, foreign body, inflammatory granuloma, amyloidosis, and others.\[[@ref76]\] In most instance, the history and clinical findings should make the diagnosis obvious. In some instances, however, excision of the mass may be necessary in order to exclude a neoplasm. Method of Literature Search {#sec1-19} =========================== A comprehensive literature search over the past 30 years was derived from PubMed using general search words *conjunctiva, cornea, caruncle, tumor, neoplasia, cancer, and malignancy*. Additional search words were input for each of the 47 specific diagnostic entities listed in the outline from *dermoid* to *liposarcoma* to *caruncle tumor*. The search words *conjunctiva tumor* yielded 77 pages of 1,536 references. The search words *conjunctiva neoplasia* yielded 74 pages of 1,473 references, *conjunctiva melanoma* yielded 19 pages of 364 references, and *conjunctiva squamous cell carcinoma* produced 13 pages of 249 references. Additional references were gathered from published articles that provided a literature review of a topic. References used in this report included those that represented the first or second report in the literature of an entity or treatment of an entity, those that represented substantial case series of certain entities, and those that were particularly well-written, well-illustrated, or recent publication. English literature articles were used and non-English articles were included if they met the above criteria. Financial support and sponsorship {#sec2-47} --------------------------------- Nil. Conflicts of interest {#sec2-48} --------------------- There are no conflicts of interest. General ConsiderationsAnatomyDiagnostic ApproachesManagementObservationIncisional biopsyExcisional biopsyCryotherapyChemotherapyRadiotherapyModified enucleationOrbital exenterationMucous membrane graftCongenital TumorsDermoidDermolipomaEpibulbar osseous choristomaLacrimal gland choristomaRespiratory choristomaComplex choristomaBenign Tumors of Surface EpitheliumPapillomaKeratoacanthomaHereditary benign intraepithelial dyskeratosisEpithelial inclusion cystDacryoadenomaKeratotic plaqueActinic KeratosisMalignant Tumors of Surface EpitheliumConjunctival intraepithelial neoplasia (CIN)Invasive squamous cell carcinoma (SCC)Melanocytic TumorsNevusRacial melanosisOcular melanocytosisPrimary acquired melanosis (PAM)Malignant melanomaConditions that can simulate melanocytic tumorsVascular TumorsPyogenic granulomaCapillary hemangiomaCavernous hemangiomaRacemose hemangiomaLymphangiomaVarixHemangiopericytomaKaposi\'s sarcomaFibrous TumorsFibromaFibrous histiocytomaNodular fasciitisNeural TumorsNeurofibromaNeurilemomaGranular cell tumorHistiocytic TumorsXanthomaJuvenile xanthogranulomaReticulohistiocytomaMyxoid TumorsMyxomaMyogenicRhabdomyosarcomaLipomatous TumorsLipomaHerniated orbital fatLiposarcomaLymphoid TumorsLeukemiaMetastatic TumorsSecondary TumorsCaruncular Tumors and CystsMiscellaneous Lesions that Can Simulate Conjunctival NeoplasmsMethod of Literature Search

Introduction ============ Stem cells stay in their undifferentiated stage until they receive appropriate activation signals and begin the differentiation process into specific lineages, according to the type of received stimuli. Signals such as growth factors and physical cues are provided by the surrounding cellular microenvironment ([@B1],[@B6]). Cell density and cell-cell interactions play major roles in the differentiation process ([@B7]). Some chemical compounds such as dexamethasone are osteogenic supplements and, like growth factors, they play essential roles in osteogenic differentiation of mesenchymal stem cells (MSCs) ([@B8]). In this study, we have used a pulsed electromagnetic field (PEMF) as the biophysical guide to create waves with constant properties. Such waves are non-ionizing and create non-thermal fields with high rates of amplitude changes ([@B9]). The applied frequency of the extremely low frequency electromagnetic field is under 300 Hz and the amplitude ranges from 0.2 to 20 millitesla (mT) ([@B10]). PEMF is clinically used to treat osteoporosis by increasing bone mass in women with menopause and snapback in patients with osteotomies. This field also acts to reduce the bone resorption activity of osteoclasts ([@B11]) as well as increase calcium content and other bone minerals ([@B12]). As a biophysical factor, PEMF motivates the release of Ca^2+^ions from the smooth endoplasmic reticulum as the starting point of signaling pathways that activate osteogenic differentiation. The increase in intracellular Ca^2+^level also triggers enzymatic cascades, resulting in the secretion of growth factors such as bone morphogenetic proteins (BMPs), expression of osteoblast-specific genes, and cell proliferation ([@B13]). The interaction between an electromagnetic field and biological tissue is related to the amplitude, frequency, and form of the wave in addition to the time duration of the exposure ([@B9]). Until now, modern medicine has used extremely low frequency PEMFs to treat non-union bone fractures, pseudarthrosis, osteoporosis, and periodontal disease ([@B9]). Interaction of electromagnetic fields with the extracellular matrix can increase cytosolic Ca^2+^and then promote the proliferation of osteoblastic cells ([@B14]). It has been proven that the expression of osteoblastic marker genes is upregulated in response to a combination of specific PEMFs and chemical compounds such as BMPs or other inductive factors ([@B9], [@B15]). In this study, we researched the effect of PEMF on MSCs proliferation and differentiation toward osteoblasts along with the amount of expression of osteoblastic marker genes such as osteocalcin (*Ocn*) and runt-related transcription factor 2 (*Runx2*). Our objective was to analyze the effects of an electromagnetic field on osteogenic differentiation of stem cells. In addition, we assessed the influence of chemical factors when combined with PEMF. Materials and Methods ===================== This was an experimental animal study conducted on rat bone marrow derived MSCs. Mesenchymal stem cell isolation and culture ------------------------------------------- All animal experiments were performed according to approved guidelines of the Ethics Committee at Pasteur Institute of Iran. A total of 9 male, 4-week-old Wistar rats (weights: 230-250 g) were anesthetized in order to obtain bone marrow aspiration from their iliac crests under sterile conditions. After isolation of bone marrow stem cells according to the Ficoll-Paque technique, we cultured these cells in Minimum Essential Medium Eagle Alpha Modification (α-MEM medium, Sigma, NY, USA) supplemented with 15% fetal bovine serum (FBS), 1% penicillin/ streptomycin \[100 U/ml of penicillin and 100 µg/ml of streptomycin (Sigma, NY, USA)\] and 1% L-glutamine (Gibco, NY, USA). The medium was changed every 3 days ([@B8]). Cells at passage-3 (P3) were used for the experiments. The rat osteosarcoma cell line (UMR106) provided by National Cell Bank of Iran (C586) was the positive control group and stem cells comprised the negative control group. Multipotential assay -------------------- We performed chondrogenic, osteogenic, and adipogenic differentiation experiments to examine the multipotential differentiation ability of the isolated cells. For osteogenic differentiation P3 cells were exposed to osteogenic medium that contained Dulbecco's Modified Eagle's Medium (DMEM), 10% FBS, 100 nM dexamethasone, 10 mM β-glycerol phosphate, and L-ascorbic acid 2-phosphate for 21 days. The medium was changed every 3 days. Thereafter, the cells were fixed and stained with Alizarin red S. In order to induce adipogenesis, the cells were subjected to DMEM that contained 10% FBS, 0.5 μM of 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 10 μg/ml insulin, and 100 μM indomethacin for 15 days. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with Oil red O. For directing cells toward chondrogenic differentiation, the cell pellets were prepared and incubated with DMEM that contained 50 mM ascorbic acid-2 phosphate, 10 ng/mL transforming growth factor b1 (TGF b1, R&D Systems, USA), 100 nM dexamethasone, 1% ITS-Premix (BD Biosciences, USA), and 1 mM sodium pyruvate (Gibco, NY, USA) for 28 days. Chondrogenic differentiation was examined by fixing the cells with 10% formalin, followed by sectioning the pellets and staining them with Alcian blue. All chemicals unless otherwise indicated were purchased from Sigma, USA ([@B16]). Immunophenotyping ----------------- Bone marrow derived MSCs were studied for the expression of CD45 as the hematopoietic marker, along with CD73 and CD90 as the MSC surface markers. CD73, a PE conjugated antibody, (BD Biosciences, CA, USA) and FITC-conjugated goat anti-mouse IgG antibodies for CD45 and CD90 (FITC conjugated) were used. Mouse IgG1 K isotype control (eBiosciences, CA, USA), mouse IgG2a K isotype control FITC (eBiosciences, CA, USA), and donkey anti-mouse IgG (H+L) PE (eBiosciences, CA, USA) were used as secondary antibodies for detection of the selected markers. Unstained cells were used for gating in the flow cytometric analysis. We counted 15000 events for each antibody. Data were analyzed by FlowJo software version 7.6.4 ([@B17]). Pulsed electromagnetic field exposure ------------------------------------- PEMF stimulation was performed using Helmholtz coils of copper wire ([@B18]). A pair of 12.7 cm-diameter circular coils was placed opposite to each other within the incubator and the cell flask located in the uniform field area of the coil center. The proper shielding and utilization of Plexiglass was performed to guarantee the prevention of any disturbance to the applied stimulating magnetic field. The electromagnetic field generator, named the Helmholtz coil, consists of two solenoid electromagnets on the same axis. The non-sinusoidal magnetic field is generated by an electric current through the coils. This device is used to produce uniform electromagnetic waves in order to create a uniform magnetic field. These coils cancel the interference of external magnetic fields generated by nearby electrical devices or the Earth's magnetic field. The employed device had three parts: a stimulator, the coils, and a control box. Intensity of the created field was regulated by changing the voltage of the stimulator. The apparatus was ordered by the National Cell Bank of Iran and Behi Afzar Saz Pooya Company of Iran fabricated the entire system ([@B10]). Intensity of the field was 0.2 mT with a 15 Hz frequency. We used a pulse on time of 40 mseconds and pulse off time of 27 mseconds. A tesla meter (Lutron) measured the magnetic flux at the center of the coil. At first, we analyzed three differentiation periods of daily exposure in order to determine the most effective duration of exposure. PEMF was used to stimulate the cells with 0.2 mT and 15 Hz for 10 consecutive days with 2, 4, and 6 hours of exposure per day. The cells from all the groups were exposed to PEMF at 0.2 mT intensity and 15 Hz frequency for 10 consecutive days and 6 hours of exposure per day. This study had three experimental groups: i. Cells incubated with regular culture medium and exposed to the field, ii. Cells stimulated with simultaneous application of the electromagnetic field and chemical differentiation medium (50 μM ascorbate-2 phosphate, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone) for 7 consecutive days, and iii. Cells subjected to a combination of the mentioned electromagnetic field and chemical differentiation medium for 10 consecutive days. Upon completion of the tests, we performed real- time polymerase chian reaction (PCR) analysis to quantify the expressions of the marker genes ([@B15], [@B19]). Untreated MSCs were utilized as the negative control and UMR-106 was the positive control. MTT assay --------- The tetrazole 3-([@B4],[@B5]-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), as a histomorphological stain, was used to study the effect of PEMF on MSC proliferation. MTT is reduced to purple formazan by viable cells. Hence, the number of living cells can be determined based on the absorbance of the formazan solution ([@B20]). We have performed the MTT assay on cells subjected to the 0.2 mT electromagnetic field (6 hours of exposure per day) on the 5^th^, 10^th^, and 14^th^days. Immunocytochemistry ------------------- Immunocytochemistry assay was used to scan the influence of electromagnetic field exposure. Antibodies were used against two osteogenic markers, anti *Runx2* and anti osteocalcins. Immediately after exposure to the field, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma, NY, USA) for 20 minutes at 4˚C. Next, they were permeabilized with 0.5% Triton X100 (Merck, NJ, USA) after which 0.5% gout serum was used to block the nonspecific antibodies. Cells were incubated overnight at 4˚C with mouse monoclonal antibodies against *Runx2* and *Ocn* (both from Abcam, Cambridge, UK). Thereafter, they were incubated with FITC conjugated secondary antibody at a 1:100 dilution (Abcam, Cambridge, UK) at room temperature in the dark for 2 hours. Finally, the presence of the mentioned proteins was examined under a Zeiss fluorescence microscope (×630) ([@B21]). Real-time reverse transcriptional polymerase chain reaction assay ----------------------------------------------------------------- We used real-time reverse transcriptional polymerase chain reaction (RT-PCR) to examine the expressions of the *Ocn* and *Runx2* genes by the stimulated cells. Total RNA was extracted using the RNeasy plus Mini Kit (Qiagen, MD, USA) according to the manufacturer's instructions. The purity of extracted RNA was evaluated by means of a nanodrop spectrophotometer (Implen, Germany). High quality samples with concentrations \>400 ng/μl and A260/A280 ≥1.8 were chosen for analysis. The QuantiTect Reverse Transcription Kit (Qiagen, MD, USA) was used to synthesize complementary DNA (cDNA) from the extracted RNA. Gel electrophoresis was carried out to verify the integrity of cDNA. TaqMan real-time PCR was performed for quantitative analysis of *Ocn* and *Runx2* expressions. Reactions were carried out using an ABI StepOne system with StepOne v2.1 software (Applied Biosystems, CA, USA). All primers and probes were designed using the Primer Express software (version 3.0). The recommended sequences by this software were analyzed using gene runner software. Ribosomal protein large subunit 13a (*RPL13A*) was selected as the housekeeping gene for normalization of the obtained data that corresponded to *Runx2* and *Ocn* mRNA level quantification. Primer sequences were as follows: *Runx2* F: 5ʹ-GCCAGGTTCAACGATCTGAGA-3ʹ R: 5ʹ-GGAGGATTTGTGAAGACCGTTATG-3ʹ probe: 5ʹ-TGAAACTCTTGCCTCGTCCGCTCC-3ʹ *Ocn* F: 5ʹ-GCAGACCTAGCAGACACCATGA-3ʹ R: 5ʹ-CCAGGTCAGAGAGGCAGAATG-3ʹ probe: 5ʹ-TCTCTGCTCACTCTGCTGGCCCTG-3ʹ *RPL13* F: 5ʹ-TGAACACCAACCCGTCTCG-3ʹ R: 5ʹ-GCAGCCTGGCCTCTTTTG-3ʹ probe: 5ʹ-CCCCTACCACTTCCGAGCCCCA-3ʹ. PCR products were checked by gel electrophoresis according to the product size (data not shown). Each reaction was performed in triplicate with a total volume of 20 μl that contained 5 μl of cDNA sample, 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems, USA), 10 pmol of each primer, and 5ʹ-Fam-/3ʹ-Tamra-labeled probe. The thermal cycling profile involved an initial activation for 10 minutes at 95˚C, followed by 15 seconds at 95˚C, 1 minute at 60˚C, and running for 40 cycles. The melting curve stage was set at 95˚C (15 seconds), 60˚C (1 minute), and 95˚C (15 seconds) ([@B22]). Gene expression values were calculated using the following formula: ΔΔCT= \[minimum CT Targets-minimum CT RPL-13A\]Test samples-\[minimum CT Targets- minimum CT RPL-13A\]Stem cells Real-time PCR was performed to compare the effects of 0.1 and 0.2 mT-fields on the expressions of the osteogenic markers. In another part of this study the effects of three daily-exposure durations of 2, 4 and 6 hours for electromagnetic field application were studied after 10 days of stimulation. Real-time PCR was used to compare gene expression levels among the above mentioned groups. Surgical procedures ------------------- Each animal was anesthetized and a small incision was made on the left side of the cranium. The periosteum and soft tissues were removed to access the cranial bone. The collagen-based scaffolds ([@B23]) with dimensions of 5×5×1 mm^3^were implanted after a predrilling with a dental drill. The following three groups were defined and studied in triplicate: i. Bone sockets without any scaffolds; ii. Defects filled with scaffolds seeded with untreated MSCs, and iii. Defects filled with scaffolds seeded with electromagnetically and chemically motivated MSCs. Vicryl 3-0 suture was used to close the incisions. We performed autologous transplantation and each animal used its own stem cells. The scaffolds were retrieved after 10 weeks of implantation and fixed with 4% paraformaldehyde for 12 hours. Thereafter, decalcification of bones was carried out in 10% EDTA for two weeks followed by embedding in paraffin. Tissue blocks were sectioned into 5 µm thick sections and stained with hematoxylin and eosin (H&E) to assess bone healing ([@B24]). Statistical analysis -------------------- All data that corresponded to the three separate experiments were expressed as means ± SD. Statistical analyses were performed using oneway ANOVA and the student's t test via SPSS software version 17.0. P values lower than 0.05 were considered statistically significant. Results ======= Differentiation potential assays -------------------------------- The results of multi-lineage differentiation experiments confirmed the potential of isolated cells to differentiate into adipocytes, osteoblasts, and chondrocytes. After oil red O staining, we observed the presence of lipid vacuoles. Alizarin red S staining revealed the presence of calcified nodules and Alcian blue staining demonstrated sulfated glycosaminoglycans and chondrogenesis (data not shown). These observations indicated the multi-potent identity of the isolated cells. Characterization of mesenchymal stem cells ------------------------------------------ We used flow cytometry to characterize the mesenchymal identity of the isolated cells. According to the results, the cells were negative for the hematopoietic marker, CD45. These cells highly expressed CD73 and CD90 as MSC- associated surface proteins. The obtained results ([Fig.1](#F1){ref-type="fig"}) confirmed the mesenchymal identity of the isolated cells. {#F1} Electromagnetic field and proliferation --------------------------------------- We used the MTT assay to determine the influence of a low frequency electromagnetic field on stem cell proliferation after 5, 10, and 14 days of cell exposure to PEMF (0.2 mT, 15 Hz for 6 hours exposure/day). Unstimulated MSCs were the negative control. As illustrated in Figure 2, cells stimulated with the electromagnetic field had a higher proliferation rate compared to unstimulated MSCs. Thus PEMF treatment for 14 days did not have any negative effect on MSC proliferation; rather, it enhanced the proliferative activity of these cells. {#F2} Effects of electromagnetic field intensity ------------------------------------------ We conducted real-time PCR analysis of the effects of field intensity on gene expression. MSCs from two separate groups were exposed to 0.1 mTor 0.2 mT-intensity fields with similar field parameters of 15 Hz frequency and 6 hours application of PEMF per day for 10 consecutive days. As shown in Figure 3, the 0.2 mT intensity field resulted in a greater increase in expression of osteoblastic genes compared to the 0.1 mT field. {#F3} Effects of electromagnetic field exposure duration -------------------------------------------------- We tested three different durations of daily exposure in order to find the most influential duration. Stem cells were stimulated with PEMF (0.2 mT and 15 Hz) for 10 consecutive days with daily exposure durations of 2, 4, or 6 hours. We observed the highest expression levels of *Runx2* and *Ocn* in the group that received 6 hours of daily exposure to PEMF ([Fig.4](#F4){ref-type="fig"}). {#F4} Combination of electromagnetic field and chemical induction ----------------------------------------------------------- Simultaneous application of chemical supplements and the electromagnetic field was carried out to assess the effects of combined treatment on expressions of the osteogenic genes. Real-time PCR was performed after electromagnetic field exposure at 6 hours daily for a 10-day period along with concurrent incubation with chemical factors in order to quantify mRNA levels of the osteogenic markers. MSCs were incubated for 7 and 10 days in induction medium. We compared the results with cells stimulated only with PEMF. The results showed that *Runx2* and *Ocn* had the highest expression levels 10 days after cells were subjected to the combination of induction medium and PEMF waves ([Fig.5A, B](#F5){ref-type="fig"}). Immunocytochemistry for pulsed electromagnetic field stimulation ---------------------------------------------------------------- Immunocytochemistry results demonstrated a slight expression of *Runx2* protein in stem cells ([Fig.6A](#F6){ref-type="fig"}) and presence of higher amounts of *Runx2* in cells stimulated only with the electromagnetic field ([Fig.6B](#F6){ref-type="fig"}). We observed no osteocalcin expression in unstimulated stem cells ([Fig.6C](#F6){ref-type="fig"}) and large amounts of osteocalcin in cells stimulated only with the electromagnetic field ([Fig.6D](#F6){ref-type="fig"}). In vivo studies --------------- Histological analysis was performed to assess bone and tissue ingrowth by differentiated MSCs stimulated by the electromagnetic field. After 10 weeks of implantation, we observed no signs of any inflammatory cells such as macrophages, lymphocytes, or giant cells in the different experimental groups. In all test groups, new osteoid areas formed adjacent to the pre-existing bones ([Fig.7](#F7){ref-type="fig"}). As a result of osteoblast activity, osteoids were produced on the surface of the new bone. Implanted scaffolds underwent degradation and no signs of the scaffold residues could be observed. In general, after 10 weeks the created defects had evidence of new bone in all three test groups. {#F5} {#F6} {#F7} Discussion ========== The present study evaluated the effects of electromagnetic field application and biochemical stimulation on MSCs and their gene expression patterns. Electromagnetic field parameters were selected such that the effect of PEMF on the expression of osteogenic markers and osteogenic differentiation could be assessed. We used the MTT assay, immunocytochemistry, TaqMan real- time PCR, and histological analysis to study the behavior of stem cells in response to this exposure. Flow cytometry analysis confirmed the identity of the cells .The MTT assay was carried out to investigate the effects of the low electromagnetic field on MSCs. The results indicated a progressive increase in the proliferation rate of MSCs due to the application of the extremely low frequency PEMF. A 20-60% increase in cell density due to exposure to the field was previously reported ([@B15], [@B19]). It has been suggested that the healing effects of the electromagnetic field on fractures may be related to its effects on promoting proliferation and growth acceleration in stem cells and preparing more progenitor cells for differentiation toward osteoblasts ([@B19]). Previous studies have suggested that PEMF may activate free ions on the cell surface. K^+^and Ca^2+^currents affect the activated K^+^channels when progressing from the G~1~-to S-phase and this mechanism may promote the proliferation of undifferentiated stem cells ([@B14]). In another study, electromagnetic fields have been shown to alter membrane functions by opening or closing ion channels, bind ligands, and the numbers and distribution of receptors ([@B11]). Thus PEMFs affect the molecular currents and cause a specific transmembrane signaling which can promote osteogenic differentiation. There are contradictions in terms of the duration of daily exposure and its consequences among different studies. Although Matsumoto et al. ([@B25]) have reported that longer stimulation durations per day resulted in higher bone contents, they did not observe any significant difference in terms of bone formation between two groups stimulated for 4 or 8 hours per day. However, the results of the present experiment revealed that 6 hours of stimulation per day showed greater benefit in enhancing the mRNA level of *Ocn*, as a late osteogenesis marker. Previous studies have demonstrated that compared to higher intensity values such as 0.8 mT, low electromagnetic intensities (0.2 and 0.3 mT) are more effective in promoting bone formation ([@B25]). In the present study, we have compared low electromagnetic intensities in order to determine which one more effectively promoted osteogenesis. Among the 0.1 and 0.2 mT intensities, we determined that the latter intensity level led to higher expression levels of early and late osteoblastic genes. These findings supported the results of previous reports ([@B26], [@B27]). *Runx2* has an inconsistent expression pattern during differentiation. This disharmony in mRNA levels was first reported by Tsai et al. ([@B15]). However, an overall up-regulation of this gene during osteogenic culture has been observed. Jansen et al. ([@B28]) previously reported that bone marker genes reached their highest expression levels between days 5 and 9 of exposure to PEMF. In other words, these genes reached their maximum expression levels just before and around the onset of cell mineralization. This result was also observed in the current study, in which we documented the highest expressions of *Runx2* and *Ocn* on day 10 of exposure to the electromagnetic field. *Runx2* and *Ocn* expressions downregulated between days 10 and 14 which indicated a transition to the mineralization stage. According to this finding and in agreement with previous reports, we concluded that PEMF treatment affected osteogenic differentiation of stem cells and stimulated mineralization at a time period just prior to the mineralization stage. Downregulation of osteogenic genes after an initial upregulation has been reported in previous works ([@B15], [@B28]). Multiple signaling pathways promote osteogenic differentiation of stem cells, some of which such as the canonical Wnt signaling pathway are triggered by PEMF application. The canonical Wnt pathway results in β-catenin stability, which goes to the nucleus and leads to the expression of target genes subsequently resulting in osteogenic differentiation and bone formation ([@B20], [@B29]). The chemical induction medium that contained ascorbic acid, β-glycerophosphate, and dexamethasone has promoted mineralization of the extracellular matrix through activation of different signal transduction pathways ([@B8]). Thus PEMF waves and the utilized biochemical factors reinforced the effects of each other. Implantation of differentiated cells on prefabricated scaffolds to defective areas of the bone and following the changes in the tissue has not been previously considered. According to the *in vivo* results of this study, differentiated osteoblasts seeded on scaffolds promoted filling of the incision and healing of the defects, after H&E staining of the sections related to different implant types, we observed the formation of new bone tissues throughout the scaffold structures. There was no fibrous tissue formation or inflammatory response observed in the different groups. The new osteoblasts produced osteoids on the surface of the pre-existing bone. This research intended to find the optimized parameters of the electromagnetic field in order to achieve an osseous tissue that could be implanted into the stem cell donor. In this process certain defects or malformations would be treated, therefore PEMF could be used to treat some osteogenic disorders via promoting osteogenic differentiation. In similar studies, no *in vivo* analysis was used to estimate the efficiency of the new osteoblasts and their life-time. Some of the field parameters utilized in those studies were slightly different. Conclusion ========== The induced electric currents by electromagnetic fields have the potential to induce osteogenesis in MSCs. Therefore, PEMF has modulating effects on stem cell proliferation and promotion of osteogenic differentiation. PEMF is a potentially low cost tool for tissue engineering which can construct new bone. This tool can be applied for fabrication of autografts in orthopedic surgeries as well as for treatment of maxillofacial disorders. This work was financially supported by the Iran National Science Foundation ( INSF ) grant number 89004147. There is no conflict of interest in this study.

胸腺肿瘤是一类少见疾病，发病仅为0.17/10万，亚裔人种略高于白种人，约0.3-0.4/10万。胸腺瘤研究与投入均较少，诊断与治疗缺乏统一的指南，尤其在术前定性诊断、组织学分型、术前治疗、手术方式、术后治疗以及对术后复发的治疗等一系列问题上均处于不规范状态。很多医生也对该疾病的治疗经验有限，而且研究进展缓慢，但是对于患者和家属，这是不可接受的理由。2003年美国患者Barbara Neibauer身患胸腺肿瘤，虽经积极的治疗，但仍于两年后去世。Barbara Neibauer去世后，为促进胸腺瘤的研究，其家族出资于2005年成立了全球首个关于胸腺肿瘤的基金会即FTCR（Foundation for Thymic Cancer Research）。2010年5月5日由美国NIH（National Institutes of Health）牵头，在FTCR的基础上于纽约成立了专业胸腺肿瘤学术组织，即ITMIG（International Thymic Malignacy Interest Group）。随后，日本也成立了相应的胸腺肿瘤学术组织即JART（Japanese Association for Research on the Thymus）。2012年6月由方文涛、陈克能等教授发起，在上海成立了中国胸腺肿瘤协作组（ChART），并与JART一同加入到ITMIG。 对胸腺瘤的深入研究，显然要寻找有效的方法来综合全世界的数据，这也是ITMIG（International Thymic Malignancy Interest Group）的主要目标。想要合作，就必须对该疾病使用相同的术语，但令人惊讶的是，现行的胸腺瘤实践中存在众多模糊定义和对相同概念有众多不同的解释，使得数据之间无法进行比较。因此，ITMIG联合全球的胸腺医务工作者对关键定义和方法共同讨论并达成共识，旨在促进胸腺瘤的研究。这项工作的结果收录在《胸部肿瘤杂志》（Journal of Thoracic Oncology）。ITMIG在建立这一广泛接受的共识时，首先由小组集中讨论提出初步建议，之后由扩大工作组（约80人组成）修订。然后将这些工作集中后于2010年11月15日-16日在耶鲁大学召开了为期2天的会议，对推荐的定义和方法进行讨论，并将结果分发给ITMIG的每位成员进一步讨论并反馈意见，最终修订版本经ITMIG批准并被采用。希望将来不管是单中心研究还是ITMIG的合作研究，都能采用这些标准。其中，因为胸腺瘤有一些不同于其它恶性肿瘤的特点，如除发病率低外，胸腺瘤还表现为惰性生物学行为，这对预后的临床评估有着诸多影响，因此，ITMIG特地撰写了"Standard Outcomes for Thymic Malignancies"，以供大家参考。 ITMIG共识中多数涉及患者的临床治疗，包括影像、活检、手术、放疗和化疗。对应用最广泛的Masaoka-Koga分期作了详细的细节规定。统一的定义将推动ITMIG的两个主要项目，即建立全球合作数据库和与IASLC的合作项目，为AJCC第8版肿瘤分期手册制订一个正式有效的胸腺瘤分期系统。必须强调的是，尽管要求统一的定义和方法，但并不意味它们是一成不变的，希望将来的研究能改善临床实践和共同使用的术语。同时鼓励对这些原则进行质疑和改进。 在ChART成立短短不到一年的时间里，已向ITMIG提供了信息较为完整的胸腺瘤病例资料近900例，占ITMIG目前所收集病例的四分之一，得到了ITMIG的充分肯定。2012年11月在日本福冈举办的第三届胸腺肿瘤会议上，ChART的4篇文章参加会议口头和壁报交流，并获得本次大会的Barbara Neibauer奖。同时积极参与了ITMIG目前正在筹划开展的两项前瞻性多中心国际合作课题，其一是前瞻性观察研究，其二是Ⅲ期胸腺瘤的全球多中心随机对照研究。 然而，与ITMIG一样，我国胸腺肿瘤学术界长期以来同样存在定义不规范，名词不统一等一系列问题，为此，经ChART研究，有必要就胸腺肿瘤的影像诊断规范、活检标本及切除标本的处理规范、临床及病理分期、预后评估标准、微创的标准用语及术式、放疗及化疗的定义及报告规范等问题做深入讨论，旨在进一步规范胸腺肿瘤的诊疗。首届论坛由北京大学肿瘤医院承办，邀请了影像学专家，病理学专家探讨胸腺肿瘤的诊断问题，同时邀请了外科专家，放疗科专家及内科专家，就治疗问题进行探讨，并将ITMIG提出的相关规定综合成册，供大家讨论应用。 2013年3月

Background ========== Surface layers (S-layers) are cell envelope structures ubiquitously found in Gram-positive and Gram-negative bacterial species as well as in *Archaea*. They are composed of numerous identical (glyco)protein subunits, 40--200 kDa in molecular weight, which completely cover the cell surface forming a two-dimensional, regular array having either oblique (p1, p2), square (p4) or hexagonal (p3, p6) symmetry. The subunits are held together and attached to the underlying cell surface by noncovalent interactions, and they have an intrinsic ability to spontaneously form regular layers either in solution or on a solid support under suitable conditions \[[@B1]\]. Functions of S-layers are poorly known thus far. They include the determination and maintenance of cell shape, action as a protective coat, molecular sieve or ion trap or as a mediator of adhesion or surface recognition. The contribution of S-layers to virulence has been reported \[[@B1],[@B2]\]. In general, S-layer proteins have two structural regions in which two essential functions reside: a region involved in the attachment of the S-layer subunit to the cell envelope and a region involved in S-layer assembly. These regions have been characterized in several Gram-positive and some Gram-negative bacteria. In many Gram-positive bacilli and in *Thermus thermophilus*so called SLH (S-layer homology) motifs \[[@B3]\], 55--60 amino acids long and often located in the N-terminal part of the protein, are responsible for the attachment of the subunit proteins to the cell wall through a pyruvylated polysaccharide receptor in the cell wall \[[@B4]\]. In S-layers of Gram-positive bacteria not having SLH-motifs the attachment to the cell wall has been proposed to be mediated by an interaction between basic amino acids in the cell wall binding region and negatively charged secondary cell wall polymers. The cell wall receptors of such S-layers in *Geobacillus*species characterized so far contain mannuronic acid and can be classified as acidic oligosaccharides other than teichoic or teichuronic acids, while teichoic and lipoteichoic acids have been shown to be the cell wall receptors of the S-layer proteins of *Lactobacillus acidophilus*and *Lactobacillus crispatus*. However, some cell wall polysaccharides of Gram-positive bacteria proposed to be involved in S-layer binding have a net neutral charge \[[@B1],[@B5]-[@B7]\]. Among Gram-positive bacteria, the self-assembly regions of S-layer proteins have so far been studied in the S-layers of lactobacilli (see below), and in the S-layers of *Bacillus anthracis*, *Lysinibacillus sphaericus*and *Geobacillus stearothermophilus*. These studies rely on electron microscopy of recombinant S-layer protein fragments, and the self-assembly region has been shown to be of either central or C-terminal location \[[@B8]-[@B11]\]. In addition to *L. brevis*, S-layers have also been found in *Lactobacillus helveticus*as well as in several *Lactobacillus acidophilus*group bacteria \[[@B12]\] including *L. acidophilus*, *L. crispatus*and *L. gallinarum*. The overall sequence similarity between characterized *Lactobacillus*S-layer protein genes is low and similarity is usually found only between related species. The presence of multiple S-layer protein genes in a single strain is common in lactobacilli. For example, *L. brevis*ATCC 14869 has three S-layer protein genes, two of which are expressed under different environmental conditions and one is silent under laboratory conditions \[[@B13]\]. Other typical features of *Lactobacillus*S-layer proteins include their relatively small size and a high predicted overall pI \[[@B7]\]. Self-assembly and cell wall binding regions have been characterized in the S-layer protein S~A~of *Lactobacillus acidophilus*ATCC 4356 \[[@B14]\] and CbsA of *L. crispatus*JCM 5810 \[[@B15]\]. The sequences of S~A~and CbsA are homologous especially in the C-terminal region, which mediates the attachment to the cell wall, and the more variable N-terminal part is responsible for the self-assembly of the S-layer subunits. The S-layer protein of *Lactobacillus brevis*ATCC 8287, SlpA \[[@B16]\], is a 435 amino acid, 46 kDa protein, which assembles on the bacterial cell forming an oblique lattice \[[@B17]\] and for which a fibronectin-binding function has been described \[[@B18]\]. *L. brevis*is phylogenetically distant from *L. acidophilus*group \[[@B19]\], and this is reflected in the unique amino acid sequence of SlpA compared to *L. acidophilus*group S-layer proteins \[[@B7]\]. Foreign epitopes up to 11 amino acids long have been expressed in SlpA in order to develop tools for mucosal immunization \[[@B17]\]. *L. brevis*ATCC 8287 would be a suitable strain to be used as a live oral vaccine, as it has a GRAS (Generally Recognized As Safe) status and it has been shown to possess probiotic properties \[[@B20]\]. For vaccine development, as well as for nanobiotechnological applications, for which regularly arranged S-layers are especially well-suited \[[@B21],[@B22]\], knowledge about the structure-function relationships of SlpA, presented in this study, is essential. In this work, we have characterized the two-domain structure of the S-layer protein SlpA of *L. brevis*ATCC 8287 with its C-terminal self-assembly and N-terminal cell wall binding domains. Conserved carbohydrate binding motifs were detected in the N-terminal, positively charged regions of six *L. brevis*S-layer proteins; however, the cell wall receptor of SlpA was found to be different from the receptors of previously characterized *Lactobacillus*S-layer proteins. Methods ======= Bacterial strains, plasmids and culture conditions -------------------------------------------------- The strains and plasmids used in this study are listed in Table [1](#T1){ref-type="table"}. *Lactobacillus brevis*ATCC 8287 and *Lactobacillus acidophilus*ATCC 4356 were grown in MRS (Difco, Detroit, MI, USA) at 37°C. *E. coli*strains were grown in Luria-Bertani medium or M9ZB-medium \[[@B23]\] at 37°C under aeration. When appropriate, kanamycin, 30 μg/ml, was used for *E. coli*. ###### Strains and plasmids used in this study --------------------------------------------------------------------------------------------------------------------------------------------- Strain or plasmid Relevant properties^a^ Reference or source -------------------------------------- -------------------------------------------------------------------------------- --------------------- Strains *Lactobacillus brevis*ATCC 8287 ATCC *Lactobacillus acidophilus*ATCC 4356 ATCC *Escherichia coli*DH5αF\'\ F\' *endA1 hsd17*(r~k~^-^m~k~^+^) *supE44 thi-1 recA1 gyrA*(NaI^r^) *relA1Δ*\ 58\ *(lacIZYA-argF) U169 deoR*\[ϕ80 d *lacΔ(lacZ)*M15\] *Escherichia coli*BL21(DE3) F^-^*ompT hsdS*~*B*~(r~B~^-^m~B~^-^) *gal dcm*(DE3) Novagen Plasmids pET-28a(+) Km^r^, *E. coli*expression vector Novagen pET-28b(+) Km^r^, *E. coli*expression vector Novagen pKTH5198 Km^r^, pET-28b(+)(*Nco*I/*Xho*I::SlpA~1--435~-linker~thrombin~-Tag~*his*6~) This study pKTH5199 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~1--435~) This study pKTH5200 Km^r^, pET-28b(+)(*Nco*I/*Xho*I::SlpA ~146--435~-linker~thrombin~-Tag~*his*6~) This study pKTH5201 Km^r^, pET-28b(+)(*Nco*I/*Xho*I::SlpA~291--435~-linker~thrombin~-Tag~*his*6~) This study pKTH5203 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~1--145~) This study pKTH5204 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~1--290~) This study pKTH5258 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~190--423~) This study pKTH5259 Km^r^, pET-28a(+)(*NheI*::Tag~*his*6~-linker~thrombin~-SlpA~210--423~) This study pKTH5260 Km^r^, pET-28a(+)(*NheI*::Tag~*his*6~-linker~thrombin~-SlpA~1--189~) This study pKTH5261 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~190--435~) This study pKTH5262 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~210--435~) This study pKTH5264 Km^r^, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~167--435~) This study pKTH5325 Km~r~, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~179--435~) This study pKTH5333 Km~r~, pET-28a(+)(*Nhe*I::Tag~*his*6~-linker~thrombin~-SlpA~149--435~) This study --------------------------------------------------------------------------------------------------------------------------------------------- ^a^Km^r^, resistance to kanamycin DNA manipulations and transformation ------------------------------------ Routine molecular biology techniques were used essentially as described previously \[[@B24]\]. Plasmid DNA of *E. coli*clones was isolated by using the Wizard Minipreps kit (Promega, Madison, WI, USA). Chromosomal DNA of *L. brevis*was isolated essentially as described before \[[@B16]\]. PCR products were purified with the QIAquick PCR purification kit (Qiagen). DNA restriction and modification enzymes were used as recommended by the manufacturers (New England Biolabs Inc., Beverly, MA, USA; Promega). PCR was carried out with DyNAzyme II DNA polymerase as recommended by the manufacturer (Finnzymes, Helsinki, Finland). *E. coli*cells were transformed by standard methods \[[@B24]\]. Oligonucleotides and DNA sequencing ----------------------------------- Oligonucleotides (Oligomer, Helsinki, Finland) used in this work are listed in Table [2](#T2){ref-type="table"}. Nucleotide sequencing was performed by the dideoxy chain termination method of Sanger *et al*. \[[@B25]\] by using an ABI Prism 310 Genetic analyzer (Applied biosystems, Foster City, CA, USA) in combination with the DNA sequencing kit for BigDye Terminator cycle sequencing (Applied Biosystems). ###### Oligonucleotides used in this study. Oligonucleotide Nucleotide sequence (5\'→3\')^a^ ----------------- ------------------------------------------------------------------- 1594 GTCAT[CCATGG]{.ul}GCAAGTCATACGCTACTGCAGG 1595 TCGCA[CTCGAG]{.ul}GCTGCCGCGCGGCACCAGGCCGCTGCTGTTGAACCAAGTAGTACCGT 1596 TCGTA[TCTAGA]{.ul}AAGTCATACGCTACTGCAGG 1597 TCGCA[TCTAGA]{.ul}TTATTAGTTGAACCAAGTAGTAC 1602 GTCAT[CCATGG]{.ul}GCCTTTATGGTGTTGCTAAGGAC 1603 GTCAT[CCATGG]{.ul}GCTCCCAAGCAGCTACTTCTAAG 1604 TCGCA[CTCGAG]{.ul}GCTGCCGCGC 1628 GTCAT[GCTAGC]{.ul}AAGTCATACGCTACTGCAGG 1629 ATTCC[GCTAGC]{.ul}TTATTAAACAGTAGCGTAAACTGTGTT 1630 TGATA[GCTAGC]{.ul}TTATTAGCTAACTTTACTTGCCTTGTAT 1635 ATTCC[GCTAGC]{.ul}GGCTTCAGTACTACTGCTACT 1636 TCGCA[GCTAGC]{.ul}TTATTAGTTGAACCAAGTAGTAC 1637 ATTCC[GCTAGC]{.ul}GTTACAGCAACCAACGATAAC 1638 TGATA[GCTAGC]{.ul}TTATTACTTACCAGCGTAAATCC 1639 TGATA[GCTAGC]{.ul}TTATTACTTACCCATAACAAGGGT 1644 ACTAC[GCTAGC]{.ul}GGTTCATTATACTATCACGTAAC 1776 GCGG[GCTAGC]{.ul}AGTGGTATTAGTGGTTGGATTT 1777 GCGG[GCTAGC]{.ul}GTTGCTAAGGACACCAAGTTT ^a^recognition sites of restriction enzymes are underlined Protein analysis ---------------- Protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli \[[@B26]\] and stained with Coomassie brilliant blue. Construction of plasmid vectors ------------------------------- For the expression of the mature SlpA protein, SlpA~1--435~, the gene was amplified by PCR from the chromosomal DNA of *L. brevis*ATCC 8287 using primer pairs 1594/1595 or 1596/1597 (Table [2](#T2){ref-type="table"}). The PCR fragment obtained with primer pair 1594/1595 was digested with *Nco*I and *Xho*I and ligated with *Nco*I-*Xho*I digested pET-28b(+), resulting in plasmid pKTH5198 encoding rSlpA with a C-terminal His-tag (Table [1](#T1){ref-type="table"}). The PCR product amplified with primers 1596 and 1597 was digested with *Xba*I and cloned into the *Nhe*I site of plasmid pET-28a(+). The resulting plasmid, encoding rSlpA with an N-terminal His-tag, was named pKTH5199. Three C-terminal truncations, seven N-terminal truncations and two N-and C-terminal truncations of SlpA were constructed, each with a His-tag sequence at either N- or C-terminus. For a summary of the plasmid constructs, see Table [1](#T1){ref-type="table"}. For cloning the C-terminal truncations, primer pairs 1628/1630 (for SlpA~1--145~), 1628/1629 (for SlpA~1--290~) and 1628/1638 (for SlpA~1--189~) (Table [2](#T2){ref-type="table"}) were used to amplify the *slpA*sequences with plasmid pKTH5199 as a template. PCR fragments obtained were digested with *Nhe*I and cloned into *Nhe*I-digested pET-28a(+). The resulting plasmids were named pKTH5203, pKTH5204 and pKTH5260, respectively (Table [1](#T1){ref-type="table"}). Cloning of the N-terminal truncations of SlpA with the His-tag sequence at the 3\'-terminus was carried out with primer pairs 1602/1604 (for SlpA~146--435~) and 1603/1604 (for SlpA~291--435~) (Table [2](#T2){ref-type="table"}) and pKTH5198 as a template. The resulting PCR fragments were cloned as *Nco*I-*Xho*I fragments into pET-28b(+), giving plasmids pKTH5200 and pKTH5201, respectively (Table [1](#T1){ref-type="table"}). For cloning the N-terminal truncations with the His-tag sequence at the 5\'-terminus, primer pairs 1777/1636 (for SlpA~149--435~), 1644/1636 (for SlpA~167--435~), 1776/1636 (for SlpA~179--435~), 1635/1636 (for SlpA~190--435~), or 1637/1636 (for SlpA~210--435~) were used to amplify the *slpA*sequences with plasmid pKTH5199 as a template. The PCR fragments obtained were digested with *Nhe*I and cloned into *Nhe*I-digested pET-28a(+) resulting in plasmids pKTH5333, pKTH5264, pKTH5325, pKTH5261 and pKTH5262, respectively (Table [1](#T1){ref-type="table"}). Sequences encoding N-and C-terminally truncated SlpA were PCR amplified with primers 1635/1639 (for SlpA~190--423~) and 1637/1639 (for SlpA~210--423~), using plasmid pKTH5199 as a template. The resulting PCR fragments were cloned as *Nhe*I-fragments into pET-28a(+), giving plasmids pKTH5258 and pKTH5259, respectively (Table [1](#T1){ref-type="table"}). All constructs were sequenced to verify the correct open reading frames. Heterologous expression of the sequences encoding mature SlpA and its truncated forms ------------------------------------------------------------------------------------- Gene expression was carried out as described in the pET System Manual (Novagen, Madison, WI, USA) by using *Escherichia coli*strain BL21(DE3). Briefly, expression of recombinant SlpA proteins was induced by adding isopropylthiogalactoside (IPTG) at a concentration of 0.5 to 1.0 mM to the medium of exponentially growing *E. coli*strains harboring one of the expression plasmids listed in Table [1](#T1){ref-type="table"}. After IPTG was added, the incubation was continued for one to five hours, depending on the protein to be purified. Recombinant SlpA proteins were purified in the presence of 4 M guanidine hydrochloride (GHCl) or 6 M urea with a His Trap HP column according to the instructions given by Amersham Biosciences (Uppsala, Sweden). After purification the fractions containing the recombinant SlpA protein were dialyzed overnight at +4°C against distilled water. Purity of the recombinant proteins, present as a precipitate and/or as soluble proteins after dialysis, was checked by SDS-PAGE. Isolation of SlpA protein from *L. brevis*ATCC 8287 --------------------------------------------------- The S-layer protein was extracted from *L. brevis*cells grown to an OD~600\ nm~of 1.0 in MRS broth. Cells from 1 l of culture were harvested and washed twice with distilled water. The pellet was resuspended in 15 ml of 2 M GHCl and incubated for 30 min at +4°C followed by centrifugation (15,000 × g for 20 min). The supernatant was concentrated with Centricon Plus-20 centrifugal filter (Millipore, Bedford, MA, USA) before dialysis against distilled water supplemented with 5 mM CaCl~2~overnight at +4°C, followed by dialysis against distilled water overnight at 4°C. Before dialysis the SlpA protein concentration was adjusted to 1 mg/ml. After dialysis a centrifugation step (20,000 × g for 20 min) was performed. The pellet containing the S-layer self-assembly products was resuspended in 25 mM Tris-HCl buffer (pH 8.0). Proteolytic degradation of SlpA with trypsin and peptide mapping ---------------------------------------------------------------- Isolated SlpA at a concentration of 1 mg/ml was dialyzed against distilled water supplemented with 5 mM CaCl~2~overnight followed by a second overnight dialysis against distilled water. The dialysis was followed by a centrifugation step (16,000 × g for 30 min). The S-layer monomers, present in the supernatant, were digested with trypsin under the following conditions: 300 ng SlpA protein and 3 μg trypsin (Sigma-Aldrich, St. Louis, MO, USA) in 300 μl of 25 mM Tris-HCl (pH 8.0) for 10 to 30 min at 37°C. The reaction was stopped by heating the samples for 10 min at 100°C and the samples were subjected to SDS-PAGE. N-terminal sequencing was performed by a gas-pulsed liquid sequencer as described previously \[[@B27]\] and peptide mapping by a Biflex matrix-assisted laser desorption ionization-time of flight mass spectrometer (Bruker-Franzen Analytic, Bremen, Germany) as described by \[[@B28]\]. Investigation of the self-assembly properties of purified truncated S-layer proteins ------------------------------------------------------------------------------------ To assess the ability of the recombinant S-layer proteins to self-assemble, affinity purified proteins were dissolved in 5 M GHCl at a concentration of 1 mg/ml and the solutions were dialyzed against phosphate-buffered saline (PBS) in Slide-A-Lyzer Mini Dialysis Units (Pierce, Rockford, IL, USA) for two hours at 4°C. Dialysis was followed by a centrifugation step (16,000 × g for 30 min) and the formation of a precipitate was checked by SDS-PAGE. For transmission electron microscopy, 1 mg of the purified, lyophilized proteins were dissolved in 1 ml 5 M GHCl in 50 mM Tris-HCl buffer (pH 7.2) and the solution was dialyzed against 10 mM CaCl~2~in distilled water for 18 h. Samples were transferred onto carbon-coated electron microscope grids rendered hydrophilic by glow discharge, negative stained with 2,5% uranyl acetate as described previously \[[@B29]\], and electron micrographs were taken with Philips CM 12 transmission electron microscope (Philips Eindhoven, the Netherlands) operated at 80 kV in a low-dose mode. Freeze-etched preparations of *L. brevis*ATCC 8287 cells were prepared as previously described \[[@B30]\], and lattice constants of the S-layer formed by SlpA were determined as described by \[[@B31]\]. Isolation of native cell wall fragments (CWF) from *L. brevis*ATCC 8287 ----------------------------------------------------------------------- *L. brevis*ATCC 8287 cells were cultivated overnight in 1 l of MRS broth, collected and washed three times with distilled water. Cells were suspended in 30 ml of 2 M GHCl, incubated shaking for 30 minutes at +4°C, collected and washed once with 50 mM Tris-HCl (pH 7.4). Cells were disrupted by French Pressure Cell Press (SLM Instruments Inc, IL, USA) in 50 mM Tris-HCl (pH 7.4), and the lysate was centrifuged at 3000 g for 5 minutes at +4°C. Cell wall fragments were collected, washed five times with 50 mM Tris-HCl (pH 7.4) and treated with DNAase I (25 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) and RNAase I (25 μg/ml, Roche Diagnostics GmbH, Mannheim, Germany) in 50 mM Tris-HCl (pH 7.4), 10 mM MgCl~2~for 30 minutes at 37°C. Cell wall fragments were collected, treated with 1% SDS for 30 minutes at 100°C, washed extensively with distilled water at room temperature and lyophilized. Treatment of native cell wall fragments with TCA ------------------------------------------------ 0.5 mg or 0.25 mg of isolated CWF in water were incubated in the presence of 5% (V/V) TCA either at +4°C or at +37°C for 24 h in a rotary shaker. The cell walls were collected, washed three times with distilled water at +4°C and suspended in distilled water. The treatment at +4°C was performed twice. Organic phosphorous was measured from native and treated CWF and from the supernatant obtained in the extraction by the method described by \[[@B32]\]. Binding of the truncated S-layer proteins to bacterial cells and isolated cell wall fragments --------------------------------------------------------------------------------------------- Binding assays of recombinant S-layer proteins to LiCl-extracted *L. brevis*ATCC 8287 and *L. acidophilus*ATCC 4356 cells were performed essentially as described previously \[[@B14]\]. The amount of recombinant S-layer protein used in one binding reaction was 50 μg and the buffer was 50 mM Tris-HCl (pH 7.5) with 150 mM NaCl. The presence of cell-bound S-layer protein in the samples was verified by SDS-PAGE. In binding assays with isolated cell wall fragments, monomeric truncated S-layer proteins, present in the supernatant after centrifugation (20 minutes at 16 000 g at +4°C), were used. 20 μg CWF and 10 μg full length recombinant SlpA or an equimolar amount of truncated S-layer proteins were combined in 50 μl of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. After incubation (1 hour at room temperature) cell wall fragments were collected, washed once with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and analyzed by SDS-PAGE. Analysis of primary amino acid sequences ---------------------------------------- The isoelectric point (pI) values of the *L. brevis*S-layer proteins as well as those of the constructed rSlpA proteins were obtained by ProtParam \[[@B33]\], and the analyses of the hydrophobicity patterns of S-layer proteins of *L. brevis*were performed by the Kyte-Doolittle method \[[@B34]\] with ProtScale \[[@B35]\] on the ExPASy server. Repeat structures from S-layer proteins were localized by REPRO \[[@B36],[@B37]\]. The comparison matrix used in the protein repeat analysis was blosum62 (gap open penalty, 12; gap extension penalty, 1). Sequence alignment analyses of *L. brevis*S-layer proteins were performed by ClustalW \[[@B38]\] using gonnet as a comparison matrix (gap open penalty, 10; gap extension penalty, 0.2). Pairwise comparison analyses were performed by SIM \[[@B39],[@B40]\] using blosum62 as a comparison matrix (gap open penalty, 12; gap extension penalty, 4). From the complete genome sequence of ATCC 367, deposited under GenBank accession number [CP000416](CP000416), the hypothetical S-layer proteins of *L. brevis*ATCC 367 were identified by BLAST \[[@B39]\] using complete SlpA, SlpB, SlpC and SlpD sequences. The identified hypothetical S-layer proteins of ATCC 367 have been deposited in Swiss-Prot under accession numbers Q03P39 and Q03NT3. Results ======= Primary amino acid sequence analysis of the S-layer proteins of *L. brevis* --------------------------------------------------------------------------- The only thus far characterized S-layer proteins in *L. brevis*are the SlpA protein of *L. brevis*ATCC 8287 \[[@B16]\] and the SlpB, SlpC and SlpD proteins of *L. brevis*ATCC 14869 \[[@B13]\]. By performing a homology search for the recently sequenced genome of *L. brevis*ATCC 367 \[[@B41]\] with the BLAST program, two new putative S-layer proteins, Q03P39 and Q03NT3, were identified in the genome. The amino acid sequences encoding the mature S-layer proteins of *L. brevis*ATCC 8287 (SlpA) and ATCC 14869 (SlpB, C and D) and the putative mature S-layer proteins of ATCC 367 (Q03NT3 and Q03P39) were subjected to a number of analyses. A multiple alignment of SlpA, SlpB, SlpC, SlpD, Q03NT3 and Q03P39 amino acid sequences revealed significant conservation in the N-terminal regions (see Fig. [1a](#F1){ref-type="fig"} and additional file [1](#S1){ref-type="supplementary-material"}: Multiple amino acid sequence alignment of the *L. brevis*S-layer proteins). Analysis of the distribution of the isoelectric point values in *L. brevis*S-layer proteins revealed a distinction between the N-terminal region with a high predicted pI and a C-terminal region with a low predicted pI in each of the proteins (Fig. [1c](#F1){ref-type="fig"}). In SlpA and SlpB the region of a high predicted pI comprises approximately two fifths of the protein, while in SlpD and its homolog, Q03P39, as well as in SlpC and its homolog, Q03NT3, the region of an overall high pI extends further towards the C-terminus and the most distinct boundary between the differently charged regions is located around residue 260 in SlpC and Q03NT3 and around residue 290 in SlpD and Q03P39. Hydrophobicity analysis performed for the S-layer protein sequences of *L. brevis*showed a similar distribution of hydrophilic and hydrophobic amino acid residues along the mature proteins with evenly alternating hydrophobic and hydrophilic residues, as exemplified by the hydrophobicity plot of SlpA in Fig. [1b](#F1){ref-type="fig"}. ![**1(a) -- Alignment of *L. brevis*S-layer protein sequences**. The mature forms of S-layer proteins were aligned by ClustalW and the alignments were divided into stretches of 10 amino acids, from which the percentage of identical amino acids and amino acids with conserved substitutions were calculated. The following colours are used to indicate the percentages of identical and similar amino acids in each calculated stretch: white, 0--20%, light gray, 21--40%, medium gray, 41--60%, dark gray, 61--80%. 1(b) &\#8211 Hydrophobicity of mature SlpA. The hydrophobicity was calculated according to Kyte and Doolittle \[[@B34]\] with a window of seven amino acids. 1(c) &\#8211 Predicted pI values of the N- and C-terminal regions of *L. brevis* S-layer proteins. The lengths of the N- and C-terminal regions as well as the full lengths of the mature forms of the proteins are indicated in brackets.](1471-2180-8-165-1){#F1} To predict the domain organization of SlpA, the analyses performed were compared with similar analyses of the S-layer proteins of *L. acidophilus*group organisms \[[@B14]\]. In each of the *L. acidophilus*S-layer proteins, one region is found which is conserved, located at the C-terminus and contains positively charged and hydrophilic sequences. In S~A~of *L. acidophilus*ATCC 4356 and CbsA of *L. crispatus*JCM 5810 these domains mediate the binding to the cell wall, while the variable N-terminal regions are responsible for the assembly of the S-layer \[[@B14],[@B15]\]. In CbsA the variable N-terminal domain is also responsible for collagen binding \[[@B42]\]. The only function characterized for SlpA so far, binding to fibronectin and human epithelial cells \[[@B18]\], resides in the conserved N-terminal region. However, the pattern of sequence conservation and the distribution of charge in the six *L. brevis*S-layer proteins strongly suggested a domain organization similar to that found in *L. acidophilus*-group S-layer proteins with the functional domains in a reverse order. Cloning and expression of gene sequences encoding the mature or truncated forms of the S-layer protein SlpA and purification of the recombinant proteins -------------------------------------------------------------------------------------------------------------------------------------------------------- PCR products encoding the mature SlpA and the various N- or C-terminal SlpA truncations were cloned in *E. coli*DH5αF\' and expressed in *E. coli*BL21(DE3). The proteins produced are summarized in Fig [2](#F2){ref-type="fig"}. After induction of expression by the addition of IPTG, samples of cultures from *E. coli*BL21(DE3) were harvested and analyzed by SDS-PAGE. On SDS-gels, each of the recombinant SlpA proteins became visible as an additional protein band, corresponding approximately to the calculated molecular mass of the rSlpA proteins (data not shown). The proteins were purified in a large scale, and total protein yields varied from 5 to 25 mg per a batch cultivation of 200 ml. {#F2} Proteolytic degradation of SlpA protein with trypsin and peptide mapping ------------------------------------------------------------------------ To gain insight to the domain structure of SlpA, wild type SlpA was digested with trypsin. This revealed two protease resistant fragments with apparent molecular masses of 25 kDa and 23 kDa (Fig. [3](#F3){ref-type="fig"}). The N-terminal sequences of these peptides were determined to be GFSTTAG (larger peptide) and SVTATND (smaller peptide). These correspond to amino acids starting from 190 and 209 of mature SlpA, respectively. Peptide mass mapping of the protease resistant fragments obtained after trypsin digestion revealed that the peptide encompassing the last 12 amino acids of full length SlpA is lacking from these fragments (data not shown). The protease resistance of the regions 190 to 423 and 209 to 423 in mature SlpA strongly suggested the existence of a compact domain structure most likely representing a region exposed on the outer surface of the S-layer. {#F3} Investigation of the self-assembly properties of the truncated S-layer protein forms ------------------------------------------------------------------------------------ As a preliminary test for the putative self-assembly properties of the truncated S-layer proteins, the formation of a precipitate after dialysis from GHCl was inspected. As shown in Fig. [2](#F2){ref-type="fig"}, C-terminally truncated proteins rSlpA~1--145~, rSlpA~1--189~and rSlpA~1--290~had lost the ability to form precipitates. N-terminally truncated forms rSlpA~179--435~and rSlpA~167--435~precipitated efficiently after dialysis, while rSlpA~146--435~, rSlpA~149--435~as well as rSlpA~190--435~and rSlpA~190--423~showed a reduced precipitation. The last twelve residues in the C-terminus of SlpA had no effect on the precipitation, as rSlpA~190--423~showed a precipitation similar to rSlpA~190--435~. The removal of 209 residues or more from the N-terminus of SlpA abolished the ability of the truncated proteins to form precipitates. Precipitate-forming N-terminally truncated SlpA proteins were chosen for transmission electron microscopical analysis of lattice formation. In these studies, rSlpA formed an oblique lattice that was identical with that formed by SlpA isolated from wild type *L. brevis*ATCC 8287 cells (compare Figures [4a](#F4){ref-type="fig"} and [4c](#F4){ref-type="fig"}), as well as with the lattice seen on *L. brevis*ATCC 8287 cells in the freeze-etched preparation (Fig. [4d](#F4){ref-type="fig"}), proving the native conformation of recombinant SlpA. In accordance, lattice constants for the self-assembly products of rSlpA (a = 10.38, b = 6.36 and 72.7°) and for the self-assembly products of SlpA isolated from *L. brevis*ATCC 8287 cells (a = 9.39, b = 6.10 and 79.8°) were practically identical. The recombinant protein SlpA~179--435~(Fig. [4b](#F4){ref-type="fig"}) was found to form a regular, oblique lattice indistinguishable from that formed by full length rSlpA, but the removal of eleven residues more from the N-terminus resulting in rSlpA~190--435~prevented lattice formation (Fig. [2](#F2){ref-type="fig"}). Surprisingly, the two larger N-terminally truncated proteins, rSlpA~167--435~and rSlpA~149--435~, were unable to form regular lattice structures. Thus, residues 179--435 in mature SlpA define the region responsible for the crystallization of SlpA monomers. {#F4} Isolation of native cell wall fragments and binding of the truncated S-layer proteins to cell wall fragments ------------------------------------------------------------------------------------------------------------ Cell wall fragments were purified from a stationary phase culture of *L. brevis*cells by differential centrifugation of GHCl-treated, mechanically disrupted cells followed by treatments with nucleases and boiling SDS as described in Materials and methods. This method efficiently removes membrane fragments and noncovalently bound cell wall components like lipoteichoic acids (LTAs), but leaves covalently bound secondary cell wall polymers, like wall teichoic acids and other carbohydrates, essentially intact. The purity of the cell wall preparation was checked by light and transmission electron microscopy (Fig. [5](#F5){ref-type="fig"}). From 1.6 g of wet cells approximately 28 mg of cell wall fragments (dry weight) were recovered. {#F5} To test the hypothesis that the N-terminal, positively charged region of SlpA is responsible for anchoring the S-layer to the cell wall, truncated recombinant SlpA-proteins encompassing the N- and C-terminal regions of SlpA were tested for binding to isolated *L. brevis*cell wall fragments. As shown in Fig. [6a](#F6){ref-type="fig"}, full length rSlpA, rSlpA~1--145~and rSlpA~1--189~localized in the pellet fraction after incubation with the cell wall fragments, while rSlpA~190--435~and rSlpA~167--435~were unable to bind to CWF and were found in the supernatant. Truncated S-layer proteins incubated alone mainly localized to the supernatant, although small amounts were found in the pellets due to the inherent tendency of S-layer proteins to aggregate. These results are in good accordance with our results from similar experiments with whole, LiCl-treated *L. brevis*ATCC 8287 and *L. acidophilus*ATCC 4356 cells. In these experiments, full length rSlpA, rSlpA~1--145~and rSlpA~1--189~as well as rSlpA~1--290~bound to *L. brevis*cells, while rSlpA~190--435~, rSlpA~167--435~and rSlpA~210--435~were unable to bind. Full-length rSlpA and its cell wall binding fragment rSlpA~1--145~also bound to LiCl-treated *L. acidophilus*ATCC 4356 cells (data not shown). {#F6} To get preliminary information about the cell wall component interacting with the N-terminal region of SlpA, binding tests with full length rSlpA and CWF treated with TCA at +4°C or at +37°C were performed. rSlpA bound to CWF treated with TCA at +4°C as efficiently as to native CWF, while the treatment of CWF with TCA at +37°C substantially reduced the binding of rSlpA (Fig. [6b](#F6){ref-type="fig"}). Repeated TCA-extraction at +4°C and a change in the rSlpA: CWF-ratio from 1:2 to 1:1 did not change the result (data not shown). TCA extracts carbohydrate polymers, and the treatment at +4°C has been reported to selectively remove teichoic acids, while the treatment at +37°C removes teichuronic acids and polysaccharides \[[@B43]\]. The efficiency of the extraction was confirmed by the measurement of organic phosphorous from native and TCA-treated CWF as well as from the supernatant obtained in the extraction, which indicated the loss of approximately 75% of the organic phosphorous from the CWF by the treatment at +4°C (data not shown). These results suggest that the binding component in the *L. brevis*ATCC 8287 cell wall is other than teichoic acid, and that cell wall components extractable by TCA at +37°C, presumably polysaccharides, participate in the binding. The amino acid sequence analysis of SlpA and other *Lactobacillus brevis*S-layer proteins revealed sequences in the N-terminal regions with apparent similarity to the repetitive carbohydrate binding motifs of clostridial toxins and streptococcal glucosyltransferases \[[@B44],[@B45]\]. These regions were found within amino acids 60--90 and 165--192 in each of the mature proteins (Fig. [7](#F7){ref-type="fig"}). In the N-terminal parts of SlpC and Q03NT3 as well as SlpD and Q03P39 additional regions with less obvious similarity were detected as well. Similar motifs have also been detected in the C-terminal cell wall binding regions of *L. acidophilus*ATCC 4356 S~A~protein and *L. crispatus*JCM 5810 CbsA protein as well as in the S-layer protein of *L. helveticus*CNRZ 892 and in several other bacterial cell surface-associated proteins, in which they were located in two tandemly repeated sequences of 65--72 amino acids \[[@B14]\]. A protein repeat analysis of the *L. brevis*S-layer proteins did not indicate the presence of the carbohydrate binding sequences in obvious repeat sequences, and despite the similar carbohydrate binding motifs in SlpA and in *L. acidophilus*group S-layer proteins, the cell wall receptor of SlpA apparently is dissimilar. ![**Similarity of the N-terminal regions of *L. brevis*S-layer proteins with carbohydrate binding motifs**. The motifs were determined by Wren \[[@B45]\] and von Eichel-Streiber *et al*\[[@B44]\]. In the consensus sequences upper case letters indicate highly conserved residues \[[@B44]\] or residues with an identity of 50% or higher \[[@B45]\]. X, variable residue. A broken underline indicates potential carbohydrate-binding motifs at different locations: YFRAYG of SlpA corresponds to YFDxNG of the consensus sequence of Ref \[[@B44]\]; KAYRGW of SlpB corresponds to KAVTGW of the consensus sequences of References \[[@B44]\] and \[[@B45]\]; LSNKSYY of SlpD and Q03P39 corresponds to IDGkwYY of the consensus sequence of Ref \[[@B44]\].](1471-2180-8-165-7){#F7} Discussion ========== In this study, we have identified the cell wall binding and self-assembly domains in the S-layer protein SlpA of *L. brevis*ATCC 8287, a strain phylogenetically distant from *L. acidophilus*group organisms, the S-layer proteins of which have previously been functionally characterized. Two new putative S-layer proteins, Q03P39 and Q03NT3, were identified in the recently sequenced genome of *L. brevis*ATCC 367 \[[@B41]\] and compared with SlpA and other *L. brevis*S-layer proteins characterized thus far. Q03P39 is almost identical (99% identity) with SlpD of *L. brevis*ATCC 14869 and relatively dissimilar (\<40% identity) from the SlpA, SlpB and SlpC sequences. Q03NT3 is highly similar with SlpC of *L. brevis*ATCC 14869 (87% identity) whereas not that similar with the other characterized *L. brevis*S-layer proteins (\<44% identity). Similarity of the mature forms of the new putative S-layer proteins or the mature forms of SlpA, SlpB, SlpC or SlpD proteins with *L. acidophilus*group S-layer proteins is negligible. Analysis of the six *L. brevis*S-layer protein sequences deposited in databanks indicated the subdivision of each sequence into two regions: a conserved N-terminal region characterized by a high predicted pI and potential carbohydrate binding motifs, and a more variable C-terminal region with an acidic predicted pI, with the N-terminal region corresponding for 40--75% of the sequence lengths. The observed high predicted overall pI values of *Lactobacillus*S-layer proteins \[[@B7]\] thus seem to be due to the concentration of basic amino acids to a defined region, as is also the case in the S-layer proteins of *L. acidophilus*group, which have cationic, cell wall binding C-terminal regions. The presence of a conserved N-terminal region with a high predicted pI in *L. brevis*S-layer proteins strongly suggested an N-terminal cell wall binding domain. This was confirmed for SlpA of *L. brevis*ATCC 8287 by interaction studies performed with truncated rSlpA proteins and LiCl-treated *L. brevis*cells or isolated *L. brevis*cell wall fragments. In these studies, truncated proteins encompassing the whole positively charged region of SlpA bound to the cell wall; however, the first 145 residues in mature SlpA contained sufficient information for cell wall binding. An assay suitable for measuring the binding strength would be needed to detect the putative difference between the cell wall binding affinities of SlpA~1--145~and SlpA~1--189~. In S-layer proteins of lactobacilli, no SLH motifs have been detected. Instead, interactions between a positively charged S-layer protein region and negatively charged secondary cell wall polymers have been shown to mediate the cell wall binding in the case of S~A~of *L. acidophilus*ATCC 4356 \[[@B46]\] and CbsA of *L. crispatus*JCM 5810 \[[@B41]\]. S~A~and CbsA were shown to bind teichoic acids, and CbsA bound also to lipoteichoic acids purified from *Staphylococcus aureus*and *Streptococcus faecalis*, but not to the teichuronic acid/polysaccharide fraction of the cell wall of *L. crispatus*JCM 5810. In contrast, the results of this study suggest the involvement of another cell wall structure than teichoic acid or lipoteichoic acid in the interaction between SlpA and the cell wall, as the purification process of the CWF used efficiently removed LTAs, and the extraction of CWF with TCA at +4°C to remove teichoic acids had no effect on the binding of SlpA. The chemical nature of the cell wall component interacting with the S-layer protein has been determined in *Geobacillus stearothermophilus*strains \[[@B47]-[@B49]\], in *Lysinibacillus sphaericus*\[[@B50]\] and in *Aneurinibacillus thermoaerophilus*\[[@B6]\]. In *G. stearothermophilus*and *L. sphaericus*S-layers, which possess SLH-domains, the component is a pyruvylated GlcNac and GalNac-containing polysaccharide not groupable as a teichoic or lipoteichoic acid. In other *G. stearothermophilus*strains the component is a negatively charged mannuronic acid-containing cell wall polymer, and in *A. thermoaerophilus*the cell wall receptor is a neutral biantennary oligosaccharide. The secondary cell wall polymers of lactobacilli are poorly characterized. The detailed structure of a wall polysaccharide of *L. casei*has been determined \[[@B51]\], but no precise structures for polysaccharides of *L. brevis*strains are available at present. In early studies, the cell walls of *L. buchneri*\[[@B52]\] and *L. brevis*ATCC 8287 \[[@B53]\] were shown to contain neutral polysaccharides, which were suggested to be involved in the anchoring of the S-layer protein to the cell wall through hydrogen bonding \[[@B54],[@B55]\]. These results are in agreement with the data presented in this study, which suggest a non-teichoic acid polysaccharide, either neutral or anionic, involved in the cell wall binding of SlpA, but the detailed structure of this polysaccharide remains to be elucidated. Interestingly, despite the fact that polysaccharides rather than (lipo)teichoic acids of *L. brevis*ATCC 8287 are involved in the cell wall binding of SlpA, the C-terminal cell wall binding region of the S-layer protein CbsA of *L. crispatus*JCM 5810 bound to GHCl-treated *L. brevis*ATCC 8287 cells \[[@B15]\]. Using the same experimental design we showed that rSlpA and its cell wall binding fragment rSlpA~1--145~bind to LiCl-treated *L. acidophilus*ATCC 4356 cells. The interaction between the S-layer protein and the secondary cell wall component is supposed to be lectin-like and highly specific \[[@B56]\], and in artificial experimental procedures the lack of a specific interaction between two complementary surfaces may be masked by unspecific charge interactions with lower affinity. To obtain information about specific interactions, competition experiments with fragments of S~A~, CbsA and SlpA and the corresponding bacterial strains, or direct determinations of the K~D~values of the interactions, e. g. by surface plasmon resonance studies, are needed. Amino acid sequence analysis of the *L. brevis*S-layer proteins revealed motifs with similarity to repeated C-terminal carbohydrate binding sequences detected in clostridial toxins, streptococcal glucosyltransferases and the S-layer proteins of *L. acidophilus*group organisms \[[@B14],[@B44],[@B45]\]. These motifs are supposed to play a general role in protein-carbohydrate interactions by acting as initial attachment sites and thus enabling the specific interactions to occur \[[@B44]\] and may thus be partly responsible for the observed positive cross-binding results between SlpA and *L. acidophilus*ATCC 4356 cells, and between the cell wall binding domain of CbsA and *L. brevis*ATCC 8287 cells (see above). The sequences of the potential *L. brevis*carbohydrate-binding motifs deviated to some extent from the consensus sequences determined for clostridial toxins and streptococcal transferases \[[@B44],[@B45]\]. The divergence of the sequences in distantly related organisms and different macromolecular structures is apparently allowed as long as the basic function of the motif, bringing the interacting partners to initial contact, is preserved. The self-assembly domain of SlpA was shown to comprise residues 179--435 in mature SlpA, as truncated proteins encompassing this region were able to form a periodic structure indistinguishable from that formed by full length SlpA, as detected by electron microscopy. The length of the truncated protein was critical, since rSlpA~167--435~and rSlpA~149--435~as well as N-terminal truncations shorter than rSlpA~179--435~were unable to form regular lattices. Apparently, the region, or part of the region, encompassing amino acids 149--178 disturbs the lattice formation of the truncated proteins either by steric hindrance and/or by preventing the acquisition of a native conformation. Trypsin degradation experiments revealed two protease resistant peptides encompassing residues 190--423 and 209--423 in mature SlpA supporting the hypothesis about a morphologically separate, compact C-terminal unit, which most probably corresponds to the round, periodically arranged structures seen in electron microscope pictures of self-assembly products of SlpA (Fig. [4](#F4){ref-type="fig"}). Similar trypsin degradation experiments with whole *L. brevis*cells resulted in identical fragments but at a very low efficiency (data not shown), indicating poor accessibility of the enzyme to the N-terminal domain through the pores in the S-layer, and further supporting the presumption about a protease sensitive, more flexible N-terminal domain shielded from the environment beneath the C-terminal self-assembly domain. A protease-resistant, surface-located self-assembly domain has also been observed in the N-terminal part of the S-layer protein S~A~of *L. acidophilus*ATCC 4356 \[[@B14]\]. The results of the present study are supported by the report of Hynönen *et al*\[[@B18]\], in which an antiserum specific for the recombinant peptide SlpA~66--215~, originating from the cell wall binding region, was not able to recognize polymerized SlpA on *Lactobacillus brevis*ATCC 8287 cells. In the same report, whole S-layered *L. brevis*ATCC 8287 cells as well as the N-terminal part of SlpA, residues 66--146 of mature SlpA in minimum, were shown to bind to immobilized fibronectin. Fibronectin is highly glycosylated, and the binding of fibronectin to a region of SlpA shielded beneath the C-terminal domain may be explained by an interaction between the protruding oligosaccharide moieties of fibronectin and the identified N-terminal carbohydrate binding sequences of SlpA. In this respect the identification of human blood group A-trisaccharide as the receptor for the S-layer protein of a human *L. brevis*isolate \[[@B57]\] is of interest, especially considering that the nine N-terminal amino acids of the S-layer protein of this strain were identical with the N-terminus of SlpA. Conclusion ========== In this work SlpA of *L. brevis*ATCC 8287 was shown to be a two-modular protein in which the domains responsible for the self-assembly (C-terminal) and cell wall binding (N-terminal) are located in a reverse order compared to those in all other *Lactobacillus*S-layer proteins characterized thus far, reflecting the unrelatedness of SlpA with previously characterized *Lactobacillus*S-layer proteins. The study confirms the role of conserved, repeated, general carbohydrate binding sequences in the cell wall binding domains of *Lactobacillus*S-layer proteins, but in contrast to the *Lactobacillus acidophilus*-group organisms, the specific cell wall component interacting with the S-layer protein in *L. brevis*ATCC 8287 was shown to be other than (lipo)teichoic acid. As SlpA is a potential tool for mucosal immunization, the data presented in this study forms a basis for further studies concerning vaccine development. The mapping of surface exposed residues in the self-assembly domain of SlpA is currently in progress. Authors\' contributions ======================= SÅJ and UH performed the experiments (excluding electron microscopy, N-terminal sequencing and peptide mass mapping), analyzed the results, carried out the sequence analyses and prepared the manuscript. NI carried out electron microscopy, DP determined the lattice constants and performed the statistical analysis, UBS coordinated the EM studies, AP participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ###### Additional file 1 **Multiple amino acid sequence alignment of the *L. brevis*S-layer proteins**. ClustalW -- alignment of the predicted mature forms of SlpA, SlpB, SlpC, SlpD, Q03NT3 and Q03P39 proteins. Asterisks, colons and dots indicate identical, strongly similar and weakly similar amino acids, respectively. A primary consensus sequence is shown below the alignment. ###### Click here for file Acknowledgements ================ We thank Ilkka Palva for valuable discussions and critical reading of the manuscript, Esa Pohjolainen, Outi Immonen and Sinikka Ahonen for skillful technical assistance and Nisse Kalkkinen for amino acid sequencing and mass mapping. This work was supported by the FP6 EC-STREP project NAS-SAP 13523. The work was performed in the Centre of Excellence on Microbial Food Safety Research, Academy of Finland.

INTRODUCTION ============ *Acinetobacter baumannii* is considered as an important cause of healthcare associated infections, particularly in the intensive care units (ICU), and many reports have indicated that antibiotic misuse leads to appearance of resistance strains \[[@R1], [@R2]\]. So, a lot of efforts have been contributed to find out a solution in order to treat theses nosocomial pathogen, one of which is combined therapy \[[@R3], [@R4]\]. The combinations of two antibiotics have shown different effects on each other and in many cases the effect is synergistic or strengthening but in some cases, antagonism is observed \[[@R5], [@R6]\]. combination therapy is usually used in life-threatening infections, to cover a wide spectrum broad against all potential pathogens, to induce synergistic effects of the combination against a specific pathogen to prevent resistance emergence, or to combat a polymicrobial infection not easily treated with a single medication. In combination therapy the balance in its potential disadvantages, including, the possible increase in side effects, superinfection, antagonistic activity and increase in cost should be considered. Combination therapy might also be used to prevent the side effects of a specific drug \[[@R7]\]. Rifampin is an antibiotic which is frequently used with other antibiotics, and the results have shown synergy with colistin; however, the result of this combination is dependent on various conditions such as the rifampin's MICs or the methods used. For example, the enhancement effects have not been observed in the strains with MIC higher than 256µg/ml \[[@R8], [@R9]\]. We focused on the studies that examined the combination effects of rifampin and colistin against *Acinetobacter in vitro*. The aim of this study was to obtain the information about the activity of rifampin and colistin and their effects on *A. baumannii* isolates through a review of existing literature and data analysis. MATERIALS AND METHOD ==================== Data Source and Criteria for Selecting Articles ----------------------------------------------- In the period from December 2014 to January 2015 the detailed databases (PubMed, Scopus, EMBASE and ISI Web of Sciences) search was performed. The inclusion criteria for meta-analysis were the studies that had examined the interactions of the two antibiotics (rifampin and colistin) and all *in vitro* combination therapies (Checkerboard, Time-kill). The posters printed (ECCMID) from 2007 to 2014 were also checked (the ISI Web of Science web site) (flow chart. **[Chart 1](#FC1){ref-type="fig"}**. The key words used were \" *A. baumannii* + colistin, *A. baumannii* + rifampicin, \" *A. baumannii* + colistimethate \" and *A. baumannii* + colistin and rifampicin \". In order to avoid bias, the search was performed by 2 independent researchers. Interaction Analysis and Data ----------------------------- The following information-researcher's name, year, country, number of strains, strain's resistance and sensitivity to rifampin and colistin, MIC and type of interaction including additive, partially synergism, synergism and antagonism and applied methods were extracted. The interaction between two antibiotics was evaluated by two methods (Checkerboard, Time-kill) and the initial results of the *in vitro* effects on the bacteria were based on inhibition or killing. In the time-kill method the synergism was defined as the reduction by the amount of log 2 of CFU/ml of the bacteria in the presence of antibiotics' combination compared to the single state and as antagonism, if it showed an increase. For the checkerboard method the index FICI was applied and the amount of it was obtained by the sum of the two drugs MIC divided by each drugs individual MIC. FICI = ( MIC of drug A in combination MIC of drug A alone ) \+ ( MIC of drug B in combination MIC of drug B alone ) The results were interpreted according to the following crieteria: FICI≤0.5, synergistic; 0.5 \< FICI \< 1, partially synergistic; FICI = 1, additive; 1\> FICI≤4, indifferent; and FICI \> 4, antagonistic. Data Analysis ------------- The heterogeneity between studies was assessed by Chi-squared test with significance level of 0.05. and *I* ^2^ test. Heterogeneity was considered as *I* ^2^ 50%. The random effects model was applied to combine the studies\' results. The data analysis was performed by STATA software version 11.1. RESULT ====== A total of 104 articles were found in the initial search. The titles and abstracts were reviewed and after the exclusion of unrelated articles, 17 studies entered the meta-analysis. The included articles were 1 poster from ECCMID, 1 short communication, 1 letter to editor, 1 brief report and 13 original articles (Table **[1](#T1){ref-type="table"}**) \[10 - 22\]. The sensitivity of 448 strains was analyzed and 2% and 72% resistance to colistin and rifampin when administrated individually were observed, respectively. We also analyzed the abundance of the strains\` MIC comparing these two antibiotics. The range of MIC for rifampin and colistin was 0.25 μg/ml to 64 μg/ml and 0.25 μg/ml to 16 μg/ml, respectively. The majority of strains (42%) demonstrated MIC= 4 and 30% had MIC= 2 for rifampin and colistin, respectively. The interaction between these two antibiotics was analyzed by the Time-kill method (11 studies) and the Checkerboard method (6 studies). The results demonstrated synergy in 63%, while, partial synergy, additive effect and no effect were present in 7, 3 and 14% of the cases, respectively. No combinations were antagonistic. Results and its details were shown in Figs. (**[1](#F1){ref-type="fig"}**-**[6](#F6){ref-type="fig"}**). DISCUSSION ========== Recent increase of the healthcare associated infections caused by MDR strains of *A. baumannii* is becoming a serious problem. The combination antibiotic therapy is proven to reduce the resistance and increase the efficiency of antibiotics. Two antibiotics that are commonly used in combination therapy are colistin and rifampin. There is no reliable information about the amount of resistance to these two antibiotics, especially to rifampin, and resistance pattern varies from hospital to hospital. Therefore, we analyzed the amounts of synergism and resistance to colistin and rifampin and their relationship with the MIC. After analyzing 448 strains, it was identified that 72% of the strains were resistant towards rifampin. Interestingly, in 4 studies the amount of resistance to rifampin was 100%. In one of the studies no synergy was observed, while in another study in 50% of the strains, the two antibiotics had no effect on each other. This shows that the relationship between MIC and the interactions of the two antibiotics are very important \[[@R5]\]. On the contrary, another study showed that 49% of the strains were sensitive to rifampin and in 100% of the cases synergism was observed. It seems that higher MIC and the consequent increase of the resistance to the antibiotic, caused the decrease of the antibiotic\`s efficiency and amount of synergism. It is notable that in one of the studies, 6% of the XDR strains were antagonistic towards antibiotics and no synergism was observed in this study. In this study, in 88% of the strains the combination of the two antibiotics was ineffective (13). These results suggested that the increase in resistance to rifampin reduces synergism. The synergic mechanism of the two antibiotics is based on colistin's effect to the outer membrane of gram-negative bacteria which causes increasing penetration of rifampin into the bacterial cell. One of the mechanisms of resistance to rifampin is *rpo* gene mutation, which causes high-level of resistance, with the concentration of bacterial MIC reaching 256 μg/ml. In Zarrilli and colleagues study the synergy of two antibiotics on the strains of *Acinetobacter* was examined and in one of the strains, where the synergism was not found, the study had identified mutations in the *rpo* gene with MIC higher than 512 μg/ml \[[@R9]-[@R18]\]. The resistance to colistin, observed in 3 studies, was found in 2% of the strains. In one of the studies that took place in Taiwan, from the total of 134 MDR strains of *A. baumannii*, 6 strains (10.2%) were resistant to colistin. The same strains were destroyed in the synergistic test \[[@R19]\]. In a study conducted in China, from the 25 strains of XDR *A. baumannii* 3 strains were resistant to colistin. The synergism test results showed 56%, 36% and 0.8% relative to synergism, additive effect and indifference to the two antibiotics, respectively \[[@R20]-[@R22]\]. These cases demonstrated the importance of combination therapy against MDR strains, and that the use of combination of two antibiotics can also control the strains resistant to colistin. The mechanism of synergy between two antibiotics in strains resistant to colistin is unknown and it needs further studies. The limitations of the current study were the inclusion of some studies that only mentioned synergism and not referring to interactions such as indifferent or antagonism. Also, some of the studies did not mention the strains' MIC fully and accurately, however, we tried to do calculation and analysis with less bias possible. In addition, in some cases the breakpoint for rifampin in *A. baumannii* was not defined, and in some articles the break point of *Staphylococcus aureus* for rifampin was used. In this study, the different effects of two antibiotics and their relationship with MIC were conducted in a systematic review method. As a result, in 63% of the strains synergy was observed. This study investigated the interaction of two antibiotics and recommend the use of combination therapy in the treatment of infection caused by *A. baumannii,* especially by MDR strains. The situation in these two phases is not the same, due to different pharmacodynamic and pharmacokinetic effects of different drugs in the host and drug concentration at the site of infection. However, for further simulation of the 2-phase in the time-kill method we used 6 mg/l for colistin and 5mg/l for rifampin, which are suitable concentrations for the human body. Greater use of this method seems to be due to the similarity in the concentration. In addition, the results of a study indicated that the resistance to rifampin occurs after 48 to 72 hours \[[@R11]\]. By increasing the time of the Time-Kill test we can compare the interaction of the two antibiotics better. *In vitro* studies were not able to evaluate the toxicity of the drug, this issue being more important for colistin. The other problem of *in vitro* studies is the lack of generalization and its use in treatment. In detailed *in vitro* studies by examining the MIC of antibiotics in 2 phases (single and in combination), the resistance was evaluated, but in the body phase, we were not able to check the resistance. Thus, more research should be done to fix these problems. The combination therapy has several advantages and one of them is that by using a combination of 2 antibiotics the concentration of each antibiotic can be reduced. This issue, as mentioned above, is more important for colistin. On the other hand, in this study, most of the strains showed MIC=4 for rifampin (42%) and MIC=2 for colistin (30%). In the observed studies, MIC of each antibiotic was calculated individually and then for the combination of two antibiotics the sub inhibitory concentration or a concentration lower than MIC was applied. We suggest an experimental basis to combine two antibiotics with a concentration of 2ug/ml for rifampin and 1 ug/ml for colistin, based on our study results. However, depending on the resistance of the strains (XDR or MDR) these concentrations could be different. It is recommended to use an accurate test such as E-test or micro dilution to measure the MIC of each of the 2 antibiotics first, because the synergy depends on rifampin. Examination of the strains' MIC can help predict the effect of the two antibiotics on each other. CONCLUSION ========== In this study, based on a systematic review and analysis of the existing studies we have shown that rifampin and colistin had a significant synergy in the in-vitro phase. Declared None. FUNDING ======= Clinical Microbiology Research Center, Department of Microbiology, School of Medicine, Ilam University of Medical Sciences, Ilam, Iran. CONFLICT OF INTEREST ==================== The authors confirm that this article content has no conflict of interest. ETHICAL APPROVAL ================ Not required. {#FC1} {#F1} {#F2} {#F3} {#F4} {#F5} {#F6} ###### Characteristics of the Studies Included in Meta-analysis. -------------------------------------------------------------------------------------------------------------------------------------------- **Reference** **Country** **Published year** **No. of isolates** **Susceptibility**\ **Synergy method(s)** **test** --------------------------------- ----------------- -------------------- --------------------- --------------------- ----------------------- **Timurk \[**[@R10]**\]** **Turkey** **2005** **60** **Agar dilution** **Checkerboard** **Tripodi \[**[@R6]**\]** **Italy** **2007** **9** **Micro dilution** **Time-kill** **Ibanez \[**[@R11]**\]** **Spain** **2010** **4** **Agar dilution** **Time-kill** **Lee \[**[@R12]**\]** **USA** **2013** **2** **Micro dilution** **Time-kill** **Shields \[**[@R13]**\]** **USA** **2010** **17** **E-test** **Checkerboard** **Rodriguez \[**[@R14]**\]** **Argentina** **2010** **14** **Agar dilution** **Time-kill** **Giamarel \[**[@R8]**\]** **Greece** **2001** **39** **Micro dilution** **Time-kill** **Song \[**[@R15]**\]** **South Korea** **2007** **8** **Micro dilution** **Time-kill** **Montero \[**[@R5]**\]** **Spain** **2004** **2** **Micro dilution** **Time-kill** **Hogg \[**[@R16]**\]** **England** **1998** **13** **Micro dilution** **Checkerboard** **Liang \[**[@R17]**\]** **China** **2011** **4** **Micro dilution** **Time-kill** **Dizbay \[**[@R18]**\]** **Turkey** **2009** **25** **E-test** **Checkerboard** **Chang \[**[@R19]**\]** **Taiwan** **2010** **134** **Micro dilution** **Checkerboard** **Dong \[**[@R20]**\]** **China.** **2014** **25** **Micro dilution** **Checkerboard** **Jian \[**[@R21]**\]** **Australia** **2007** **17** **Micro dilution** **Checkerboard** **Giannouli \[**[@R9]**\]** **Italy** **2011** **57** **Micro dilution** **Time-kill** **Antonopoulou \[**[@R22]**\]** **Greece** **2007** **18** **Micro dilution** **Time-kill** --------------------------------------------------------------------------------------------------------------------------------------------

1. Introduction {#sec1} =============== The cholesteatoma of the middle ear is a chronic otitis described as dangerous because of the evolutionary risks and the potentially serious complications. The posttraumatic cholesteatoma is recognized like a rare late complication of various types of the temporal bone damage. We report in this study a case of posttraumatic cholesteatoma revealed by a facial paralysis occurring several years after a crash of the external auditory canal. 2. Case Report {#sec2} ============== A patient, 51 years old, has consulted for a progressive right facial paralysis evolving for two years. In his antecedents there is a war wound caused by a splinter at the right temporomandibular area dating back over 23 years. The examination, carried out on a patient in good general state, finds at the inspection signs of a right complete peripheral facial paralysis, with presence in the ipsilateral preauricular area of an old traumatic scar. The otoscopy objectifies a cicatricial total stenosis of the right external auditory meatus. The instrumental acoumetry is in favour of a right transmission deafness confirmed by the liminary tonal audiometry. The stapedial reflex is absent on the affected side. The remainder of ENT and somatic examination is strictly normal. A CT-scan of the temporal bone objectifies a crash of the right external auditory canal, quasitotal lysis of the mastoïd, tissue filling the middle ear cavities with total destruction of the ossicular chain, and erosion of 2nd and 3rd portions of right facial nerve canal (Figures [1](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}). The surgery confirmed the CT-scan data and the patient benefited from an open technique of tympanoplasty (canal wall down) aimed at eradication of the cholesteatoma and decompression of the facial nerve with repermeabilisation of the external auditory meatus. The postoperative courses were simple but no improvement of facial nerve function was noted. 3. Discussion {#sec3} ============= The cholesteatoma is the result of development in the middle ear of a keratinized malpighian epithelium endowed with a potential of desquamation, migration, and erosion \[[@B1], [@B2]\]. There are two main types of cholesteatomas: the primitive or congenital cholesteatoma developing behind an intact tympanic membrane, and the acquired cholesteatoma whose origin can be an invasion by progressive colonization of the mucosae after crossing the free edge of a tympanic perforation, an evolution of a retraction pocket, or an iatrogenic or posttraumatic epidermal inclusion by skin incarceration during a fracture involving the external auditory canal \[[@B1]\]. The posttraumatic cholesteatoma is a rare and often very delayed complication of temporal bone trauma and can remain undetected for years allowing it to develop intensively \[[@B3]\]. The time interval between the temporal bone damage and the diagnosis of the posttraumatic cholesteatoma is very variable and may range from 1 to 25 years. In the majority of the cases reported in the literature, the interval was more than 10 years \[[@B4], [@B5]\]. In our case, the latent interval is 23 years. In most cases, the evocative clinical signs of a cholesteatoma are fetid otorrhea, otalgy, and hearing loss \[[@B1]\]. In our case, the otorrhea is absent because there is a complete meatal stenosis. In the evolved forms, the diagnosis can be revealed by labyrinthian, neuromeningeal, or facial complications, sources of cholesteatoma severity \[[@B1]\]. The facial paralysis is a rare complication of cholesteatoma with a frequency estimated at 1-2% \[[@B1], [@B6]\]. Generally, his installation is rapid during an infection but sometimes, it is progressive during an erosion of the facial canal, as in the case of our patient \[[@B7]\]. The CT-scan currently occupies an essential place in the diagnosis of middle ear cholesteatoma \[[@B8], [@B9]\] because it can provide semiological arguments in favour of the positive diagnosis with tissue filling the middle ear cavities and signs of osteolysis, specify the extensions, and seek possible complications like a lysis of the tegmen tympani and/or antri, a labyrinthine fistula, or an erosion of the facial nerve canal, what is essential for presurgical planning. The MRI occupies the second place. However, it may provide additional information on the delineation and extension of cholesteatoma and on potential complications \[[@B10]\]. The therapeutic management is surgical consisting primarily of a canal wall up (closed) or a canal wall down (open) tympanoplasty \[[@B11], [@B12]\] with treatment of the complications if it is necessary. 4. Conclusion {#sec4} ============= The posttraumatic cholesteatoma is a rare complication of the temporal bone trauma. Its diagnosis is often done in the majority of cases after several years of evolution, sometimes even at the stage of complications that can compromise the functional and even vital prognosis, hence the need for regular monitoring, by otoscopy and/or CT-scan of the temporal bone, of any patient with a temporal bone trauma in order to make an early diagnosis and lead to a therapy as soon as possible. {#fig1} {#fig2} [^1]: Academic Editors: M. B. Naguib and Y. Orita

Introduction {#s0001} ============ The cellular injury caused by oxidative stress and excess of free radicals such as reactive oxygen species has been associated with ageing and great number of clinical disorders including age-associated disorders such as neurodegenerative diseases. For example, Parkinson\'s disease, which is the second most common neurodegenerative disorder after Alzheimer\'s disease (AD), has been linked to increased levels of oxidative stress.\[[@cit0001]\] Also oxidative stress has been postulated to have an important role in the pathogenesis of schizophrenia, bipolar disorder and major depression.\[[@cit0003]\] It is also discussed, that the radical production and lipid peroxidation are involved in the pathogenesis of other diseases, including atherosclerosis, cardiac and cerebral ischemia, carcinogenesis, diabetes, alcohol induced liver disease, ulcerative colitis and rheumatic disorders.\[[@cit0005]\] Antioxidant-based drugs and formulations for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, AD, cancer and some psychiatric disorders have appeared during the last decades.\[[@cit0008]\] This has attracted a great deal of research interest in the field of antioxidants. Piperazine nucleus is one of the most important heterocycles exhibiting remarkable pharmacological activities. Piperazine is consisting of a six-membered ring containing two opposing nitrogen atoms. Slight change in substitution pattern in piperazine nucleus causes distinguishable difference in their pharmacological activities, such as antipsychotic, anticonvulsant, antiarrhythmic, antimicrobial, antimalarial, citotoxic and antioxidant effects.\[[@cit0010]\] Thus, in this study some new aryl/aralkyl substituted piperazine derivatives, containing methylxanthine moiety were synthesized and their antioxidant activity was established. Materials and methods {#s0002} ===================== Materials {#s0002-0001} --------- Synthetic grade chemicals procured from Acros organics, Belgium, were used for the synthesis of the target compounds, as received. Non-commercially available intermediates required for the synthesis of novel derivatives of arylpiperazine were prepared according to the literature procedures without modifications: 1-(3-iodopropyl)-3,7-dimethylxanthine.\[[@cit0013]\] The completion of reactions and the purity of the final products was monitored and confirmed through thin layer chromatography (TLC) using Kieselgel 60 F~254~ plates (Merck) and solvent system: ethanol:chloroform:acetone (4:3:3 ). The spots were detected at UV~254~ nm. The reagents: 2,2′-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzo thiazine-6-sulfonic acid) (ABTS), sulfanilamide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride × 6H~2~O, sodium acetate and potassium persulphate were purchased from Sigma-Aldrich. All the other chemicals including the solvents were of analytical grade. Apparatus {#s0002-0002} --------- Melting points were determined on an electrothermal apparatus (Büchi 535, Switzerland) in an open capillary tube and are not corrected. The IR spectra 400--4000 cm^−1^ were recorded on an FT-IR Nicolet iS10 spectrophotometer (Thermo Scientific, USA). The ^1^H NMR spectra were measured on a Bruker Avance DRX 250 (250 MHz) spectrometer (Germany). ^1^H NMR spectra were measured for approximately 0.03 mol/L while using CDCl~3~ and D~2~O as solvents and chemical shifts, and were expressed as δ values in ppm against TMS as an internal standard. All names were generated by using structure-to-name and name-to-structure algorithms included with ChemBioDraw Ultra 11.0 (CambridgeSoft). Antioxidant assays were carried out using a Shimadzu UV-1203 spectrophotometer (Japan). Synthetic procedures for obtaining of the target compounds 3a--f {#s0002-0003} ---------------------------------------------------------------- ### General procedure {#s0002-0003-0001} A mixture of 1-(3-iodopropyl)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione (1, 1 mmol), the appropriate 1-substituted piperazine (2a--f, 1 mmol) and triethylamine (1 mmol) in acetone (10 mL) was refluxed till exhaustion of the starting products. The progress of the reaction was monitored by TLC. Then the acetone was evaporated to dryness. The reaction mixture was cooled overnight if it was necessary. The precipitated crude product was filtered off and purified by recrystallization from *i*-propanol. ### 1-(3-(4-benzylpiperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione (3a) {#s0002-0003-0002} Reaction time 10 hours. Yield: 77%, melting point (m.p.) 191--193 °C, Rf = 0.45, IR: 3070 (CH aromatic), 2911--2833 (CH~3~ and CH~2~), 1702 (CO xanthine), 1654 (CO xanthine), 1602 (C = N xanthine), 1552 (C = C xanthine), 762 (mono-substituted aromatic ring), ^1^H--NMR (CDCl~3~, δ, ppm): 7.54 (s, 1H, C^8^--[H]{.ul}); 7.36--7.37 (m, 4H, aromatic ring); 7.27 (bs, 1H, aromatic ring); 4.09 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.98 (s, 3H, N^7^--[CH~3~]{.ul}); 3.84 (bs, 2H, N^1^(piperazine)--[CH~2~]{.ul}); 3.56 (s, 3H, N^3^--[CH~3~]{.ul}); 3.03 (bs, 8H, piperazine); 2.17 (s, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}, 1.26, 1.22 (d, 2H, CH~2~--N^1^(piperazine), J = 8.43 Hz). ### 1-(3-(4-(4-fluorophenyl)piperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6 (3H,7H)-dione (3b) {#s0002-0003-0003} Reaction time 10 hours. Yield: 67%, m.p. 156--158 °C, Rf = 0.64, IR: 3054 (CH aromatic), 2966--2817 (CH~3~ and CH~2~), 1709 (CO xanthine), 1651 (CO xanthine), 1604 (C = N xanthine), 1577 (C = C xanthine), 1143 (C--F), 820 (1,4-disubstituted aromatic ring), ^1^H--NMR (CDCl~3~, δ, ppm): 7.51 (s, 1H, C^8^--[H]{.ul}); 6.96 (t, 2H, aromatic ring); 6.85--6.88 (m, 2H, aromatic ring); 4.10 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.99 (s, 3H, N^7^--[CH~3~]{.ul}); 3.58 (s, 3H, N^3^--[CH~3~]{.ul}); 3.11 (s, 4H, piperazine); 2.63 (s, 4H, piperazine); 2.54 (bs, 2H, [CH~2~]{.ul}--N^1^ piperazine[)]{.ul}, 1.41 ( t, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}. ### 1-(3-(4-(4-hydroxyphenyl)piperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6 (3H,7H)-dione (3c) {#s0002-0003-0004} Reaction time 10 hours. Yield: 93%, m.p. 263--264 °C with decomposition, Rf = 0.43, IR: 3202 (Aryl-OH), 3075 (CH aromatic), 2926--2815 (CH~3~ and CH~2~), 1701 (CO xanthine), 1657 (CO xanthine), 1609 with shoulder 1595 (C˭N xanthine), 1550 (C˭C xanthine), 1359 (δOH), 1186 (C--OH), 828 (1,4-disubstituted aromatic ring), ^1^H--NMR (D~2~O, δ, ppm): 8.39 (s, 1H, OH-aromatic ring) 7.85 (s, 1H, C^8^--[H]{.ul}); 6.97--6.85 (m, 2H, aromatic ring); 6.85--6.88 (m, 2H, aromatic ring); 4.04 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.89 (s, 3H, N^7^--[CH~3~]{.ul}); 3.48 (s, 3H, N^3^--[CH~3~]{.ul}); 3.34, 3.28 (d, 8H, piperazine, J = 13.08); 3.13 (s, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}, 2.06 (bs, 2H, [CH~2~]{.ul}--N^1^(piperazine[)]{.ul}. ### 1-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-3,7-dimethyl-1H-purine 2,6 (3H,7H)-dione (3d) {#s0002-0003-0005} Reaction time 5 hours. Yield: 75%, m.p. 177--179 °C, Rf = 0.56, IR: 3125 (CH aromatic), 2931--2817 (CH~3~ and CH~2~), 2817 (O--CH~3~), 1694 (CO xanthine), 1652 (CO xanthine), 1602 (C˭ N xanthine), 1548 (C˭C xanthine), 1454 (δCH~3~ O--CH~3~), 1154 (C--O--C), 745 (1,2-disubstituted aromatic ring), ^1^H--NMR (CDCl~3~, δ, ppm): 7.50 (s, 1H, C^8^--[H]{.ul}); 7.00--6.97 (m, 1H, aromatic ring); 6.93--6.90 (m, 2H, aromatic ring); 6.86--6.84 (m, 1H, aromatic ring); 4.10 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.99 (s, 3H, N^7^--[CH~3~]{.ul}); 3.85 (s, 3H, OCH~3~--aromatic ring); 3.58 (s, 3H, N^3^--[CH~3~]{.ul}); 3.06 (bs, 4H, piperazine); 2.66 (bs, 4H, piperazine); 2.53 (t, 2H, [CH~2~]{.ul}--N^1^(piperazine[)]{.ul}, 1.90 (t, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}. ### 1-(3-(4-(4-methoxyphenyl)piperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione (3e) {#s0002-0003-0006} Reaction time 13 hours. Yield: 64%, m.p. 154--156 °C, Rf = 0.58, IR: 3120 (CH aromatic), 2946--2813 (CH~3~ and CH~2~), 2813 (O--CH~3~), 1698 (CO xanthine), 1651 (CO xanthine), 1604 (C˭N xanthine), 1548 (C˭C xanthine), 1456 (δCH~3~ O--CH~3~), 1125 (C--O--C), 844 (1,4-disubstituted aromatic ring), ^1^H--NMR (CDCl~3~, δ, ppm): 7.50 (s, 1H, C^8^--[H]{.ul}); 6.90--6.88 (m, 2H, aromatic ring); 6.84--6.82 (m, 2H, aromatic ring); 4.09 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.99 (s, 3H, N^7^--[CH~3~]{.ul}); 3.76 (s, 3H, OCH~3~-aromatic ring); 3.58 (s, 3H, N^3^--[CH~3~]{.ul}); 3.06 (bt, 4H, piperazine); 2.62 (bt, 4H, piperazine); 2.52 (t, 2H, [CH~2~]{.ul}--N^1^(piperazine[)]{.ul}, 1.90 ( t, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}. ### 1-(3-(4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)propyl)-3,7-dimethyl-1H-purine-2,6 (3H,7H)-dione (3f) {#s0002-0003-0007} Reaction time 5 hours. Yield: 60%, m.p. 144--146 °C, Rf = 0.74, IR: 3070 (CH aromatic), 2962--2825 (CH~3~ and CH~2~), 1703 (CO xanthine), 1655 (CO xanthine), 1602 (C˭N xanthine), 1551 (C˭C xanthine), 1223 (C--F), 826 (p- di-substituted aromatic ring), ^1^H--NMR (CDCl~3~, δ, ppm): 7.53 (s, 1H, C^8^--[H]{.ul}); 7.35--7.27 (m, 4H, aromatic ring); 7.02--6.93 (m, 4H, aromatic ring); 4.36 (s, 1H, N^1^(piperazine)--C[H]{.ul}); 4.08 (t, 2H, N^1^(xanthine)--[CH~2~]{.ul}); 3.96 (s, 3H, N^7^--[CH~3~]{.ul}); 3.56 (s, 3H, N^3^--[CH~3~]{.ul}); 3.14 (t, 4H, piperazine); 2.94 (bs, 4H, piperazine); 2.33 (t, 2H, [CH~2~]{.ul}--N^1^(piperazine[)]{.ul}, 1.48 (t, 2H, N^1^(xanthine)--CH~2~--[CH~2~)]{.ul}. Antioxidant assays {#s0002-0004} ------------------ ### DPPH radical scavenging activity {#s0002-0004-0001} Free radical scavenging activity was measured by using the DPPH method.\[[@cit0014]\] Different concentrations (6.25--1000 μmol/L) (400 μL) of compounds in methanol were added to the 400 μL methanol solution of DPPH (0.5 mmol/L). After 30 min incubation at room temperature, their absorptions were read at 517 nm against a control sample containing the methanol solution of DPPH. Percentage of the DPPH radicals scavenged by the studied concentration was calculated according to equation: where Abs~control~ is the absorbance of DPPH radical in methanol, Abs~sample~ is the absorbance of DPPH radical solution mixed with sample. IC~50~ value (concentration of sample where the absorbance of DPPH decreases 50% with respect to the absorbance of blank) of the sample was determined. Butylated hydroxytoluene (BHT) was used as positive control. All determinations were performed in triplicate (*n* = 3). ### ABTS-radical scavenging assay {#s0002-0004-0002} For ABTS assay, the procedure followed the method of Arnao et al. \[[@cit0015]\] with some modifications.\[[@cit0014]\] The stock solutions included 7 mmol/L ABTS solution and 2.4 mmol/L potassium persulphate solution. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 14 hours at room temperature in dark. The solution was then diluted by mixing 2 mL ABTS solution with 50 mL methanol to obtain an absorbance of 0.305 ± 0.01 units at 734 nm using a spectrophotometer. A fresh ABTS solution was prepared for each assay. Different concentrations (400 μL) of compounds were allowed to react with 400 μL of the ABTS solution and the absorbance was taken at 734 nm after 5 min. The capability to scavenge the ABTS radical was compared with that of BHT and was calculated using the following equation: where Abs~control~ is the absorbance of ABTS radical in methanol; Abs~sample~ is the absorbance of an ABTS radical solution mixed with sample. IC~50~ value (concentration of sample where the absorbance of ABTS decreases 50% with respect to the absorbance of blank) of the sample was determined. BHT was used as positive control. All determinations were performed in triplicate (*n* = 3). ### Ferric reducing/antioxidant power (FRAP) {#s0002-0004-0003} The FRAP assay was done according to the method described by Benzie and Strain \[[@cit0016]\] with some modifications.\[[@cit0014]\] The stock solutions included 300 mmol/L acetate buffer (3.1 g C~2~H~3~NaO~2~ × 3H~2~O and 16 mL C~2~H~4~O~2~), pH 3.6, 10 mmol/L TPTZ solution in 40 mmol/L HCl and 20 mmol/L FeCl~3~ × 6H~2~O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl~3~ × 6H~2~O solution and then warmed at 37 °C before using. 0.15 mL of compound in methanol was allowed to react with 2.8 mL of the FRAP solution for 30 min in the dark condition. Readings of the coloured product (ferrous tripyridyltriazine complex) were then taken at 593 nm. Results are expressed in μg Trolox equivalent (μM TE/mmol/L\]). BHT was used as reference. All determinations were performed in triplicate (*n* = 3). ### Determination of antioxidant activity in linoleic acid system by the ferric thiocyanate (FTC) method {#s0002-0004-0004} The antioxidant activity of studied compounds against lipid peroxidation was measured through ammonium thiocyanate assay, as described by Takao et al., \[[@cit0017]\] with some modifications.\[[@cit0020]\] The reaction solution, containing 0.2 mL of compound (1 mmol/L in methanol), 0.2 mL of linoleic acid emulsion (25 mg/mL in 99 % ethanol) and 0.4 mL of 50 mmol/L phosphate buffer (pH 7.4), was incubated in the dark at 40 °C. A 0.1 mL aliquot of the reaction solution was then added to 3 mL of 70 % (v/v) ethanol and 0.05 mL of 30% (w/v) ammonium thiocyanate. Precisely 3 min after the addition of 0.05 mL of 20 mmol/L ferrous chloride in 3.5% (v/v) hydrochloric acid to the reaction mixture, the absorbance of the resulting red colour was measured at 500 nm. Aliquots were assayed every 24 hour until the day after the absorbance of the control solution (without compound) reached maximum value. BHT (1 mmol/L) was used as a positive control. All determinations were performed in triplicate (*n* = 3). Results and discussion {#s0003} ====================== Chemistry {#s0003-0001} --------- The target compounds have been synthesized according to the above-presented synthetic procedure. The general synthetic scheme is presented in [Scheme 1](#c0001){ref-type="fig"}. The readily available compound 1-(3-iodoropropyl)-3,7-dimethylxanthine (1) was aminated with 1-aryl/aralkylpiperazines (2a--f) in the presence of triethylamine to give the target compounds 3a--f in high yields. In this reaction scheme, it is possible to use 1-(3-chloropropyl)-3,7-dimethylxanthine as the starting compound instead of 1. When it was used as a starting material to obtain 3a--f, their yield was found to be remarkably lower and the reaction time about two times longer. This was probably due to low reactivity of this compound in comparison with 1. We also found that if there is no protection from the atmospheric moisture whilst obtaining the target compounds from both of 1 and 1-(3-chloropropyl)-3,7-dimethylxanthine, the hydrolysis took place and the yield of the new compounds decreased. The hydrolysis product is 1-(3-hydroxypropyl)-3,7-dimethylxanthine (13) and was detected by TLC. Scheme 1.General synthesis of the target compounds 3a--f. All the synthesized compounds were freely soluble in chloroform, dichloromethane, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), but insoluble in non-polar solvents like *n*-hexane, except 3c which is soluble only in boiling water. The structures of the synthesized compounds were confirmed by IR and ^1^H NMR. The results were consistent with the assigned structures. The FT-IR spectra of the studied compounds 3a--f in the region of the 4000--400 cm^−1^ exhibit several characteristic bands. Vibrations at 3054--3125 cm^−1^ belong to stretching vibrations of aromatic C--H bonds. The stretching vibration of phenolic group in the spectrum of 3c appears at 3202 cm^−1^. The two strong bands at about 1680--1702 cm^−1^ are ascribable to the stretching vibration of two carbonyl groups in the xanthine ring. The band at about 1550 cm^−1^ belongs to the stretching vibration of C˭C bonds in xanthine ring. More detailed information about the structure of compounds 3a--f was provided by the ^1^H NMR spectra. The ^1^H NMR spectra of the compounds 3a--f showed the signals of the respective protons of the synthesized compounds, which were verified on the basis of their chemicals shifts, multiplicities and coupling constants. Thus, the strong singlets at 3.48\\3.58 and 3.89\\3.99 ppm in the spectrum of the studied compounds correspond to *N*-methyl protons at positions 3 and 7. The signal of the proton at position 8 appears at 7.50\\7.54 ppm as strong singlet. The signals of methylene protons from the benzyl side chain at position 1 form strong singlet at 4.80 ppm. The signals of the aromatic protons from substituted benzyl side chain in the spectra of 3b--e correspond to complicated multiplets between 6.82 and 6.97 ppm, while the same protons in unsubstituted benzyl moiety (3a) appear as multiplet 7.27 and 7.37 ppm. The aromatic protons in benzhydryl group in 3f form multiplet at 7.27 and 7.35 ppm. The methylene proton from the benzhydryl residue forms a singlet with weak to medium intensity at 4.36 ppm. The integral curves correspond to the exact number of the protons. The values of the chemical shifts of the protons registered by ^1^H NMR spectra were compared with simulated values.\[[@cit0018]\] Due to an impossibility to render an account of influence of the solvent, we observed only small deviations of the computed values from the experimental values. Regardless, the simulated ^1^H NMR spectra are in good correlation with experimental ones. Antioxidant assays {#s0003-0002} ------------------ In this paper, we apply four common methods \[[@cit0014]\] for the determination of free radicals scavenging activity, total antioxidant capacity and antioxidant activity against lipid peroxidation to both lipophilic and hydrophilic antioxidants in an attempt to establish the most appropriate one for evaluation of possible antioxidant effects, demonstrated from the newly synthesized arylpiperazine derivatives. The described methods were chosen for their user-friendly mechanisms for antioxidant activity determination since they require a simple machine like a spectrophotometer, which is commonly available in most laboratories. The free radicals scavenging activity was measured by using DPPH and ABTS methods with slight modifications.\[[@cit0020]\] The inhibitory effect of different concentrations of compounds (6.25--1000 μmol/L^2^) on DPPH ([Figure 1](#f0001){ref-type="fig"}) and ABTS ([Figure 2](#f0002){ref-type="fig"}) was determined by recording the absorbance of the reaction mixture at 517 and 734 nm, respectively. The IC~50~ values (concentration of sample where the absorbance of DPPH and ABTS decreases 50% with respect to the absorbance of blank) of the samples were determined. Figure 1.DPPH radical scavenging activity of studied compounds 3a, 3c, 3f and BHT. Figure 2.ABTS-radical scavenging activity of studied compounds (a) 3a, 3c, 3f and BHT and (b) 3b, 3e, 3d and BHT. The applied FRAP assay was done according to the method described previously.\[[@cit0014]\] The obtained results are expressed in μmol/L Trolox equivalent for mmol/L compound (μmol/L TE/\[μmol/L TE/mmol/L\]) for 1000 μmol/L^2^ solutions of piperazines. The corresponding radical scavenging activities against DPPH, ABTS and FRAP of the compounds were compared with those of BHT, used as positive control. All determinations are performed in triplicate (*n* = 3), and the results are expressed as IC~50~ μmol L^−2^ of inhibition in DPPH and ABTS determinations and as mmol/L TE mM^−1^ in FRAP determination. The obtained values are presented in [Table 1](#t0001){ref-type="table"}. Table 1.DPPH, ABTS-radical scavenging and FRAP-activities of studied compounds.Sample IDDPPH IC~50~ (μmol/L^2^)ABTS IC~50~ (μmol/L^2^)FRAP (μmol/L TE/mmol/L)3a371.9755.87--3b--345.2527.22 ± 1.353c189.42243.45173.99 ± 1.503d--1028.99--3e--242.4883.10 ± 1.843f420.5741.04--BHT113.1726.2923.26 ± 0.45 Among the analysed structures, DPPH radical scavenging activity was shown by compounds 3a (IC~50~ 371.97 μmol/L), 3c (IC~50~ 189.42 μmol/L) and 3f (IC~50~ 420.57 μmol/L), with the highest antioxidant activity demonstrated by compound 3c. However, the obtained IC~50~ values of all piperazines were lower than that of BHT (IC~50~ 113.17 μmol/L). All studied compounds demonstrated ABTS-radical scavenging decreasing in order: 3c (IC~50~ 3.45 μmol/L) \> BHT (IC~50~ 26.29 μmol/L) \> 3f (IC~50~ 41.04 μmol/L) \> 3a (IC~50~ 55.87 μmol/L) \> 3e (IC~50~ 242.48 μmol/L) \> 3b (IC~50~ 345.25 μmol/L) \> 3d (IC~50~ 1028.99 μmol/L). In the performed FRAP assay, the reduction of ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form (which has an intense blue colour) at low pH was monitored by measuring the change in the absorption at 593 nm, which is considered to be directly related to the combined or 'total' reducing power of the electron donating antioxidants present in the reaction mixture. Among the synthesized compounds only 3b, 3c and 3e (with p-substitution in phenyl ring) manifest some activity in the FRAP method, while compound 3c demonstrated the highest activity (173.99 ± 1.50 μmol/L TE/mmol/L), followed by 3e (83.10 ± 1.84 μmol/L TE/mmol/L) and 3b (27.22 ± 1.35 μmol/L TE/mmol/L). However, piperazines 3b, 3c and 3e exhibited stronger activity compared to BHT (23.26 ± 0.45 μmol/L TE/mmol/L). These effects are probably due to the possibility of the analytes to break up the free radical chain by donating a hydrogen atom. As seen from the presented results for the three discussed methods used for evaluation of the free radicals scavenging activity and FRAP of the newly synthesized structures, the highest antioxidant activity was demonstrated by compound 3c. We believe that this result is due to the presence of hydroxyl group in the structure of this product. Furthermore, the difference in the chemical structure of the newly obtained compounds will reflect to their relative ability to quench aqueous peroxyl radicals and to reduce the ABTS+, the DPPH free radical and the ferric ion in *in vitro* systems. Moreover, ABTS, DPPH and FRAP assays gave comparable results for the antioxidant activity measured for all compounds, whereas the ABTS technique was simple, rapidly performed and was applicable for the determination of the oxidative properties for all the analysed structures. An additional advantage of the ABTS to DPPH is that the reaction is more rapid, whereas the DPPH reaction takes much longer. Therefore, it would be an appropriate technique for antioxidant evaluation of structural derivatives with piperazine residue. In this study, the antioxidant activity of the studied compounds (1000 μmol/L^2^) against lipid peroxidation was also measured through ammonium thiocyanate assay, as described previously.\[[@cit0017]\] The inhibition of lipid peroxidation of the analysed structures (1 mmol/L) was determined in the linoleic acid system using the FTC method ([Figure 3](#f0003){ref-type="fig"}). The obtained results revealed that compound 3c demonstrated the highest significant diminution, whereas it hindered the oxidation of linoleic acid for five days with an effect comparable with the BHT effect, used as a control. However, all other analytes did not show significant inhibition of lipid peroxidation compared to the control. Figure 3.Antioxidant activity of structures 3a--f in the linoleic acid system 1 μmol/L^2^. Conclusion {#s0004} ========== A series of six piperazine like derivatives of 3,7-dimethylxanthine (theobromine) was synthesized. The structures of the new compounds were confirmed by IR and ^1^H NMR. The results were consistent with the assigned structures. All compounds were *in vitro* screened for their activity as antioxidants. It was established that the difference in the chemical structure of the compounds reflects to their relative ability to quench aqueous peroxyl radicals and to reduce the ABTS+, DPPH free radical and ferric ion in *in vitro* systems. For the three discussed methods, the highest antioxidant activity was demonstrated by compound 3c. The antioxidant activity of the studied compounds against lipid peroxidation was also measured. No significant inhibition of lipid peroxidation compared to the control has been observed for the set of compounds, except for compound 3c**,** which demonstrated the highest significant diminution of linoleic acid with an effect comparable with the BHT effect, used as a control. From the performed evaluations, we may conclude that the presence of a hydroxyl group in the structure is essential for the antioxidant properties and should be taken into consideration in further design of structures with potential antioxidant properties.

Oviraptorosaurs are some of the most unusual dinosaurs. These bird-like, feathered theropods diverged dramatically from their close cousins, evolving shortened toothless skulls with a staggering diversity of pneumatic cranial crests in derived forms[@b1]. Unlike the stereotypical view of theropods as stealthy carnivores, oviraptorosaurs probably had more varied diets. Previous studies have suggested that oviraptorosaurs may have eaten such foods as eggs, mollusks, plants, shellfish, and nuts[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9][@b10][@b11], but these hypotheses remain to be more conclusively tested. The bizarre body plan and diets of oviraptorosaurs were clearly successful, as these dinosaurs were highly diverse in the Cretaceous of Asia and North America, where they ranged from the size of a turkey to nearly the length of 7 meters[@b12]. These theropods, once so poorly understood, are now recognized as important components of terrestrial food webs in the northern continents during the final \~15 million years of the Age of Dinosaurs[@b13]. Our understanding of oviraptorosaur anatomy and evolution has greatly increased with a bounty of new discoveries over the past decade. In total, more than 35 oviraptorosaur genera are now known. Many of the recent discoveries have come from China, particularly from three areas of the country: northern China (including Inner Mongolia and western Liaoning Province[@b8][@b12][@b14][@b15][@b16][@b17][@b18][@b19][@b20]), central China (Henan Province[@b21][@b10]), and southern China (including Guangdong and Jiangxi provinces[@b9][@b22][@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30]). The southern Chinese oviraptorosaurs are especially diverse. Over the last five years alone, five distinct taxa have been described from the Ganzhou area of Jiangxi Province, all of which are highly derived, toothless oviraptorids represented, thus far, by a single holotype individual. Because of these discoveries, the latest Cretaceous (Maastrichtian) deposits of the Ganzhou area becoming critical for understanding the evolution of this aberrant dinosaur subgroup. We here report a sixth diagnostic oviraptorosaur from the Ganzhou area ([Fig. 1](#f1){ref-type="fig"}), represented by a remarkably well-preserved specimen in an unusual posture with its limbs splayed to the side, its neck outstretched, and its head raised. This specimen is described as a new species, *Tongtianlong limosus* gen. et sp. nov., based on its unique dome-like skull roof, highly convex premaxilla, and many additional features setting it apart from other oviraptorosaurs, both from Ganzhou and globally. The discovery of yet another diagnostic specimen from Ganzhou begs the question of why so many oviraptorosaurs are found here. We argue that the high variability of oviraptorosaurs documents an evolutionary radiation of these dinosaurs in the very latest Cretaceous of Asia, perhaps enabled by differences in skull morphology related to feeding. This flurry of evolution helped establish some of the final dinosaur faunas before the end-Cretaceous extinction. Results ======= Systematic Paleontology ----------------------- Dinosauria Owen, 1842[@b31]. Theropoda Marsh, 1881[@b32]. Maniraptora Gauthier, 1986[@b33]. Oviraptorosauria Barsbold, 1976[@b34]. Oviraptoridae Barsbold, 1976[@b34]. *Tongtianlong limosus* gen. et sp. nov. ([Figs 2](#f2){ref-type="fig"}, [3](#f3){ref-type="fig"} and [4](#f4){ref-type="fig"}) Etymology --------- Tongtian, Chinese Pinyin, refers to Tongtianyan of Ganzhou, the first grotto south of the Yangtze River. Tongtian also means the road to heaven, a fitting epitaph for a deceased dinosaur preserved with outstretched arms. Long, Chinese Pinyin for dragon. Limosus, Latin for muddy, refers to the holotype specimen being found in an unusual posture in a mudstone ([Fig. 5](#f5){ref-type="fig"}). Holotype -------- A nearly complete, three-dimensionally preserved skeleton with skull and lower jaws (DYM-2013-8). The specimen is accessioned at the Dongyang Museum, Dongyang City, Zhejiang Province. Type locality and horizon ------------------------- The building site of the No. 3 high school of Ganxian (GPS coordinates are provided on request from the first author); Nanxiong Formation (Maastrichtian, Upper Cretaceous)[@b35]. Diagnosis --------- Oviraptorid dinosaur with the following unique combination of characters, with autapomorphies among all oviraptorosaurs indicated with an asterisk and autapomorphies among oviraptorids indicated with a double asterisk (these latter features are present in some caenagnathids): dome-like skull roof with highest point located above the posterodorsal corner of the orbit\*; anterior margin of the premaxilla highly convex in lateral view\*; distinct process at the middle of the anterior margin of the parietal on the skull roof\*; plate-like lacrimal shaft that is anteroposteriorly long in lateral view, with a flat lateral surface\*; foramen magnum smaller than the occipital condyle\*\*; absence of symphyseal ventral process of the dentary\*\*; absence of distinct lateral xiphoid process of the sternum posterior to the costal margin\*\*. *Tongtianlong* differs from other Ganzhou oviraptorids with preserved skull material (*Banji*, *Huanansaurus*) in that the anteroventral corner of the external naris is far above a horizontal line tangent with the posterodorsal corner of the antorbital fenestra, an unusual feature otherwise only seen in *Nemegtomaia*[@b36][@b37][@b38] and *Rinchenia* (Barsbold[@b39]) (=*Oviraptor mongoliensis*[@b1][@b40]). *Tongtianlong* also differs from other Ganzhou oviraptorids in numerous ways that are encapsulated in the character scores in our phylogenetic analysis. *Tongtianlong* differs from *Banji*[@b26] in possessing a postorbital process of the jugal that is posterodorsally inclined relative to the ventral ramus (not perpendicular), lacking a downturned symphyseal portion of the dentary (see also [Supplementary Information Fig. S1a](#S1){ref-type="supplementary-material"}), lacking a prominent process on the posteroventral surface of the dentary symphysis, and possessing a more anteroposteriorly elongate external mandibular fenestra. *Tongtianlong* is distinguished from *Ganzhousaurus*[@b27] in lacking a downturned symphyseal portion of the dentary, possessing a more anteroposteriorly elongate external mandibular fenestra, having a dentary that contributes to the ventral border of the external mandibular fenestra (not excluded from the border by the anterior extension of the angular), lacking a depression on the lateral surface of the dentary immediately anterior to the external mandibular fenestra, and possessing a metatarsal III that is anteroposteriorly flattened with a concave posterior surface (not ovoid or subtriangular in cross section). *Tongtianlong* is different from *Jiangxisaurus*[@b28] in lacking a downturned symphyseal portion of the dentary. Furthermore, the ratio of radius length to humerus length (78%) and the height-to-length ratio of the lower jaw (34%) are greater than those of *Jiangxisaurus* (70% and 20%, respectively). *Tongtianlong* differs from *Nankangia*[@b9] in lacking a prominent process on the posteroventral surface of the dentary symphysis, lacking a deep fossa on the lateral surface of the dentary, having a dentary that contributes to the dorsal border of the external mandibular fenestra (not excluded from the border by the anterior extension of the surangular), and lacking a depression on the lateral surface of the dentary immediately anterior to the external mandibular fenestra. *Tongtianlong* differs from *Huanansaurus*[@b29] in skull morphology and forelimb proportions. There is a crest in *Huanansaurus* but not in *Tongtianlong*. The dorsal margin of the lower jaw, from the anterior tip to the coronoid eminence, is smoothly convex in *Tongtianlong*, whilst it is wave-like in *Huanansaurus*. The antorbital fenestra is sub-oval in *Tongtianlong*, but triangular in *Huanansaurus*. The ratio of radius length to humerus length in *Tongtianlong* (0.78) is much smaller than that of *Huanansaurus* (0.97). The anteroventral corner of the external naris is slightly below the horizontal line projected through the posterodorsal corner of the antorbital fenestra in *Huanansaurus*, whilst it is far above this line in *Tongtianlong* ([Fig. 6](#f6){ref-type="fig"}). Description =========== The specimen is very well preserved in three dimensions, with the bones in natural articulation ([Figs 2](#f2){ref-type="fig"} and [3](#f3){ref-type="fig"}a; see also [Supplementary Information Table S1](#S1){ref-type="supplementary-material"}). The limbs are splayed out sideways relative to the trunk, and the neck is curved upwards, such that the head is elevated relative to the remainder of the body. Because the specimen was collected by a farmer and construction workers, and it was not mapped *in situ* while being excavated, it is difficult to interpret what biological and/or taphonomic processes caused this strange posture. Judging by the fine state of preservation, the specimen probably was originally complete or nearly complete. However, some portions of the skeleton are missing, such as the distal regions of the arms, the right pelvic girdle and hind leg, and parts of the tail. This is because the specimen was collected by workers at an active construction site. The specimen was exposed after workmen blasted away some of the surrounding rocks with TNT; a drill hole where TNT was placed can be seen near the pelvic girdle. The skull is almost completely preserved. It is missing only small portions of the anterior end of the premaxilla and nasals, and a small part of the right lower jaw. The most salient feature of the skull is that the cranial roof is dome-like, with its highest point above the posterodorsal corner of the orbit. Many other oviraptorosaurs possess cranial ornaments, which in some cases are elaborate and highly pneumatic ([Fig. 4](#f4){ref-type="fig"}). However, in other taxa these crests are usually thinner (such as in *Nemegtomaia*[@b36][@b37][@b38], it is 6 mm) than the dome-like condition in *Tongtianlong*. Furthermore, in other taxa these crests are peaked further anteriorly relative to *Tongtianlong*, either at the anterior end of the snout above the external naris and antorbital fenestra (as in *Banji*, *Citipati*, *Oviraptor*, and *Nemegtomaia*), or at approximately the midpoint of the cranium above the orbit (as in *Rinchenia*, *Huanansaurus*, and caenagnathids like *Anzu*[@b41]) ([Fig. 6](#f6){ref-type="fig"}). Therefore, the posteriorly-peaked dome-like crest of *Tongtianlong* is autapomorphic among oviraptorosaurs, and a novel type of cranial ornamentation in this highly variable clade. The fine three-dimensional preservation of the specimen ensures that the shape of the dome-like crest is not an artefact of crushing or deformation. There are five main openings in the cranium, as is standard for oviraptorosaurs and other dinosaurs. Anteriorly a large, oval-shaped external naris is positioned above a slightly smaller, triangular antorbital fenestra ([Fig. 4](#f4){ref-type="fig"}). The anteroventral corner of the naris is located far above the level of the posterodorsal corner of the antorbital fenestra, which is also the case in *Nemegtomaia* and *Rinchenia*, but differs from the condition in most other oviraptorosaurs, in which the naris extends further ventrally so that it reaches past the posterodorsal corner of the antorbital fenestra ([Fig. 6](#f6){ref-type="fig"}). The orbit is large and nearly circular, as is typical for oviraptorosaurs. The lateral temporal fenestra is the largest opening in the skull. It is rectangular, with a long axis that extends slightly anteroventrally, which differs from the more circular or square fenestrae of many other oviraptorosaurs. The supratemporal fenestra is positioned above the lateral temporal fenestra and is partially visible in lateral view. It is much smaller than the orbit and lateral temporal fenestra. The premaxilla is toothless like in all derived oviraptorosaurs. The left and right premaxillae appear to be unfused to each other, based on open sutures in the region of the broken dorsal surfaces of both bones, but the premaxilla and maxilla are fused together without a clear sutural trace. The anterior margin of the premaxilla is highly convex, which is an autapomorphy of *Tongtianlong*. Most other oviraptorosaurs have a straight anterior premaxilla (e.g., *Citipati*[@b42], *Khaan*[@b42]), and this is also the case in the Ganzhou oviraptorid *Huanasaurus*[@b29]. *Yulong* and the Ganzhou oviraptorid *Banji* have a slightly rounded anterior margin of the premaxilla in lateral view, but it is not nearly as convex as in *Tongtianlong*. The premaxilla is divided into two branches, both of which extend posterodorsally. The upper one forms the anterodorsal margin of the external naris, whereas the much wider lower one forms most of the anterodorsal margin of the antorbital fenestra, thus separating the naris from the antorbital fenestra and completely excluding the maxilla from the narial border. The posterior end of this branch overlaps the lateral surface of the lacrimal. The divergence of the two branches defines the shape of the external naris. Anteroventral to the naris, a deeply concave fossa extends on the lateral surface of the premaxilla, as in *Huanasaurus*[@b29], *Yulong*[@b10], and *Nemegtomaia*[@b37][@b38], but unlike the slightly concave surface in *Citipati*[@b6]. The maxilla is very small and exposed only as a tiny sliver of bone in lateral view. It forms the ventral margin of the antorbital opening and lacks teeth, but has a small triangular 'tooth-like' process on its ventral surface. The lacrimal is divided into three branches: a short anterior process that is covered by the premaxilla, a bulbous posterior process that extends dorsally to define the anterodorsal corner of the orbit, and a large ventral shaft. The shape of the shaft is unique: whereas in other oviraptorosaurs the lacrimal shaft is gracile (thin anteroposteriorly) and has at least a partially convex lateral surface ([Fig. 6](#f6){ref-type="fig"}), in *Tongtianlong* it is robust (thick anteroposteriorly) with a flat lateral surface ([Fig. 4](#f4){ref-type="fig"}). In effect, the lacrimal shaft of *Tongtianlong* is plate-like, which is considered an autapomorphy of the taxon. In the region where the three processes meet, the lateral surface of the lacrimal is penetrated by a large opening (called the nasopharyngeal canal by Balanoff and Norell[@b43]) that leads into an internal recess, which is further subdivided internally. Anteroventral to this pneumatic opening is an ovoid fossa on the lateral surface of the lacrimal, which is probably also pneumatic in origin, and which may also invade the bone internally, although poor preservation makes this difficult to confirm. Complex pneumaticity in this region is common in oviraptorosaurs[@b6][@b43]). However, the pneumatic openings in *Tongtianlong* are much larger and more elaborate than the corresponding pneumaticity in the two Ganzhou oviraptorids with well-preserved cranial material, *Huanansaurus*[@b29] and *Banji*[@b26]. The postorbital is triradiate, with short anterior and posterior processes and a very long ventral process that projects anteroventrally, terminating at the floor of the orbit. The slender and elongate jugal is divided into three branches. The rod-like anterior process contacts the lacrimal and maxilla. The short ascending process extends posterodorsally to make up approximately half of the postorbital bar separating the orbit and lateral temporal fenestra. The posterior process contacts the quadratojugal underneath the lateral temporal fenestra. Here, the jugal overlaps the quadratojugal laterally, and the two bones are sutured but not fused. The quadratojugal is tightly appressed to the lateral surface of the quadrate, and it does not appear that the two bones could move relative to each other. There is, however, a small fenestra between the small dorsal process of the quadratojugal and the lateral margin of the quadrate. The dorsal part of quadrate is bent backwards, and there is an opening on the anterior surface of the quadrate which indicates that the bone is pneumatized. On the skull roof, the dorsal surface of the posterior portion of the nasal is smoothly convex. The left and right nasals are fused, without any sign of a suture between them. The lateral surface of the nasal is strongly concave, and although the surface is not well preserved, visible regions of original bone texture indicate extreme pneumaticity in this region, as is standard for derived oviraptorosaurs. The nasal-frontal suture is V-shaped in dorsal view. The frontals are short anteroposteriorly, and the left and right elements are not fused on the midline. The two parietals are fused to each other, but not to the frontals and there is no parietal crest (see also [Supplementary Information, Fig. S1b](#S1){ref-type="supplementary-material"}). The frontal-parietal suture is mostly straight, but there is a distinct process extending forwards from the middle of the anterior margin of the parietal ([Fig. 3d](#f3){ref-type="fig"}, pp). This process is wedged between the frontals. It is considered an autapomorphy of *Tongtianlong*, as it is absent in other oviraptorosaurs. Portions of the braincase are visible in lateral and posterior view. The supraoccipital is triangular, which a concave posterior surface. The exoccipital-opisthotic forms the dorsal margin of the foramen magnum, thus separating the supraoccipital from the foramen margin. The exoccipital tapers as it extends lateroventrally. The occipital condyle is larger than the foramen magnum, a condition that is seen in some caenagnathids (e.g., *Anzu*[@b41]), but differs from the proportionally smaller occipital condyles of other oviraptorids. The occipital condyle is located posterior to the articular condyles of the quadrate. The mandible is nearly complete. In lateral view, the ventral margin of the lower jaw is straight. The anterior end of the dentary is not as strongly downturned as in other derived oviraptorids. There is no depressed fossa on the lateral surface of the dentary immediately anterior to the external mandibular fenestra, and there are no articular grooves for the dentary on the ventrolateral edge of the angular and the dorsal surface of the surangular. The dentary contributes widely to the dorsal and ventral margins of the external mandibular fenestra, which is more anteroposteriorly elongated than the circular fenestrae of many other oviraptorids. The posterior part of the surangular is strongly concave laterally, and is pierced by a small opening. Postcranially, the neck is comprised of 11 cervical vertebrae. The first nine of these are preserved in natural articulation, with their dorsal surfaces exposed. In dorsal view the anterior-middle cervicals are roughly square shaped, as defined by lines drawn between the posterior margins of the postzygapophyses, the anterior margins of the prezygapophyses, and the lateral edge of the vertebra ([Fig. 3e](#f3){ref-type="fig"}). They become more rectangular in shape, longer than wide, more posteriorly in the neck. The neural spines are very small, as they are reduced to tiny peg-like projections at the center of the neural arches. The epipophyses are well developed in the second, third and fourth cervical vertebrae, but they become smaller in the middle cervicals and then disappear posterior to the sixth vertebra. There is a pneumatic opening (=pleurocoel) visible on the slightly exposed lateral centrum surface of the second cervical, but the lateral surfaces of the remaining cervicals are covered by matrix. The isolated posterior cervical vertebra, which is not in close articulation with the rest of the neck and therefore more widely exposed than the others, has a concave anterior articular surface of the centrum and a slightly convex posterior articular surface. The dorsal vertebrae were heavily damaged during collection, so few details of their morphology can be observed. The neural spines of the posterior dorsals are tall and slightly expanded anteroposteriorly. Some dorsal ribs are present on both sides of the specimen, none of which exhibit any pneumatic openings on their proximal ends. The sacrum is not well preserved, but the anterior neural spines are clearly unfused to each other and were closely appressed to the medial surface of the ilium in dorsal view. There appears to be a pneumatic foramen (=pleurocoel) on the final sacral vertebra, and the lateral ends of the fused transverse processes and sacral ribs are strongly expanded anteroposteriorly, with rounded dorsal surfaces. Part of the distal tail is missing, but there are at least 19 caudal vertebrae. The caudals are rectangular in dorsal view, with elongate transverse processes that extend laterally and slightly posteriorly. One laterally exposed anterior caudal has a small opening that appears to be pneumatic in nature. The haemal arches are very long. Portions of the shoulder girdles and proximal forearms are present on both sides of the specimen. The scapula is slender and curved medially. Its proximal end is expanded but not fused to the coracoid, the two bones forming an angle of approximately 130 degrees when in articulation. The coracoid is quadrangular in shape and has a large distally tapering posteroventral process, which extends slightly past the glenoid and is rounded at its end. The lateral surface of the coracoid is convex, the distinct biceps tubercle is located anterior to the glenoid, and the small and elongated coracoid foramen is positioned between the dorsal margin of the bone and the biceps tubercle. The medial surface of the coracoid is deeply concave and the coracoid foramen is expressed as a much larger, more circular opening than on the lateral surface. The thin sternum is a single element consisting of fused left and right components. It lacks a lateral xiphoid process and there is no groove for the coracoids along its anterior margin. The furcula is a broadly U-shaped, with a short ventral process on the midline and flattened distal ends ([Fig. 3b](#f3){ref-type="fig"}). The humerus has a long deltopectoral crest, which extends distally for nearly half the length of the shaft ([Fig. 3c](#f3){ref-type="fig"}). The shaft is slightly twisted as in *Heyuannia*[@b22] and *Nankangia*[@b9]. Part of the radius is preserved on the left side, but the ulnae and more distal forelimb elements are missing. Very few details of the pelvic girdle are apparent, due to damage that occurred during collecting. Parts of the ilium and pubis are present but little can be said of their morphology, although the preserved portions indicate that the pelvis is mesopubic and the distal ends of the left and right pubes are not fused together. The ischia are better preserved on the left side. The posterior margin of the shaft is deeply concave, the distal margin of the obturator process is straight, and the lateral surface of the bone is concave. The tibia is longer than the femur. It has a straight shaft, a well-developed cnemial crest, and an expanded distal end with a concave posterior surface. The astragalus is tightly appressed to the distal tibia. In posterior view, the ventral margin of the astragalus is concave dorsally, and in anterior view the ascending process is taller than wide. Two flattened distal tarsals are fused to each other and the proximal metatarsals. Distal tarsal III, which covers the proximal ends of metatarsals II and III, is larger than distal tarsal IV, which covers the proximal end of metatarsal IV. The left pes is partially preserved (See also [Supplementary Information Fig. S2](#S1){ref-type="supplementary-material"}). Metatarsal III is longer than metatarsal II, which is longer than metatarsal IV. Metatarsal III remains visible along the length of the metatarsaus, with only a slight constriction near its proximal end. Metatarsal V is short and rod-like with a pointed distal end. It is approximately 35% of the length of metatarsal V. The single visible pedal ungual is slightly curved. Phylogenetic analysis --------------------- *Tongtianlong* can clearly be assigned to Oviraptoridae based on numerous characters that are diagnostic of the clade (or proximal nodes within Oviraptorosauria), including: a pneumatic premaxilla; a medially inset subantorbital portion of the maxilla; fused nasals; a laterally projecting medial part of the lacrimal shaft that forms a flattened transverse bar in front of the eye; pneumatic skull roof bones; left and right iliac blades closely approaching or contacting each other on the midline[@b1]; and proximal caudals with pneumatized centra[@b44]. We added *Tongtianlong* to a modified version of the phylogenetic dataset of Lü *et al*.[@b29], which itself was an updated version of the dataset of Lamanna *et al*.[@b41]. We changed some characters that were previously multistate characters combining absence/presence and morphological differences into two separate characters, and also ordered multistate characters that describe a progressive sequence of size or morphological change. The data matrix now includes 43 taxa scored for 237 characters (see Methods and [Supplementary Information](#S1){ref-type="supplementary-material"}). The strict consensus of the 33,104 most parsimonious trees recovers *Tongtianlong* as deeply nested within Oviraptoridae (synapomorphies for Oviraptoridae and other major clades largely follow previous analyses of this dataset, and won't be repeated here) ([Fig. 7](#f7){ref-type="fig"}). *Tongtianlong* is the sister taxon to a sister-taxon pair of the Ganzhou oviraptorid *Banji*[@b26] and *Wulatelong* from the Campanian of Inner Mongolia[@b18]. The subclade comprised of these three taxa is united by three synapomorphies: the lack of a sagittal crest along the interparietal contact (character 30), a jugal process of the postorbital that extends far ventrally (character 36), and the presence of a surangular foramen (character 94). *Tongtianlong* is not recovered as a particularly close relative of any of the four other Ganzhou oviraptorids. Of these, *Nankangia* is placed within a polytomy as one of the most basal oviraptorids, *Huanansaurus* is recovered as an 'intermediate' grade oviraptorid that is outside of the clade consisting of *Tongtianlong* and more derived oviraptorids, and *Jiangxisaurus* and *Ganzhousaurus* are positioned as very highly nested oviraptorids, as successive outgroups to the specialized subclade centered on *Ingenia*. The phylogenetic separation between *Tongtianlong* and other Ganzhou oviraptorids provides further evidence for their generic separation. It is not outside of the realm of possibility, however, that future work on oviraptorosaur ontogeny may show that *Tongtianlong* is synonymous with another Ganzhou taxon. If this is the case, we suggest that *Banji* would be the most likely con-specific, as it is the most closely related to *Tongtianlong* and is based on a much smaller holotype that conceivably could belong to a juvenile[@b26]. With that said, we consider the phylogenetic separation of *Tongtianlong* and *Banji*, the possession of numerous autapomorphies in *Tongtianlong* that are not seen in *Banji*, and the many character differences between the holotypes of *Tongtianlong* and *Banji* to be strong evidence that the two are distinct taxa, based on our current understanding of oviraptorosaur ontogeny and morphology. Discussion ========== *Tongtianlong* is the sixth oviraptorosaurian taxon named from the Nanxiong Formation of the Ganzhou area of Jiangxi Province, southern China. All of these have been described over the past five years, and include: *Banji*[@b26], *Ganzhousaurus*[@b27], *Jiangxisaurus*[@b28], *Nankangia*[@b9], and *Huanansaurus*[@b29]. Additionally, another oviraptorosaur taxon is known from the Nanxiong Formation in neighboring Guangdong Province, *Shixinggia*[@b24]. Because of these discoveries, this part of southern China has rapidly become one of the best areas in the world for oviraptorosaur fossils, and therefore a keystone area for understanding the evolution of this highly aberrant group of bird-like feathered dinosaurs. The recent discoveries beg the question: why are so many oviraptorosaur taxa found in southern China? There are at least two possible explanations for the pattern, which are not mutually exclusive. First, it could be that the rush of recent discoveries has led to taxonomic over-inflation, and some of the specimens described as new species may be ontogenetic or sexually dimorphic forms of previously recognized species. Second, the Nanxiong Formation may be documenting a genuine radiation of oviraptorosaurs, an evolutionary event in which these small-to-mid-sized animals blossomed into many species and during the final few million years of the Age of Dinosaurs. Testing these two scenarios is currently difficult, but evidence is emerging that we argue favors the second scenario. Regarding the first explanation, it may be that some of the six named Ganzhou oviraptorids are based on holotypes that actually belong to one of the other species. There is no doubt that the six oviraptorosaurs are anatomically distinct from each other, as each can be diagnosed by autapomorphies and/or a unique combination of characters. The new taxon *Tongtianlong*, for example, has four unique features that are not seen in any other oviraptorosaur, three additional features that are not seen in any other oviraptorid, and numerous differences with the other five Ganzhou taxa. However, not all morphological differences must be due to taxonomic separation. Differences between specimens could be caused by ontogeny, sexual dimorphism, or random variation. If the differences between the Ganzhou oviraptorids are not entirely taxonomic in nature, we suggest that the most likely culprit is ontogeny, particularly because some of the specimens differ in size (e.g., the *Banji* holotype is considerably smaller than the *Tongtianlong* holotype). At the present time, all we can do is suggest that ontogenetic differences could potentially explain some of the variation seen in Ganzhou oviraptorids, but we cannot assess this with much certainty. Unfortunately, very little is known about how the anatomy of theropod dinosaurs changed during ontogeny, because most species are represented by very few fossils that fall far short of forming an ontogenetic sequence of hatchling to adult. The one exception is derived tyrannosaurids[@b45][@b46], but these theropods are drastically different from oviraptorosaurs in their phylogenetic position, body sizes, diets, and ecological habits. A better parallel may be herbivorous dinosaurs like ceratopsids and hadrosauroids, which although distantly related to oviraptorosaurs were similar in possessing often gaudy head crests and other cranial ornamentation. It is well known that the ornamentation in these dinosaurs changed dramatically during ontogeny[@b47][@b48][@b49][@b50][@b51][@b52]. Therefore, we would expect the head ornaments of oviraptorosaurs to change during growth, making it potentially very difficult to distinguish ontogenetic morphs from separate species in the absence of histological data (which is currently unavailable for the Ganzhou oviraptorids because of the logistical difficulty of destructive sampling) or extremely large sample sizes. We argue, however, that ontogeny probably does not explain most of the extreme variation seen among Ganzhou oviraptorids. Rather, we hypothesize that this variation is taxonomically informative. Although oviraptorosaur cranial ornaments probably did change during ontogeny, very few of the diagnostic characters of the Ganzhou oviraptorids are based on crest morphology. Most of the anatomical differences between species described as separate taxa concern the shapes and positions of cranial openings, the shapes and orientations of facial bones, and particularly features of the beak, lower jaw, and cranial muscle attachments that are likely related to feeding. For example, the new taxon *Tongtianlong* has a highly convex anterior premaxilla that is unique among oviraptorosaurs and differs from other Ganzhou species in presenting a prominent process on the posteroventral surface of the dentary symphysis, and a deep fossa on the lateral dentary and sagittal crest on the skull roof (both muscle attachment sites). Unless cranial musculature and feeding habits changed drastically during the lifetime of an individual oviraptorosaur, we currently hold that these differences among taxa are better explained by taxonomic separation (perhaps driven by feeding-related niche-partitioning, related to the peculiar but still poorly understood diets of oviraptorosaurs) rather than ontogeny. This hypothesis is bolstered by the recent discovery of very small 'baby' oviraptorids from central China, which already exhibit classic adult features of cranial fusion and deep lower jaws that are tied to large jaw muscles and strong bite forces[@b10]. These arguments lead us to conclude that the great diversity of named oviraptorids from the Nanxiong Formation of southern China is genuine. In other words, there really was a variety of different oviraptorid species in this area during the latest Cretaceous. This is not unprecedented: one of the very few well-sampled small theropod faunas, the Yixian Formation of northeastern China, exhibits a staggering diversity of small carnivorous and omnivorous dromaeosaurids[@b53][@b54][@b55][@b56]. It may be that a high diversity of small theropods was common in individual dinosaur faunas, but has gone unrecognized because of preservational bias against small dinosaur fossils[@b57][@b58]. With that said, it is also possible that many of the various Ganzhou oviraptorids did not actually live together. The Nanxiong Formation ranges from 600 to 7300 meters thick[@b35], is very poorly dated, and its stratigraphy has not yet been studied in detail, making it difficult to determine the relative stratigraphic positions of different oviraptorid specimens. We are very much still in the initial exponential phase of collecting in the Ganzhou region: the wealth of new fossil discoveries over the past five years is the direct result of a burst of construction activity in the region. Many areas remain to be explored, many fossils remain to be collected, and much work on the local geology is clearly needed. It may turn out that the Nanxiong Formation spans a long length of time and/or that the individual oviraptorid specimens are widely separated from each other stratigraphically. We suspect that the story of dinosaur evolution in the Nanxiong Formation may turn out to be similar to that in the Horseshoe Canyon and Dinosaur Park Formations of western Canada, two fossil-rich units that were also deposited in the latest Cretaceous. Initial exploration of these units in the early-mid 20^th^ century produced a fortune of dinosaur fossils, most notably numerous species of ceratopsids and hadrosauroids. As the geology of these formations became better understood and collecting was undertaken in a more rigorous manner, it became apparent that each formation spanned a few million years of time, and that the dinosaurs were not homogeneously distributed throughout the entire sequence[@b59][@b60]. Instead, unique and often short-lived assemblages of dinosaurs evolved, went extinct, and then were replaced by another assemblage. These formations record evolutionary radiations of dinosaurs: rapid evolution of many species, most likely enabled by dietary and ecological differences and possibly driven by environmental changes. We hypothesize that the Ganzhou oviraptorids underwent their own evolutionary radiation during the latest Cretaceous, in one of the final flurries of dinosaur evolution before the end-Cretaceous asteroid impact knocked out all non-avian species and ushered in the Age of Mammals. Methods ======= Phylogenetic analysis --------------------- To determine the phylogenetic position of *Tongtianlong* within Oviraptorosauria, we added this taxon to a modified version the phylogenetic dataset of Lü *et al*.[@b29]. This is an updated version of the dataset of Lamanna *et al*.[@b41], which includes a comprehensive sample of nearly all oviraptorosaurs scored for phylogenetically informative features of the skeleton. With the addition of *Tongtianlong*, the data matrix now includes 43 taxa (40 oviraptorosaurs plus *Herrerasaurus*, *Velociraptor*, and *Archaeopteyx* as outgroups) scored for 237 characters. We slightly modified some multistate characters and character ordering relative to Lamanna *et al*.[@b41], as explained in the supplement. We subjected the dataset to a maximum parsimony analysis in TNT v1.1[@b61]. We first conducted a 'new technology' search (with default parameters for sectorial search, ratchet, tree drift, and tree fusion), which recovered a minimum length tree in 10 replicates. This procedure aims to broadly sample tree space and identify individual tree islands. We then subjected the recovered most parsimonious trees (MPTs) to a traditional search with TBR branch swapping, which more fully explores the tree islands found in the 'new technology' search. This process returned a total of 33,104 MPTs of 566 steps (consistency index = 0.484, retention index = 0.676). Bremer values were used to assess clade support. Additional Information ====================== **How to cite this article**: Lü, J. *et al*. A Late Cretaceous diversification of Asian oviraptorid dinosaurs: evidence from a new species preserved in an unusual posture. *Sci. Rep.* **6**, 35780; doi: 10.1038/srep35780 (2016). **Publisher's note**: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Material {#S1} ====================== ###### Supplementary Information We thank Zhang Yuqing for preparing the specimens. JL was funded by the National Natural Science Foundation of China (grant no. 41272022; 41672019), the Fundamental Research Funds for the Chinese Academy of Geological Sciences (grant no.: JB1504); the China Geological Survey (grant no. 12120114026801), and the Erasmus Mundus EXPERTS SUSTAIN Programme (for travel to the University of Edinburgh to work with SLB). SLB was supported by a Marie Curie Career Integration Grant (EC 630652) and the University of Edinburgh. We thank G. Funston and an anonymous reviewer for helpful comments that improved the manuscript. **Author Contributions** J.L. designed the project. J.L., R.C., Y.Z. and C.S. organized the curation and preparation of the specimen and oversaw all research at the Dongyang Museum. J.L. performed the anatomical descriptive research. S.L.B. and J.L. performed the phylogenetic analyses. J.L. and S.L.B. wrote the manuscript. ![Map of the fossil locality near Ganzhou, Jiangxi Province, southern China.\ The solid five-pointed star represents the fossil site. Modified from Lü *et al*.[@b29].](srep35780-f1){#f1} {#f2} {#f3} {#f4} {#f5} ![Skull comparisons of oviraptorosaurs showing relative positions of the posterodorsal corner of the antorbital fenestra and the anteroventral corner of the external narial opening.\ (**a**) *Incisivosaurus gauthieri*; (**b**) *Conchoraptor gracilis*; (**c**) *Wulatelong gobiensis*; (no scale) (**d**) *Banji long*; (**e**) *Anzu wyliei*; (**f**) *Khaan mckennai*; (**g**) *Citipati osmolskae*; (no scale) (**h**) *Huanansaurus ganzhouensis* (reversed); (**i**) *Yulong mini*; (**j**) *Oviraptor philoceratops*; (**k**) *Nemegtomaia barsboldi*; l: "*Oviraptor*" *mongoliensis*; m: *Tongtianlong limosus* gen. et sp. nov. (**a**,**b**,**f**,**g**,**j**,**k**) and l are from Lü[@b23]; (**c**) is modified from Xu *et al*.[@b18]; (**d**) is modified from Xu and Han[@b26]; (**e**) is modified from Lamanna *et al*.[@b41] (reversed), (**i**) is from Lü *et al*.[@b10] and (**h**) is from Lü *et al*.[@b29]. External narial opening is in red, and antorbital fenenstra is in yellow. Note: The horizontal line projected through the posterodorsal corner of the antorbital fenestra is parallel to the line linking the articular end of the quadrate and the ventral margin of the premaxilla. Modified from Lü *et al*.[@b29].](srep35780-f6){#f6} {#f7}

INTRODUCTION {#sec1-1} ============ Salivary gland infections are uncommon in neonates; however, when they occur, they most commonly involve the parotid glands. Only 44 cases have been reported till now in the English literature in the past four decades.\[[@ref1]\] Seventy-seven percent of the neonates affected with neonatal suppurative parotitis (NSP) are male, and only 23% of the neonates require surgical drainage. The most common organism isolated is *Staphylococcus aureus*. The organisms reach the parotid gland via the Stensen\'s duct and less commonly through the blood. Intravenous antibiotic therapy is recommended for treatment, and surgical drainage is needed in only a few cases.\[[@ref2]\] We report a full-term breast-fed female neonate who developed an acute neonatal parotid abscess. CASE REPORT {#sec1-2} =========== A 15-day-old full-term, breast-fed, female neonate presented with a 3 days' history of irritability, fever, poor sucking and left preauricular swelling \[[Figure 1](#F1){ref-type="fig"}\]. She was born at full-term by normal vaginal delivery in an uneventful pregnancy, and her birth weight was 2950 g. On admission, the baby was irritable and dehydrated, and her weight was 2800 g and axillary temperature was 38.5°C. Examination revealed a toxic neonate with erythema and a hot and fluctuant swelling of the left parotid gland of size 5 cm × 5 cm. Pus exuded from the left Stensen\'s duct on applying pressure on the external surface. Systemic examination was otherwise unremarkable. Aspiration of the swelling revealed thick pus. {#F1} Laboratory tests revealed haemoglobin 13 g/dl, white blood cells 17.6 × 10^9^/L, urea 4.5 mg/dl, sodium 142 mmol/dl, potassium 5.2 mmol/dl and chloride 109 mmol/dl. Ultrasound of the parotid glands demonstrated an enlarged left parotid gland with hypoechoic areas with a few pockets of thick pus which is suggestive of acute suppurative parotitis \[[Figure 2](#F2){ref-type="fig"}\]. {#F2} Rehydration of the baby was done with intravenous fluid and was started on intravenous cefotaxime (100 mg/kg/day) at the time of admission. The parotid abscess was surgically drained \[[Figure 3](#F3){ref-type="fig"}\], and the patient showed gradual improvement. The pus culture showed methicillin-resistant *S. aureus*. In spite of gradual improvement, the antibiotic treatment was switched to intravenous vancomycin (40 mg/kg/day) according to the pus culture. The treatment was continued for 10 days with complete recovery. On follow-up examination, there was no residue or abnormality of the gland, and she did not show chronic recurrent parotitis. {#F3} DISCUSSION {#sec1-3} ========== NSP is a rare disease. In infants, infection of the parotid glands is more common than infection of the sub-mandibular glands.\[[@ref3][@ref4]\] Spiegel *et al*. and Ismail *et al*. reviewed the cases of patients with NSP during the past five decades, mostly from case reports.\[[@ref1]\] All neonates showed parotid gland swelling with varying degrees of erythema, warmth and tenderness. One of the risk factors for NSP is considered to be insufficient breast-feeding. Other risk factors such as pre-maturity, environmental hot weather, excessive oral suctioning nasogastric tube feeding, maternal breast abscess in a breast-fed infant,\[[@ref6]\] cytomegalovirus parotitis and maternal treatment with methyldopa have been linked to NSP in the recent case reports. Seventy-seven per cent of the NSP are unilateral and common in male.\[[@ref5]\] Pus exudes from the Stensen\'s duct on application of pressure externally to the affected gland in most of the cases.\[[@ref7][@ref8]\] The most common organism causing NSP is *S. aureus* followed by Gram-positive, Gram-negative and rarely anaerobic organisms.\[[@ref5]\] In NSP, infection of parotid glands most commonly occurs by retrograde spread of the organisms from the oral cavity through the Stensen\'s duct and rarely by haematogenous spread.\[[@ref3]\] Infection of the parotid gland may be initiated by dehydration leading to precipitation of the mucous or stone formation in the Stensen\'s duct.\[[@ref1]\] NSP commonly presents with fever, erythema and swelling in the preauricular region. The infection may be bilateral. Purulent drainage from Stensen\'s duct is pathognomonic of this condition. Leucocytosis above 15 × 10^9^/L with neutrophil predominance was found in 71% of the cases, and the erythrocyte sedimentation rate was elevated in only 20% of the patients.\[[@ref1]\] In our patient, leucocyte counts are elevated with neutrophil predominance. Laboratory findings have been non-specific and not very helpful in the diagnosis of NSP. Ultrasonography of the parotid gland usually demonstrates enlarged parotid gland with oedema, increased vascularity and hypoechoic areas.\[[@ref3]\] Neonatal suppurative parotitis resolves with antibiotic treatment in majority of cases. The empirical antibiotics used in NSP are a combination of anti-staphylococcal agent and an aminoglycoside, or a third-generation cephalosporin along with clindamycin or a similar medication to cover possible anaerobic infection, are good initial choices until the pus culture reports are available. After starting antibiotics, fever usually settles down within 24 h and the swelling decreases within 3--5 days. Surgical drainage is needed in only a few cases (23%),\[[@ref1]\] where there is a delay in seeking medical attention, or the organism is resistant to the empirical antibiotic therapy. Facial palsy, salivary fistula, mediastinitis resulting from pus tracking down the carotid sheath and rupture into the external auditory meatus are the complications of NSP, but these are uncommon due to the prompt initiation of antibiotic therapy.\[[@ref3][@ref9]\] CONCLUSION {#sec1-5} ========== Although acute NSP is a rare disease, it should be suspected in all the patients presenting with unilateral or bilateral preauricular swelling with redness and fever. Medical treatment with antibiotics may usually suffice in most of the cases to resolve the infection, but in a few cases, surgical drainage may be needed. Declaration of patient consent {#sec2-1} ------------------------------ The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship {#sec2-2} --------------------------------- Nil. Conflicts of interest {#sec2-3} --------------------- There are no conflicts of interest.

{#sp1 .539} {#sp2 .540} {#sp3 .541} {#sp4 .542} {#sp5 .543} {#sp6 .544} {#sp7 .545}

Introduction {#s1} ============ Expanding capacity to deal with the HIV epidemic is a formidable task in low- and middle-income countries given the scale of the epidemic and the limited public health infrastructure. While much has been achieved to make antiretroviral therapy (ART) affordable, access to care is still inadequate. According to the latest UNAIDS report, only 46% of those who were in need had started ART by the end of 2010 in low- to middle-income countries [@pone.0053570-WHO1]. One way to expand access to ART and improve retention within ART care for public sector patients is to utilize the private sector. In many low- and middle-income countries a high proportion of doctors work in the private sector [@pone.0053570-Dreesch1]. Contracting private doctors to initiate ART and follow up public sector patients in their private rooms according to the public sector guidelines has been successfully implemented in Botswana [@pone.0053570-Dreesch1] and other developing country settings [@pone.0053570-WHO2]. However, there are concerns about the ability and willingness of individual private doctors to implement the public health approach to ART management, and about high costs in the for-profit private sector. To date there have been no published comparisons of clinical and economic outcomes of the provision of ART care to public patients between the private sector and public sector. In addition to the debates about public versus private ART care, there are also questions about how frequently patients should be followed up, and by whom. In the earlier years of ART provision, patients were required to attend facilities for regular consultations with doctors or nurses [@pone.0053570-Cleary1]. More recently, however, there has been a move towards less frequent follow-up, and towards task shifting from doctors to nurses, and from nurses to counselors [@pone.0053570-Cleary2]. It is however unclear whether this changing intensity in follow-up will impact negatively on patient adherence and outcomes. We assessed the costs and outcomes of providing ART care for public patients in the private versus public sector in two South African ART programs where no co-payment from patients was required: a grant-funded program providing care for public patients in private practices and a public-sector program providing care for public patients in public sector community clinics. We utilized a newly developed Markov-model, which addresses many of the limitations of existing models [@pone.0053570-Leisegang1]. Methods {#s2} ======= Study design {#s2a} ------------ We assessed the costs and outcomes of ART provision in the private-care and public-care models to provide care to public sector dependent patients. We took the provider\'s perspective and only included ART-related costs: antiretroviral drugs, CD4+ cell count (CD4) and viral load (VL) monitoring, toxicity laboratory monitoring, and public clinic or private general practitioner (GP) visits. We used Markov modeling to extrapolate primary data in order to estimate results over 10 years and lifetime for costs, rates of loss to follow-up and life years. Zero and three percent annual discount rates were used. The model was developed using data from the public-care cohort, and validated externally using data from the private-care cohort. Uncertainty was assessed using multi-way and probabilistic sensitivity analyses. Study setting {#s2b} ------------- ART care for patients in both programs followed the 2003 South African national guidelines, which were based on the 2003 World Health Organization guidelines for resource-limited settings [@pone.0053570-Organisation1]. Patients were eligible for ART when they met the following criteria: either a CD4 below 200 cells/µL or a WHO stage 4 illness (other than extra-pulmonary tuberculosis) irrespective of the CD4 count. The first line ART regimen consisted of two nucleoside reverse transcriptase inhibitors (NRTI), zidovudine (ZDV) or stavudine (D4T) with lamivudine (3TC), with a non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine (NVP) or efavirenz (EFV). Viral load and CD4 counts were monitored 6 monthly. Patients with confirmed virologic failure (two consecutive viral loads \> = 5000 copies/ml) in spite of enhanced adherence promotion, were switched to a second line regimen of two NRTIs, ZDV and didanosine (DDI), in combination with a boosted protease inhibitor, lopinavir/ritonavir (LPV/r). Safety monitoring was limited to serum alanine aminotransferase (ALT), complete blood count, and lipogram for patients on NVP, ZDV, and LPV/r respectively. Cohort Description {#s2c} ------------------ The public-care cohort was the Khayelitsha HIV treatment program, which is a public sector program operating in an urban area in Cape Town, South Africa. The program is jointly funded by the state and a donor, Medecins Sans Frontieres. ART care was provided at three primary care clinics. ART was initiated by doctors but routine follow up was largely done by nurses. The clinics operated on a queue system and therefore patients would spend between 1--4 hours at the clinic. Counselors and peer-educators played an important role in educating and encouraging patients while they waited to see clinical staff. Most patients returned to the clinic every month to collect medicines, attend group or individual counseling sessions, and/or for clinical assessments. We included data from the inception of the program on 15 January 2000 until 25 Jan 2008. The private-care cohort was the BroadReach Healthcare program, a donor-funded (President\'s Emergency Plan for AIDS Relief (PEPFAR)) managed-care ART program. Patients were recruited into the program at several urban and rural public sector clinics in the Mpumulanga, Eastern Cape and Kwazulu-Natal provinces in South Africa. ART care was provided by local contracted general practitioners (GPs) in their private practices on an appointment basis and visit frequency was pre-specified. The private doctors had to successfully complete internet-based training on the national ART guidelines before they could enroll patients. Telephonic counseling support for the patients and clinical guidance for the doctors was provided by Aid for AIDS, a private sector disease management program. Patients collected their medication from the doctors\' rooms monthly, but clinical consultations were performed less frequently. We included data from the inception of the program on 1 May 2005 until 31 July 2010. New patient enrollment was stopped in March 2008. In both cohorts severely ill or complicated patients were referred to secondary level public sector hospitals for further management and then re-integrated back into the program once their condition had stabilized. Data were entered prospectively into databases. Deaths were ascertained by several mechanisms: (1) clinic staff or private practice practitioners who learnt of a death from family members or friends, would either complete a specific form and fax it to a central office or capture it on a computer-based system onsite; (2) staff and program administrators identified patients who had missed several appointments and contacted a family member or treatment supporter of the patient to determine whether the patient was deceased and if so the date of death; and (3) the patient\'s South African identity number, where available, was used to cross-reference the South African national death register to establish whether a death was recorded. We included adult patients (19 years and older) who started first line ART within the programs and had a baseline CD4 count below 200 cells/µL. The study intervals differed somewhat for each cohort, although the median year of starting ART was 2005 in both cohorts. A patient\'s follow-up period was truncated on the date they either: transferred out of the program, died, on the study end date, or on the last date seen if they were not seen within six months of the end of the study period and their identity number was not available (and we were therefore unable to ascertain whether they had died). Healthcare utilisation and cost data {#s2d} ------------------------------------ GP or clinic utilisation was determined from the electronic database records for both cohorts. The cost in South Africa Rands (ZAR) for a public-sector clinic visit was determined from a previously published estimate [@pone.0053570-Cleary1]. In that study, the unit clinic visit costs included time allocations for nurses, doctors, and counselors, and this has changed in more recent times due to increased task shifting. Together with improved economies of scale and learning by doing, cost would have fallen substantially had it not been for substantial increases in doctor\'s salaries over the same period. We therefore decided to only use the consumer price index table [@pone.0053570-Consumer1] to inflate costs to April 2010 levels. Private GP visit costs were determined from contracted rates in April 2010. Drug utilisation was divided into first line (2NRTIs and NNRTI) and second line (2NRTIs and PI) therapy, and the average utilisation of each drug was determined within each line of therapy. Because estimates of ARV drug utilization were not available within our dataset, we conservatively assumed that all patients had received their ARVs each month and therefore allocated full monthly ARV drug costs within the ART model. ARV drug costs were set at the public sector tender prices for April 2010. There was some under-reporting of CD4 and VL monitoring, and ARV laboratory toxicity monitoring was not recorded in both programs. We conservatively assumed all patients underwent laboratory monitoring as per the South African public-sector guidelines. The guidelines recommended six monthly CD4 and VL monitoring. Laboratory toxicity monitoring, which occurred predominant in the first six months on ART, was limited to ZDV, NVP, and LPV/r. We scaled the specific toxicity monitoring utilisation associated with a specific ARV drug in accordance with its relative proportion within the two regimen lines. All laboratory costs were set at the public sector tender prices for April 2010. All costs were converted from ZAR to United States Dollars (USD) in April 2010 (7.34 ZAR per USD). The Markov model framework, development and uncertainty analysis {#s2e} ---------------------------------------------------------------- WHO stage, current CD4, and current VL were identified as key determinants of lifetime costs and outcomes [@pone.0053570-Kranzer1]. Many patients categorized as "LTFU" in studies return to ART care and therefore are not truly LTFU [@pone.0053570-Ndiaye1]. This is important as: (a) ART-related resources are not consumed while a patient is LTFU, (b) the CD4 count falls rapidly to pre-ART levels in patients who interrupt ART [@pone.0053570-Ananworanich1], (c) additional resources are consumed in patients restarting ART [@pone.0053570-Kranzer1], (d) treatment interruptions increase resistance to first line regimens [@pone.0053570-Kranzer1] [@pone.0053570-Kranzer1] [@pone.0053570-Kranzer1] [@pone.0053570-Kranzer1] [@pone.0053570-Kranzer1](9)(9)(8), and (e) treatment interruptions increase deaths [@pone.0053570-Kranzer1] and attenuate CD4 recovery [@pone.0053570-Kaufmann1]. We based the structure of the Markov model on these determinants of costs and outcomes as well as on our own analysis of the public-care program -- the larger of the two cohorts. We implemented this Markov model in Treeage 2009 [@pone.0053570-TreeageSoftware1] and populated it with parameter estimates derived in Stata 11 [@pone.0053570-Analysis1] using survival models for time-to-event analyses and generalized linear models for clinic/GP utilisation. We evaluated the model fit and adjusted the model design where appropriate. Then, using the data from the private-care program, we derived new parameter estimates and evaluated the ability of the model to predict outcomes and costs. This procedure allowed us to assess the external validity of the model [@pone.0053570-Cleary1], [@pone.0053570-Sendi1]. The model was run for two durations: 10 years and until all members of each cohort were dead (i.e. lifetime duration). Finally, we conducted probabilistic sensitivity analysis to assess uncertainty. This entailed specifying distributions on utilization and outcome parameters, where possible and propagating uncertainty through the model by way of first and second order Monte Carlo simulations. The models were run using a 1 month cycle length [@pone.0053570-Badri1], [@pone.0053570-Freedberg1]. The Markov Model {#s2f} ---------------- The overall Markov model was divided into two parts: an ART model and a LTFU model (see [figure 1](#pone-0053570-g001){ref-type="fig"}). All patients started in the ART model, and remained there until they either died or became LTFU. Healthcare utilisation and mortality has been shown to be significantly higher in the first 6 months on ART [@pone.0053570-Cleary1], [@pone.0053570-Leisegang1]. Therefore the ART model was divided into two phases: 0--6 months on starting or restarting ART and \>6 months on ART. We defined LTFU as defaulting ART for more than 6 months. Patients entering the LTFU model remained there until they either died or restarted ART. We used parametric survival analysis with an exponential distribution to determine the transition probabilities to outcomes (death, LTFU, CD4 category change, and VL category change), and generalized regression models to determine utilisation (GP and clinic visits) within the Markov states. Covariates included time on ART, on-ART CD4 category, on-ART VL category, and year of starting ART (normalizing findings to 2005). We assumed that non-HIV related deaths of a typical individual (34 years) were included in the recorded deaths. We modeled the increasing relative contribution of non-HIV related deaths over time using the mortality curves for South Africa (less the typical mortality for a 34 year old adult) before the onset of South Africa\'s HIV epidemic (prior to 1990). {#pone-0053570-g001} In the first 6 months after starting or restarting ART, patients were split according to their pre-ART CD4 count category (0--49 or 50--199 cells/µL), and remained within this CD4 category for 6 months. At the end of 6 months, the remaining patients (i.e. not LTFU or dead) were distributed into the Markov states of the \>6 months on ART model using a competing risks regression model with the pre-ART CD4 category as the only covariate. The \>6 months on ART phase was defined by fifteen Markov states. These included: five on-ART CD4 categories (0--49, 5--199, 200--349, 350--499, and ≥500 cells/µL) and three on-ART VL categories (\<1,000; 1,000--99,999; and ≥100,000 copies/mL). Within each Markov cycle, we limited transitions between these Markov states to either a CD4 or VL category change but not both, as this reduced model complexity. We distributed patients entering the LTFU model into the two pre-ART CD4 categories (0--49 and 50--199 cells/µL) with the relative proportions being derived from the observed data. Given the limited LTFU data within our cohorts, we used the transition probability from the higher to the lower pre-ART CD4 category on a previously published natural history HIV model [@pone.0053570-Cleary1], and adapted the transition probabilities from these CD4 categories to death to match the observed trends in deaths within our cohorts. We used a regression model to determine the transition probability of restarting ART for patients LTFU, with time since first starting ART as the covariate. The transition probability from first line to second line ART was determined separately within the two phases of the ART model and the covariates for the regression model included pre-ART CD4 category, on-ART VL category, on-ART CD4 category, and time since starting ART. Within the second line ART model all transition probabilities were the same as the first line ART model, but the ARV drug utilisation and therefore costs differed. Patients within the LTFU model were assigned no ART-related utilisation and therefore no costs. Uncertainty analysis {#s2g} -------------------- We assessed the uncertainty in the data and model design using probabilistic sensitivity analysis (first and second-order Monte Carlo simulations). First-order simulations were used to capture the variability in the simulated population and tracked the varying paths taken by patients moving through the model in order. Second-order simulations were used to capture the variability in the parameter estimates by randomly sampling from the triangular-shaped distribution for the parameter, which approximated the 95% confidence interval. We ran 1,000 second-order and 10,000 first-order simulations to determine the 95% uncertainty intervals around the lifetime costs and outcomes. We assessed uncertainty related to extrapolation of the data and the generalizability of the model in three ways: (1) we externally validated the model derived from public-care cohort using the private-care cohort dataset, (2) we extrapolated our estimates over 10 year and life-time durations and compared the results, and (3) we compared our outcomes and cost estimates with other published studies. Finally, we assessed the uncertainty related to analytical methods by comparing the findings with 0% and 3% annual discounting of costs and outcomes. Scenario analysis {#s2h} ----------------- Clinic visit utilisation within the public-care program was intensive due to a policy decision by the program managers that all patients should be seen by a nurse or doctor every one to two months. In more recent years, the clinic visit utilisation has been substantially reduced to accommodate the growing number of patients. We therefore explored the impact of reduced clinic visit utilisation within the public-care program on the overall results. Ethics statement {#s2i} ---------------- The study was approved by the Research Ethics Committee, University of Cape Town. All patients signed consent for their information to be entered into the central databases and analysed. Anonymity was ensured using generated identifiers and all personal data were deleted from the datasets. Results {#s3} ======= Cohorts {#s3a} ------- The characteristics and overall outcomes of the study cohorts are described in [Table 1](#pone-0053570-t001){ref-type="table"}. We included 6372 and 963 patients from the public-care and private-care programs respectively. Median follow-up time on ART was shorter in the public-care cohort. No patients were transferred out to other facilities from the private-care program. The model fit diagnostics for both the private-care and public-care programs are shown in [figures S1](#pone.0053570.s001){ref-type="supplementary-material"} and [S2](#pone.0053570.s002){ref-type="supplementary-material"} respectively. These include current CD4, current VL, line of therapy and status (current, LTFU or dead). 10.1371/journal.pone.0053570.t001 ###### Cohort characteristics. {#pone-0053570-t001-1} Characteristic Khayelitsha Broadreach ------------------------------- ---------------- ---------------- Numbers 6372 963 Age baseline (years) Median 33 34,9 IQR (28,7 to 39,3) (30,4 to 41,9) Sex (%) Female 67,7 68,3 CD4 count (cells/µl) baseline Median 99 92 IQR (44 to 161) (44 to 146) Unknown 435 3 Viral load (log~10~) baseline Median 5,1 5,1 IQR (4,6 to 5,6) (4,7 to 5,6) Unknown 2941 241 Follow-up duration (months) Median 21,3 54,6 IQR (11,7 to 33,4) (29 to 57,8) Status at end of study (%) Current 77,5 72,2 Transferred 6,3 0 LTFU 5,5 7,8 Deceased 10,6 20 Health care utilization and unit costs in Markov states {#s3b} ------------------------------------------------------- Over the study period, 212,175 clinic visits in the public-care cohort and 10,477 GP visits in the private-care cohort were recorded. The contracted rate for a GP visit was 31.04 USD and the estimated cost of 24.53 USD for a clinic visit was derived by inflating the cost estimate from a previous publication [@pone.0053570-Cleary1]. The average monthly GP/clinic utilisation (with 95% confidence intervals) and the cost estimates are shown in [table S1](#pone.0053570.s004){ref-type="supplementary-material"}. Within both cohorts, utilisation was highest in patients restarting ART and, to a lesser extent, during the 0--6 months after starting ART, compared with the \>6 months on ART phase. In this latter phase, monthly visit utilisation was lower in both cohorts. Importantly, the public-care cohort had approximately 2 to 4 times higher visit utilisation within the \>6 months on ART phase compared with the private-care cohort. The South African public sector guidelines were used for laboratory utilisation -- the costs and utilisation are shown in table S2. CD4 and VL were taken 6 monthly, whilst other laboratory utilisation related to toxicity monitoring depended on the specific antiretroviral drugs and was higher in the first 6 months on ART. The utilisation of individual drugs within the first and second line ART regimens, the ART-related costs, and the hazard coefficients and transition probabilities for the model describing the transition between first and second line ART are shown in [table S3](#pone.0053570.s006){ref-type="supplementary-material"} and [figure S3](#pone.0053570.s003){ref-type="supplementary-material"}. We assumed 100% utilisation of both ARV drugs and laboratory tests while within the ART model. The public-care cohort had higher zidovudine but lower efavirenz utilisation in the first line ART regimen. The public-care cohort had higher didanosine utilisation in the second line ART regimen. The transition probability of moving to second line ART was lowest in the 0--6 months after starting ART and highest in the 0--6 months after restarting ART. In the \>6 month on ART phase, the transition probability of moving to second line ART decreased with lower VL and higher CD4 categories respectively, increased with time on ART and plateaued at about 3 years. The transition probabilities to second line ART were generally lower in the private-care cohort. The estimated distribution of time between first and second line ART was 61% and 39% in the public-care cohort versus 66% and 34% in the private-care cohort. Effectiveness {#s3c} ------------- The transition probabilities for the CD4 and VL models on ART are shown in [table S4](#pone.0053570.s007){ref-type="supplementary-material"}. The baseline CD4 category distribution for patients starting ART was similar in both cohorts: 30% in the 0--49 cells/µL category and 70% in the 50--199 cells/µL category. A lower baseline CD4 category was associated with a lower CD4 category distribution after 6 months on ART, but lower baseline CD4 category did not impact on the VL distribution. Public-care patients were more likely than private-care patients to have VL \<1000 copies/ml (92% versus 87%) and CD4 counts ≥200 cells/µL (64% versus 42%) after the first 6 months on ART. This trend was similar for patients restarting ART, but the outcomes were worse: 61% and 43% had VL\<1000 copies/ml, and 49% and 63% had CD4 counts \<200 cells/µL for patients in the public-care and private-care cohorts respectively. The transition probabilities and hazard coefficients for deaths on ART are shown in [table 2](#pone-0053570-t002){ref-type="table"}. The transition probability to death was highest in the first 3 months on ART and in patients with a low pre-ART CD4 category. The transition probability to death was lowest for the first 6 months after restarting ART. For patients in the \>6 months on ART phase, the transition probability to death decreased with lower VL category, higher CD4 category, and time on ART (using a Gompertz time function). The median of the Gompertz time function was 20 months in both cohorts, but the scaling constant was higher in the private-care cohort (1.19 versus 1.04). Thus there were more early deaths in the private-care cohort. 10.1371/journal.pone.0053570.t002 ###### Transition probabilities and hazard coefficients for deaths on antiretroviral therapy. {#pone-0053570-t002-2} Variables Transition probabilities and hazard coefficients (95% CI) per 1 month cycle ------------------------------------------------------------ ----------------------------- ----------------------------------------------------------------------------- ------------------------ **First 6 months after starting antiretroviral therapy** **Transition probability** 3 months CD4 0--49 cells/µL 0,035 (0,029 to 0,044) 0,040 (0,029 to 0,056) 3 months CD4 50--199 cells/µL 0,010 (0,008 to 0,012) 0,017 (0,013 to 0,022) 6 months CD4 0--49 cells/µL 0,011 (0,010 to 0,014) 0,027 (0,021 to 0,036) 6 months CD4 50--199 cells/µL 0,003 (0,003 to 0,004) 0,011 (0,009 to 0,014) **First 6 months after restarting antiretroviral therapy** Transition probability: 0--6 months 0,008 (0,004 to 0,016) 0,004 (0,001 to 0,010) **\>6 months on antiretroviral therapy** **Hazard coefficient due to CD4 and VL** CD4 0--49 cells/µL VL \<1,000 copies/ml −5,01 −5,03 CD4 0--49 cells/µL VL 1,000--100,000 copies/ml −4,71 −4,69 CD4 0--49 cells/µL VL \>100,000 copies/ml −3,83 −4,13 CD4 50--199 cells/µL VL \<1,000 copies/ml −6,00 −6,5 CD4 50--199 cells/µL VL 1,000--100,000 copies/ml −5,69 −6,16 CD4 50--199 cells/µL VL \<1000 copies/ml −4,82 −5,6 CD4 200--349 cells/µL VL \>100,000 copies/ml −7,25 −7,48 CD4 200--349 cells/µL VL 1,000--100,000 copies/ml −6,94 −7,14 CD4 200--349 cells/µL VL \<1000 copies/ml −6,07 −6,58 CD4 350--499 cells/µL VL \>100,000 copies/ml −7,63 −8,53 CD4 350--499 cells/µL VL 1,000--100,000 copies/ml −7,32 −8,19 CD4 350--499 cells/µL VL \>100,000 copies/ml −6,45 −7,63 CD4 ≥500 cells/µL VL \<1,000 copies/ml −7,76 −8,16 CD4 ≥500 cells/µL VL 1,000--100,000 copies/ml −7,46 −7,82 CD4 ≥500 cells/µL VL \>100,000 copies/ml −6,58 −7,26 **Hazard coefficients for Gompertz function** alpha 0,93 (0,52 to 1,34) 1,73 (1,17 to 2,28) beta -- half-life (months) 20 20 The hazard coefficients and transition probabilities related to the LTFU model are shown in [table 3](#pone-0053570-t003){ref-type="table"}. The transition probability from ART to LTFU was lowest in the first 6 months after starting ART and highest in the first 6 months after restarting ART. Thereafter, the transition probability from ART to LTFU increased with higher VL category, lower CD4 category, and time on ART. We modeled the effect of time on ART by adapting the Gompertz function so that it plateaued. The median of the adapted Gompertz function was longer (12 months versus 8) and the scaling constant has higher (1.5 versus 0.5) in the public-care compared with the private-care cohort. We distributed patients entering the LTFU model as follows based on our analysis of the data: 30% to the 0--49 cells/µL and 70% to the 50--199 cells/µL CD4 categories. The transition probability from LTFU to restarting ART was higher in the private-care cohort (26% versus 13%) and independent of LTFU CD4 category. 10.1371/journal.pone.0053570.t003 ###### Transition probabilities and hazard coefficients related to loss to follow-up. {#pone-0053570-t003-3} Variables Transition probabilities and hazard coefficients (95%) per 1 month cycle -------------------------------------------------------------- -------------------------------------------------------------------------- --------------------------- --------------------------- **Transitions within ART model** **Transition probability to LTFU within 0--6 months on ART** On starting ART 0,0085 (0,0080 to 0,0091) 0,0006 (0,0006 to 0,0006) On restarting ART 0,0270 (0,0205 to 0,0356) 0,0251 (0,0251 to 0,0251) **Hazard coefficient to LTFU within \>6 months on ART** CD4 0--49 cells/µL VL \<1,000 copies/ml −4,7 −5,13 CD4 0--49 cells/µL VL 1,000--100,000 copies/ml −3,79 −4,16 CD4 0--49 cells/µL VL \>100,000 copies/ml −4,00 −4,37 CD4 50--199 cells/µL VL \<1,000 copies/ml −5,31 −5,44 CD4 50--199 cells/µL VL 1,000--100,000 copies/ml −4,4 −4,47 CD4 50--199 cells/µL VL \<1000 copies/ml −4,61 −4,68 CD4 200--349 cells/µL VL \>100,000 copies/ml −5,73 −4,52 CD4 200--349 cells/µL VL 1,000--100,000 copies/ml −4,82 −3,56 CD4 200--349 cells/µL VL \<1000 copies/ml −5,03 −3,76 CD4 350--499 cells/µL VL \>100,000 copies/ml −5,73 −4,52 CD4 350--499 cells/µL VL 1,000--100,000 copies/ml −4,82 −3,56 CD4 350--499 cells/µL VL \>100,000 copies/ml −5,03 −3,76 CD4 ≥500 cells/µL VL \<1,000 copies/ml −5,73 −4,52 CD4 ≥500 cells/µL VL 1,000--100,000 copies/ml −4,82 −3,56 CD4 ≥500 cells/µL VL \>100,000 copies/ml −5,03 −3,76 **Hazard coefficients for Gompertz function** alpha 1,5 0,5 beta -- half-life (months) 12 8 **Initial distribution within LTFU model** CD4 0--49 cells/µL 0,278 (0,255 to 0,302) 0,243 (0,217 to 0,269) CD4 50--199 cells/µL 0,722 (0,745 to 0,698) 0,757 (0,783 to 0,731) **Transitions within LTFU model** **Transition probability between CD4 category** CD4 50--199 to CD4 0--49 cells/µL 0,005 (0,005 to 0,005) 0,006 (0,006 to 0,006) **Transition probability back to ART** CD4 0--199 cells/µL 0,134 (0,128 to 0,141) 0,146 (0,139 to 0,154) **Transition probability to death** CD4 0--49 cells/µL 0,006 (0,005 to 0,008) 0,006 (0,005 to 0,008) CD4 50--199 cells/µL 0,001 (0,001 to 0,017) 0,001 (0,001 to 0,017) The highest death rates were observed within the first year on ART for both cohorts, especially in the private-care cohort: 8% and 15% had died by 12 months and 32% and 39% had died by 120 months in the public-care and private-care cohorts respectively. The distribution of VL categories stabilized by 3 years to 90% and 85% of patients having a VL \<1000 copies/ml within public and private-care cohorts respectively. The distribution of CD4 categories was more dynamic over time and the private -care cohort fared better with 50% versus 40% of patients having a CD4 ≥500 cells/µL by 10 years. The percentage of patients who were alive and still on ART stabilized at approximately 80% for both cohorts, although the private-care cohort achieved this earlier due to generally higher transition probabilities to and from LTFU. Ten**-**year and lifetime costs, outcomes, probabilistic sensitivity and scenario analysis {#s3d} ------------------------------------------------------------------------------------------ We ran Monte Carlo simulations for 10 years and until everyone had died to generate lifetime costs and outcomes together with their 95% confidence intervals, as shown in [table 4](#pone-0053570-t004){ref-type="table"}. The conclusions we derived from the 10 year and lifetime estimates (with and without discounting) were congruent: the private-care program was approximately as effective, but was less costly than the public-care program. These reduced costs were predominantly driven by the lower level of utilisation in the private-care program. Given that the outcomes between the two programs were not significantly different, this finding suggests that reduced visit utilization has the potential to be cost saving (reducing costs without impacting on patient outcomes). 10.1371/journal.pone.0053570.t004 ###### 10 year and lifetime estimates of cost and outcomes of the private-care and public-care programs. {#pone-0053570-t004-4} Treatment option 10 year estimates Lifetime estimates ------------------ ------------------------ -------------------- --------------------------- --------------------- **Undiscounted** Public-care 8,825 (8,614 to 9,036) 7.6 (7.4 to 7.8) 18,734 (17,385 to 20,083) 14.1 (13.2 to 15.0) Private-care 6,187 (5,997 to 6,377) 7.2 (7.0 to 7.4) 13,062 (12,077 to 14,047) 14.0 (13.1 to 14.8) **Discounted** Public-care 7,688 (7,513 to 7,863) 6.7 (6.5 to 6.8) 13,305 (12,588 to 14,022) 10.4 (9.9 to 10.9) Private-care 5,407 (5,250 to 5,564) 6.3 (6.2 to 6.5) 9,273 (8,704 to 9,842) 10.0 (9.4 to 10.5) When we reduced the frequency of clinic visits in the \>6 months on ART phase by two-thirds in the public-care program (in line with the changes introduced in late 2011 by the program administrators), the estimated 10-year and lifetime costs within the public-care program approximated the levels observed in the private-care program. In other words, the programs were equivalent in terms of costs and outcomes. Discussion {#s4} ========== We determined that the private-care program had lower costs and similar outcomes to the public-care program at the time of the study using a novel Markov model. Key differences between the programs were less frequent visits and higher rates of returning to care after loss to follow-up in the private-care program, and lower early death rates on ART, but more deaths while LTFU in the public-care program. We estimated that the recent shifts towards less frequent visits in the public-care ART program would achieve large cost savings, making the costs of the two programs similar. These findings suggest that properly managed private-care programs can ease the burden of ART care in endemic countries by looking after public sector patients without increasing costs. Further, reducing clinic visits may be a viable strategy to save costs while maintaining outcomes in public sector programs. Our Markov model included several significant improvements on previously published models [@pone.0053570-Cleary1], [@pone.0053570-Goldie1]--[@pone.0053570-Bendavid1]. First, we separated out the first six months on ART, as outcomes and costs in this period are driven by baseline CD4 count and program protocols (higher frequency of clinic visits and toxicity monitoring) [@pone.0053570-Leisegang1]. Second, we developed a novel LTFU model, in which patients transitioned between ART and LTFU, changed baseline CD4 count within LTFU, and transitioned to death within LTFU. Third, we developed Markov models to account for CD4 and VL category changes within the ART and LTFU models. Fourth, we developed a more detailed model describing the transition between first line and second line ART, which is a major cost driver [@pone.0053570-Cleary3]. Fifth, the model included the impact of time on ART on the transition to LTFU, death, and second line ART. Finally, we assessed the external validity of the model by first developing the model using the public-care program data and then validating it using private-care program data. The fact that our novel Markov model was able to describe the data from two very different models of ART care suggests that its utility may be generalizable. We are aware of one other study that compared costs and outcomes after 1 year in public-care and private-care programs for public sector patients [@pone.0053570-Rosen1]. Their private-care program had significantly lower costs due to fewer GP visits and poorer patient retention than their public care program. The costs of providing ART care were similar, although patient retention was better in our programs. Lifetime analyses using Markov models populated with data from resource-limited settings predicted varying survival on ART (6 to 13 years) and varying discounted total costs (3,000 to 9,500 USD from the provider\'s perspective) [@pone.0053570-Badri1], [@pone.0053570-Goldie1], [@pone.0053570-Bachmann1]--[@pone.0053570-Cleary3], [@pone.0053570-Freedberg2], [@pone.0053570-Wolf1]. Many of these models were developed using short term follow up data. Furthermore, retention within ART programs and cost of providing ART care in resource-limited settings varies dramatically [@pone.0053570-Rosen2], [@pone.0053570-Rosen3]. We estimated that average survival on ART was longer than most resource-limited setting model estimates. The patients included in this analysis were public-sector patients receiving ART care in accordance with WHO public sector ART program guidelines. Therefore the results from this analysis have important policy implications that are relevant to other resource-limited settings. The rapid expansion of access to ART in resource-limited settings is both needed [@pone.0053570-Walensky1] and challenging [@pone.0053570-Boulle1]. Our findings suggest that managed private-care for public sector patients could be used to increase access to ART, provided that the private practices follow national protocols and that loss to follow-up is managed -- key components of the private-care program in our study. A similar model was implemented in Botswana to expand access to ART in areas where limited public-sector resources were available, by utilising doctors working in private practice to look after public sector patients [@pone.0053570-Dreesch1]. Their findings suggested that ART care coverage was extended by 10% and public-sector programs were strengthened by the interaction [@pone.0053570-Dreesch1]. We found that reduced utilisation of clinic visits, especially after the initial six months of care, would considerable lower costs of public-care programs. Finally, our model predicted that LTFU contributed significantly to deaths, utilisation of ART-related resources (on restarting ART), and attenuated CD4 recovery. This suggests that focusing on reducing LTFU could be a cost-saving strategy. There were several limitations to our study. First, the findings in our study are based on a model that extrapolated the trends we observed over the first 3--5 years on ART predominantly. Second, we limited costs in this study to direct ART care costs, while the other components of care represent a significant portion of total costs [@pone.0053570-Leisegang2]. Data on these other cost components were not available. Third, we did not account for the impact of adherence on the total cost of ART drugs, nor the changing composition of specific drugs within the therapy lines over time [@pone.0053570-Leisegang2], [@pone.0053570-Nachega1]. Fourth, given the limited data on actual laboratory utilisation, especially for toxicity monitoring, we set the laboratory utilisation to those recommended in national guidelines. Fifth, it is likely that the patients within the public-care program had better access to HIV clinic services than typical public-sector patients in South Africa, and this would have increased costs, and possibly enhanced patient retention and improved outcomes [@pone.0053570-Boulle2]. Sixth, the relative proportions of individual drugs within the lines of therapy differed between cohorts: *the average ART costs were marginally lower in the private-care program and the different regimens may have impacted the outcomes*. Seventh, given the different models of ART care and different settings in which the programs were based, these programs were not completely comparable and therefore the overall conclusions in terms of costs and outcomes cannot be regarded as definitive. Finally, our public sector clinic visit cost was based on secondary data, which may not capture recent programmatic changes in ART provision (including task shifting) and economies of scale and scope. However, it is difficult to predict the extent to which this unit cost may under or overestimate costs. In moving towards universal access to ART, South Africa intends to offer ARVs from all primary care facilities, which will have implications for the efficiency of service provision and the resulting unit cost. Economies or diseconomies of scale can equally arise in small new facilities during start-up and in older large facilities with high patient volumes. While analyses of provider costs and patient outcomes are crucial in guiding resource allocation for HIV care, it is equally important to consider barriers to patient access, particularly within the context of lifelong care [@pone.0053570-Cleary4]. Evidence suggests that the key barriers to ongoing ART care include the cost of transport to facilities as well as the opportunity cost associated with long waiting times in facilities [@pone.0053570-Cleary4], [@pone.0053570-Rosen4]. Less frequent visits would mitigate these access barriers. One advantage of private care is that waiting times are usually shorter. Conclusions {#s4a} ----------- In conclusion, we have developed a novel Markov model that has the potential to improve the accuracy of estimations of future costs and outcomes of long-term ART care. We have used this model to evaluate two ART programs, and have shown that managed private-care ART programs have the potential to complement the public sector platform in resource poor settings, thereby enhancing and sustaining coverage of patients in need. Our findings suggest that cost savings could be achieved through reducing clinic utilization without compromising patient outcomes. Supporting Information {#s5} ====================== ###### **Model calibration curves for the public-care ART program.** (TIF) ###### Click here for additional data file. ###### **Model calibration curves for the private-care ART program.** (TIF) ###### Click here for additional data file. ###### **Survival hazard coefficient for switching from first to second line therapy over time since starting antiretroviral therapy.** (TIF) ###### Click here for additional data file. ###### **General practitioner and clinic visit utilisation and costs on antiretroviral therapy within the private-care and public-care programs respectively.** (XLS) ###### Click here for additional data file. ###### **Laboratory costs and utilisation on antiretroviral therapy.** (XLS) ###### Click here for additional data file. ###### **The composition and costs of first line and second line antiretroviral regimens, and transition probabilities and coefficients for transitioning from first line to second line regimen.** (TIF) ###### Click here for additional data file. ###### **Transition probabilities and hazard coefficients of changes in CD4+ cell counts and viral load on antiretroviral therapy.** (XLS) ###### Click here for additional data file. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: RL GM MH JS ED SC. Performed the experiments: RL GM MH JS ED SC. Analyzed the data: RL GM MH JS ED SC. Contributed reagents/materials/analysis tools: RL GM MH JS ED SC. Wrote the paper: RL GM MH JS ED SC.

Isolated left ventricular noncompaction (LVNC), also called spongiform cardiomyopathy is a rare cause of heart failure. Arrest of compaction process during normal heart development is thought to be the cause of LVNC \[[@B1][@B2]\]. Echocardiographic demonstration of the apical trabeculation of left ventricle and blood flow from ventricular cavity into the intertrabecular recesses are the main pathologic findings in the diagnosis of LVNC \[[@B3][@B4][@B5]\]. In patients with LVNC, mechanical dyssynchrony between non-compacted and compacted myocardium results in global left ventricular (LV) dysfunction \[[@B6]\] and leads to the clinical symptoms of heart failure. Other important clinical presentations of LVNC include thromboembolic events and arrhythmias due to microcirculatory dysfunction in the noncompacted myocardium and stasis of blood in the recesses \[[@B1]\]. In addition, left ventricular hypertrabeculation is observed in association with a variety of neuromuscular disease (NMD) \[[@B7]\]. Therefore, the anesthetic management of these patients requires special attention to the cardiac function and adverse reaction to general anesthesia. Perioperative echocardiography might be the method of choice to access cardiac function and thromboembolism during general anesthesia in such patients. We reported our anesthetic experience with a LVNC patient who underwent laparoscopic ovarian cystectomy under general anesthesia with real time transesophageal echocardiography. Case Report =========== A 61-year-old woman with a left ovarian cyst was scheduled for laparoscopic ovarian cystectomy under general anesthesia. At age 49, she had difficulty going up 3 stairs and started a dry cough. She was referred to our institution for further evaluation and treatment. She had no other disease including NMD. A transthoracic echocardiogram (TTE) revealed decreased LV cavity size and mild to moderate LV systolic dysfunction with an ejection fraction (EF) of 40% by visual estimation. The patient\'s LV walls, especially apical and interventricular septum (IVS), were markedly thickened, and showed abnormal relaxation that was compatible with noncompaction of LV. She was started on digitalis, diuretics and anticoagulants. Recently, her symptoms were aggravated. She was able to perform basic activities of daily life, but frequently felt chest discomfort. On physical examination, she showed regular heart beats with grade II systolic murmur. Electrocardiogram (ECG) showed sinus bradycardia with 1st degree atrioventricular block, multiple ventricular premature contractions and left bundle branch block. TTE examination demonstrated normal LV cavity size, moderate to severe LV systolic dysfunction with EF of 30% by visual estimation and diastolic dysfunction with left atrial enlargement. Consistent with noncompaction, TTE showed prominent trabeculation with deep intertrabecular recesses and increased velocity at apical trabeculation (Vmax = 3.02 m/sec, Peak pressure gradient = 36.4 mmHg) ([Fig. 1](#F1){ref-type="fig"}). We planned general anesthesia with real time TEE monitoring to access intraoperative cardiac function. We chose TEE, without placement of a pulmonary artery catheter (PAC), because we were concerned with the increased risk of life threatening complications associated with the use of PACs, such as dysrhythmias \[[@B8]\]. No premedication was administered to the patient. Continuous monitoring of blood pressure was initiated after placement of an arterial catheter at the right radial artery under local anesthesia. Anesthesia was induced with etomidate 0.25 mg/kg, midazolam 2 mg and continuous infusion of remifentanil under 100% O~2~ mask ventilation, followed by the muscle relaxant cisatracurium 0.15 mg/kg to facilitate tracheal intubation. Patient\'s lungs were ventilated at a rate of 10 breaths/min with an oxygen/air mixture using positive pressure ventilation. Anesthesia was maintained with continuous infusion of propofol and remifentanil, and standard monitoring of invasive arterial blood pressure, ECG, peripheral oxygen saturation (SpO~2~), bispectral index (BIS), train-of-four (TOF), and TEE. Intraoperative TEE confirmed the preoperative TTE findings. TEE examination showed aneurysmal change of apex, trabeculation in anterior and anterolateral wall of LV, increased velocity at apical intertrabeculation and intertrabecular recesses ([Fig. 2](#F2){ref-type="fig"}). Immediately after anesthetic induction, the patient\'s systolic blood pressure (SBP) abruptly decreased from 120 mmHg to 85 mmHg and heart rate (HR) was also decreased from 71 bpm to 45 bpm. The real time TEE examination demonstrated mild to moderate MR, global hypokinesia, visually estimated EF of 30%, and calculated cardiac output by TEE of 1.8 L/min/m^2^. Continuous infusion of the inotropic agent dobutamine was started at 3 µg/kg/min to increase cardiac output. SBP and HR were maintained during the entire operation within the range of 90 to 140 mmHg and 51 to 70 bpm, respectively. There were no significant hemodynamic changes during insufflation and desufflation of carbon dioxide. At the end of surgery, all the continuous anesthetic medications were tapered off. Soon after, TOF monitor reached 4/4 twitches, but BIS still ranged from 50 to 60 for about 50 minutes. Apnea, pinpoint pupils and lower range of BIS suggested opioid effects. Finally, naloxone, a µ-receptor antagonist that competitively inhibits opioids side effects, was administered intravenously at 120 µg. Immediately after the injection, she opened her eyes on a verbal command and tidal volume recovered to 250−300 ml. The total operation time was 120 minutes but, the total anesthesia time was 214 minutes because of delayed awakening. Total infused amounts of crystalloid were 700 ml and estimated blood loss and urine output were 100 ml and 90 ml, respectively. The patient was transferred to the intensive care unit. At postoperative day 1, she was transferred to the general ward uneventfully. The patient was discharged from the hospital on postoperative day 6 and she had regular follow up in the cardiology and obstetric departments. Discussion ========== LVNC is defined as thickened LV segments with a distinct 2-layer appearance and prominent trabecular meshwork of the apex and adjoining inferior and lateral walls \[[@B3][@B9]\]. Often the affected myocardial segments are hypocontractile and display a 2-layer appearance with a thicker, \'non-compacted\' or hypertrabeculated layer on the epicardial side \[[@B10]\]. LVNC is an uncommon condition; however, the estimated prevalence may be higher because asymptomatic individuals rarely undergo imaging studies and the condition likely remains undetected. Stanton et al. \[[@B11]\] detected isolated LVNC in 0.02% of 141,047 patients who were at least 16 years of age. However, increased awareness and improvements in imaging resolution may also contribute to an increase in the number of reported cases. Diagnosis of LVNC is based on the echocardiographic findings of at least 4 prominent trabeculation, deep intertrabecular recesses and blood flow from the ventricular cavity into the intertrabecular recesses as visualized by color flow Doppler \[[@B9]\]. The clinical presentation of patients with LVNC is variable including congestive heart failure, arrhythmias and systemic thromboembolic events. The current recommendations for treatment according to the international guidelines of heart failure management include beta blocker, angiotensin - converting - enzyme inhibitor and diuretics \[[@B12]\]. Some clinicians prescribe anticoagulants for LVNC patients who develop atrial fibrillation to reduce the risk of thromboembolism \[[@B10]\]. Patients with LVNC may require insertion of ventricular assist devices or cardiac transplantation \[[@B10][@B13]\]. In a study on 34 patients, 12% required transplant and 35% died at 44 months follow up \[[@B4]\]. ECG reveals abnormalities in a majority of patients with LVNC. Most commonly, LV hypertrophy or ST-T wave changes are noted in the ECG. However, more severely, atrial fibrillation, Wolff-Parkinson-White syndrome and other ventricular arrhythmias have also been described \[[@B2]\]. In a study by Fischer et al. \[[@B14]\], the patients were followed up for a mean of 30 months. During this period, 10 deaths were reported of which 60% occurred as a sudden event, whereas 34% of patients required hospitalization for symptoms of heart failure. Ventricular tachycardia was seen in 36% of patients and thromboembolic events in 9%. A familial occurrence was reported in 33% of patients. Although classified by the American heart association as a primary cardiomyopathy, LVNC has been described in association with extracardiac conditions, most notably NMD \[[@B7]\]. The exact incidence is unknown, but in one study, 80% of patients with LVNC were diagnosed with an NMD \[[@B9]\]. A number of genes are thought to be associated with this condition. The anesthetic management of these patients requires specific attention to heart failure, arrhythmia, thromboembolic events and NMDs \[[@B2][@B10]\]. TEE is a useful monitor to guide intraoperative anesthetic and fluid management in the presence of severely depressed LV and RV function \[[@B4]\]. Real time TEE might be the method of choice to access cardiac function and thromboembolism during general anesthesia in these patients. When the echocardiography is not suitable, non-invasive cardiac output monitoring devices might be helpful in the evaluation of cardiac function. Also patients with LVNC are susceptible to develop severe arrhythmia. Stressors that increase sympathetic tone and anesthetic drugs can provoke severe arrhythmia including fatal ventricular fibrillations. In addition, the dose of non-depolarizing neuromuscular blocking agents should be titrated under continuous monitoring of TOF \[[@B15]\]. In conclusion, echocardiography is the method of choice in the diagnosis of LVNC, a rare cardiomyopathy. Anesthesiologist should be specifically concerned with the presenting symptoms of LVNC, heart failure, thromboembolic events, arrhythmia and NMDs. To the best of our knowledge, this is a rare case report of LVNC under general anesthesia reported by a Korean anesthesiologist. This case adds to the understanding of anesthetic management of LVNC patients using TEE. Further studies on the disease and its anesthetic management are required. {#F1} {#F2}

Are Hemodialysis Patients at a Lower Risk for COVID-19 Infection? {#sec1_1} ================================================================= Hemodialysis patients are a population displaying impaired lymphocyte and granulocyte function, and, by strict definition, they are, at least theoretically, at an increased risk for infection by COVID-19 given also the characteristics of the average dialysis center, where social distancing is difficult to achieve \[[@B1], [@B2]\]. However, the scant available data indicate somewhat otherwise; in a report from a university dialysis facility (Zhongnan Hospital) in Wuhan, China, with 201 patients, the prevalence was equal to 5 (2.5%). In addition, cases had no severe symptoms or died \[[@B3]\]. According to another report, related to another university dialysis facility (Renmin Hospital) in Wuhan, China, in the period between January 14, 2020, when the first confirmed case was diagnosed, and February 17, 2020, when the epidemic was declared extinct, among 230 hemodialysis patients 37 (16%) COVID-19 cases were diagnosed. During the epidemic, 7 hemodialysis patients died (18.9%). Symptoms were mild in most surviving patients and there were no cases admitted to the intensive care unit. Laboratory exams showed an impaired cellular immune function (especially lymphocytes of T cells, Th cells, killer T cells, and NK cells) and an incapability of mounting the "cytokine storm" linked to pneumonia, compared to COVID-19 patients not on hemodialysis. The cause of death was related instead to cardiovascular complications \[[@B4]\]. In an Italian experience, among 200 patients 18 were infected and isolated (9%), and in another unit of 170 patients only 4 were infected \[[@B5]\]. In the Piedmont and Aosta regions, among 2,893 patients 98 were infected (3.4%) during the first month of the epidemic \[[@B6]\]. By the way, in none of the mentioned studies was the mode of anticoagulation during hemodialysis mentioned. While they cast doubts on the open-space hospital model now implemented in many hospitals, which is incompatible with the need to counteract epidemics \[[@B7]\], these reports also create uncertainty regarding the concept that these patients are at a particularly increased risk for COVID-19. Hemodialysis and Anticoagulation {#sec1_2} ================================ Heparin actually consists of a heterogeneous mixture of sulfomucopolysaccharides, containing also a minimum peptide component of 2 amino acids (glycine and serine). Heparin exerts a binding capacity to both the endothelial surface and various plasmaproteins (Fig. [1](#F1){ref-type="fig"}). The molecular weight range of unfractionated heparin (UFH) is 5,000--30,000. Low-molecular-weight heparin (LMWH) fractions effectively inhibit the activated factor X (Factor X~a~), while exerting a less inhibitory effect on thrombin, compared to the unfractionated forms. It has been shown that LMWH preparations retain their efficacy toward thromboembolisms and, compared to UFH, show increased bioavailability and the need for less frequent administration. Heparin biological activity crucially depends on the endogenous antithrombin anticoagulant \[[@B8], [@B9]\]. The serine protease inhibitor activity of antithrombin is exerted toward thrombin and Factor X~a~, resulting in the inhibition of both (Fig. [1](#F1){ref-type="fig"}) \[[@B10]\]. Congenital or acquired antithrombin deficiency is indeed associated with a high risk of thromboembolic complications and an impaired interaction with heparin. Administration of antithrombin is generally indicated for the prophylaxis of thromboembolic accidents in nephrology (e.g., in patients with nephrotic syndrome) \[[@B11]\]. In addition, it has been shown that during sepsis activation of the extrinsic coagulation pathway, together with a relevant decline of both coagulation inhibition and fibrinolytic mechanisms, may result in a procoagulant state, leading to microvascular thrombosis and multiorgan dysfunction \[[@B12]\]. Antithrombin levels decrease in sepsis and, when low, may predict high mortality \[[@B13]\]. In addition, heparin is utilized in this context, also for its immunomodulatory and anti-inflammatory role \[[@B14]\]. During hemodialysis it is necessary to perform anticoagulation of the dialysis circuit to avoid blood clotting in the system due to Factor VII, platelet, and leukocyte activation. Anticoagulation is usually performed utilizing heparin, often in Western Europe in the low-molecular-weight form \[[@B15], [@B16]\], which has some advantages over UFH. Relative to UFH, in fact, LMWH could induce less undesired bleeding after completion of the dialysis session and perhaps less triglyceride reduction. Evaluating the Risk of Heparin-Induced Thrombocytopenia {#sec1_3} ======================================================= Heparin-induced thrombocytopenia (HIT) is an immune complex-mediated condition defined as a decrease in platelet counts below 150,000 per mm^3^, with a median nadir of about 55,000 per mm^3^, associated with a positive test for heparin-dependent antibodies \[[@B17], [@B18]\]. The typical onset pattern (60% of the cases) results in a platelet decline 5--10 days after exposure. The rapid onset pattern (30% of cases) occurs right after the exposure. The delayed onset pattern (∼10%) occurs 9.2 days after the start of heparin administration therapy, on average, although signs and symptoms may occur up to 3 weeks after the exposure \[[@B18]\]. Careful evaluation and monitoring should be indeed applied toward the risk of HIT \[[@B19]\]. LMWH offers the advantage of a reduced binding to platelet factor 4, thus decreasing the HIT risk \[[@B20]\]. On the other hand, the shorter size of LMWH may hamper the effectiveness of protamine as an antidote \[[@B20]\]. Useful information is provided by reporting a thrombocyte count of less than 50%, which represents an important red flag \[[@B19], [@B20]\]. Laboratory tests can then be used to confirm the presence of platelet factor 4 (PF4)/heparin antibodies \[[@B21]\]. Attention should be payed when reporting the presence of activating antibodies against PF4-heparin complexes (PF4-H) to the cut-off point for a positive test (e.g., 1 U/mL) \[[@B19]\]. A score has been developed based on 8 clinical features, each scoring between −2 and +3 \[[@B22]\]. Other scoring systems are also available \[[@B18]\]. LMWH and COVID-19 {#sec1_4} ================= LMWH has some antiviral properties in vitro and it is routinely used in COVID-19 patients to prevent or circumvent the activation of the coagulation cascade induced by inflammation \[[@B23]\]. This is a particularly severe and lethal complication, leading to disseminated intravascular coagulation and venous thromboembolism. In a retrospective study, LMWH therapy reduced interleukin-6 release and activity, which is responsible of the "cytokine storm," and treated patients had also a higher percentage of lymphocytes \[[@B24]\]. LMWH therapy is also associated with better outcomes in severe COVID-19 patients with sepsis-induced coagulopathy and markedly elevated D-dimer levels \[[@B25]\]. It should be pointed out that uremia offers a unique microenvironment in which the coagulation and anticoagulation balance can be dysregulated in many ways. For example, the presence of anti-protein C and anti-protein S antibodies has been detected \[[@B26]\], which may critically underscore the acute onset of a procoagulant situation in these patients (already characterized by an increased thrombotic risk). These data indeed may prudentially pose the indication of monitoring protein C and free protein S (Fig. [2](#F2){ref-type="fig"}). It can be hypothesized that, upon SARS-Cov-2 infection, the presence of antibodies with potential inhibitory activity on protein C and protein S may even increase the functional effect of an activated protein C (APC) resistance condition, whereas already present in the patient. Testing for APC resistance is indeed advised in these patients. COVID-19, LMWH, and Hemodialysis: Future Perspectives {#sec1_5} ===================================================== It is therefore possible that hemodialysis patients could be protected with respect to COVID-19 virus infection by the LMWH used in every hemodialysis session. We propose monitoring, in these patients, of the Anti-Factor Xa activity assay \[[@B27]\], as well as antithrombin, and D-dimer levels, and not just aPTT, in order to possibly even increase the LMWH dosage, in this pandemic period (Table [1](#T1){ref-type="table"}). The availability of these exams could therefore lead to optimization of LMWH in this patient population. Disclosure Statement {#sec1_6} ==================== A.F.P. and D.I. received funding from Gnosis, SpA, and EUTox. Funding Sources {#sec1_7} =============== None. Author Contributions {#sec1_8} ==================== All of the authors contributed equally to the writing of this paper. ![Mechanism of action of UFH and LMWH with the major ligand-binding interactions involved. One of the key proteins for the activity of heparin is antithrombin, a serine protease inhibitor (SERPINSC1) that inactivates several clotting factors. Mechanistically, heparin binds to antithrombin, thus exposing the antithrombin protease inhibitor binding site and forming equimolecular complexes with its ligands. Antithrombin uses unique and extensive conformational change mechanisms to inhibit its protease ligands \[[@B8]\]. Heparin increases by orders of magnitude the tendency of antithrombin to interact with a number of plasma proteins. The resulting complex, in particular with thrombin (factor IIa) and factors IXa, Xia, and Xa, inhibits the protease activities of these clotting factors. Heparin indeed works as an antithrombin-protease-inhibiting cofactor without being consumed, and, once the antithrombin-protease complex is formed, heparin is released in its intact conformation, thus repeatedly activating new antithrombin molecules. Heparin biological activity crucially depends indeed on the endogenous antithrombin anticoagulant activity. The role of antithrombin is pivotal in coagulation regulation by shifting thrombin from procoagulant to anticoagulant activity. The presence of heparin dramatically increases the complex stability of antithrombin with the above-mentioned coagulation factors \[[@B8], [@B9]\]. One important difference between UFH and LMWH is that the interaction of antithrombin with UHF almost equally induces inhibition and degradation of both Xa and thrombin, while LMWH preferentially determines degradation of factor Xa. Various other potential benefits of heparin in COVID-19 have been put forward recently \[[@B28]\].](kbr-0045-0357-g01){#F1} ![Scheme of the anticoagulant activity of the protein C anticoagulant pathway. Protein C is activated by thrombin (not shown), forming APC \[[@B29]\]. Protein S in turn is a cofactor of APC for the inactivation of activated coagulation factors VIII~a~ and V~a~. Only free protein S, i.e., the fraction which is not bound to C4b-binding protein (not shown) in circulation, retains its anticoagulant activity. Activated factor V (factor V~a~) procoagulant activity is inactivated by APC-catalyzed cleavage at the Arg506 level, yielding factor V~i~. Beyond its intrinsic procoagulant activity, Factor V~a~ also exerts an anticoagulant activity as a cofactor for APC and protein S, in inactivation of the procoagulant factor VIII~a~ yielding factor VIII~i~ \[[@B30]\]. This function is also compromised in the presence of factor V resistance to cleavage by APC (APC-R; hatched box), mainly due to the Arg506Gln mutation (factor V~Leiden~), which indeed represents the most common cause of APC resistance in the population.](kbr-0045-0357-g02){#F2} ###### Proposed laboratory test profile to monitor LMWH therapy and the risk of adverse reactions Test Rationale/purpose References ------------------------------------------------- ----------------------------------------------- ------------ aPTT Intrinsic and common coagulation pathway 19 Antithrombin[^1^](#T1F1){ref-type="table-fn"} Involved in heparin activity and X~a~ binding 20 Platelet count[^2^](#T1F2){ref-type="table-fn"} Adverse effect monitoring (HIT) 19, 20 HIT[^2^](#T1F2){ref-type="table-fn"} Antibodies against PF4-H complexes 19--22 D-dimer Clot degradation products 23 Anti-Factor X~a~ activity assay Monitoring the clinical effectiveness of LMWH 27 Antithrombin effects are explained in Fig. [1](#F1){ref-type="fig"}. Heparin-induced thrombocytopenia is defined as a decrease in platelet count below 150,000 per mm^3^, with a median nadir of 55,000 per mm^3^, starting 5 or more days after the beginning of heparin administration, associated with a positive test for heparin-dependent antibodies \[[@B17], [@B18]\].

INTRODUCTION ============ Drug addiction involves perseverant and compulsive drug seeking and attempts to obtain and consume drugs despite aversive consequences. One leading circuit-level hypothesis for how addiction arises is that maladaptive neuroadaptations are caused by reward circuits because the dopamine system is usurped by the addictive substances ([@b21-molce-40-6-379]; [@b69-molce-40-6-379]). The main brain areas composing the reward circuits are distributed across multiple areas and include the basal ganglia (including the striatum), the limbic system (including the amygdala and the hippocampus), and the prefrontal cortex (PFC). Among these regions, the striatum is the core input nucleus and plays key roles in reward-related learning as well as in addictive behaviors. The acquisition and maintenance of addiction-like behaviors appear to arise from a series of molecular and cellular adaptations in striatal circuits ([@b28-molce-40-6-379]; [@b32-molce-40-6-379]). In fact, the striatum is composed of several subregions that exhibit distinct connectivity and consequently different functional roles. In rodents, the dorsomedial striatum (DMS) and the dorsolateral striatum (DLS) receive excitatory inputs from the limbic and sensorimotor cortices, respectively, while the intermediate region is activated by axons from the association cortex ([@b17-molce-40-6-379]). The ventral region of the striatum includes the nucleus accumbens (NAc), which consists of the core and shell subregions. The NAc is innervated by the basolateral amygdala (BLA), hippocampus, and medial PFC ([@b3-molce-40-6-379]; [@b31-molce-40-6-379]). Importantly, the striatum receives abundant dopaminergic innervation from the midbrain. The NAc receives dopaminergic inputs from the ventral tegmental area (VTA), whereas the dorsal striatum receives dopaminergic inputs mainly from the substantia nigra pars compacta (SNpc) ([@b4-molce-40-6-379]). Thus, the striatum is considered an area of convergence for various inputs from multiple cortical areas and midbrain structures ([@b6-molce-40-6-379]; [@b39-molce-40-6-379]; [@b58-molce-40-6-379]) ([Fig. 1](#f1-molce-40-6-379){ref-type="fig"}). Within striatal circuits, the integration of various synaptic contacts has been described: gamma-aminobutyric acid (GABA)-ergic innervation has been observed ([@b10-molce-40-6-379]) along with glutamatergic synapses located on the heads of spines on striatal medium spiny neurons (MSNs) and dopaminergic synapses on the necks of spines ([@b25-molce-40-6-379]). Therefore, the striatum likely enables expression through activation and integration of distinct neuronal signals, and defining the role of each pathway will substantially aid in our understanding for addictive behaviors. In addition to the striatal connectome, the unique composition of the striatal neuronal populations must also be addressed. Striatal neurons comprise mainly GABAergic MSNs but also a small population of various types of interneurons. The MSNs, which exhibit low firing rates and high spine densities, are further divided into two subtypes: dopamine receptor type 1 (D1R)-expressing and D2R-expressing MSNs ([@b29-molce-40-6-379]). The striatal interneuron population includes fast-spiking parvalbumin-positive interneurons, low threshold-spiking somatostatin-positive interneurons, and tonically active cholinergic interneurons (ChINs). Although dynamic regulation of synaptic plasticity at individual pathways appears to play a pivotal role in the expression of distinct addiction-like behavioral phenotypes, it remains unknown which striatal circuits are implicated and modulate specific forms of the behaviors. Along with other accumulating knowledge, emerging methods, such as optogenetics and chemogenetics further increase our understanding of addiction-related striatal circuits ([@b23-molce-40-6-379]; [@b63-molce-40-6-379]). Using these molecular and cellular approaches, we have just begun to characterize the causal brain regions and related circuits playing distinct roles in addiction-like behaviors. In here, we summarize recent studies examining pathway-specific regulation of inbound and outbound striatal circuits and also provide conceptual bases for future investigations. MESO-STRIATAL CIRCUIT ===================== Dopamine released in the target brain areas controls and shapes the neural circuits and addictive behaviors. A majority of dopaminergic neurons in the brain are located in the VTA and the SNpc, which project to the ventral and dorsal striatum, respectively. Psychostimulants, including cocaine and amphetamine, elevate dopamine concentrations in these target brain areas by blocking reuptake of dopamine at the axon terminal ([@b30-molce-40-6-379]; [@b35-molce-40-6-379]). As a result, accumulation of extracellular dopamine by drug intake may induce abnormal dopamine-dependent plasticity ([@b28-molce-40-6-379]). Indeed, single or repeated exposure to addictive drugs induces long-term synaptic plasticity that can persist for months ([@b7-molce-40-6-379]). Such observations have supported the view that addictive drugs hijack dopamine pathways and may account for long-lasting remodeling of synaptic transmission ([@b70-molce-40-6-379]). A physiological consequence of increased excitatory inputs to VTA dopamine neurons is the heighted activation of the mesolimbic pathway, which may in turn contribute to addiction states ([@b55-molce-40-6-379]; [@b65-molce-40-6-379]). These findings have been substantiated by recent studies using optogenetic manipulation mimicking the activity of dopamine neurons and acting as a positive reinforcer ([@b60-molce-40-6-379]). For example, activation of dopamine neurons supports operant responding, which represents reward-seeking behaviors ([@b1-molce-40-6-379]; [@b51-molce-40-6-379]), and conditioned place preference (CPP), which represents reward learning ([@b64-molce-40-6-379]), both of which are paralleled by an elevation of dopamine ([@b62-molce-40-6-379]; [@b71-molce-40-6-379]). Thus, activation of the mesostriatal dopaminergic pathway could determine dopamine-induced plasticity that is critical for setting up and maintaining drug addiction. The NAc receives not only dopaminergic but also GABAergic inputs from the mesolimbic pathway ([@b10-molce-40-6-379]). However, it is not well understood how inhibitory transmission is provided by the long-range GABAergic projections from the VTA, and whether or not the pathway modulates drug-seeking behavior. The VTA GABAergic projections synapse on the soma and proximal dendrites of ChINs in the NAc ([@b10-molce-40-6-379]). ChINs express D2Rs and also control dopamine release; thus activation of ChINs could modulate spontaneous dopamine release ([@b2-molce-40-6-379]; [@b12-molce-40-6-379]; [@b73-molce-40-6-379]). Moreover, collateral dopaminergic and GABAergic projections from the VTA to the NAc heterosynaptically induce long-term depression (LTD) in inhibitory transmission ([@b33-molce-40-6-379]). Interestingly, this LTD is occluded after withdrawal from cocaine exposure ([@b33-molce-40-6-379]). Thus, the physiological roles of the accumbal ChINs could contribute to the altered emotional and motivational states that occur during drug ([@b67-molce-40-6-379]). However, it is still unclear whether and how this cholinergic regulation is involved in controlling addiction-like behaviors. CORTICO-STRIATAL CIRCUIT ======================== The corticostriatal pathway has been extensively characterized, and its physiological relevance has long been emphasized as a part of the cortico--striato--thalamic circuit that is implicated in cognitive hierarchies ([@b31-molce-40-6-379]; [@b34-molce-40-6-379]). Specifically, the PFC participates in modulating goal-directed behaviors by re-evaluation of drug-associated instrumental response contingency ([@b18-molce-40-6-379]; [@b37-molce-40-6-379]; [@b49-molce-40-6-379]). Neuronal information from the PFC is conveyed to the striatum, which may result in habit learning ([@b72-molce-40-6-379]). Indeed, synaptic potentiation is observed in the medial PFC--striatal circuits of drug-seeking mice after sustained withdrawal. This increased synaptic strength may suggest the potential role of the medial PFC--striatal pathway for cue-induced drug-seeking responses ([@b50-molce-40-6-379]). The medial PFC can be further divided into the prelimbic cortex (PrL) and infralimbic cortex (IL), preferentially projecting to the NAc core and shell, respectively. The PrL and IL putatively exhibit opposite roles in drug addiction, especially when being subjected to changing environmental contingencies during and after extinction training. Consistent with this notion, inactivation of the PrL prevents reinstatement of drug memory ([@b27-molce-40-6-379]; [@b36-molce-40-6-379]; [@b59-molce-40-6-379]), whereas inactivation of the IL facilitates reinstatement of drug-seeking behavior ([@b53-molce-40-6-379]). However, there are incongruent studies indicating functional roles of the medial PFC in incubation of drug craving ([@b8-molce-40-6-379]; [@b40-molce-40-6-379]; [@b46-molce-40-6-379]). Therefore, it is worth investigating how distinct corticostriatal pathways control and sculpt the learning and expression of goal-directed instrumental behavior, ultimately updating the value of drug-seeking behavior. AMYGDALO--ACCUMBAL CIRCUIT ========================== Addictive drugs or psychostimulants modulate emotional states, and recreational drug use can induce positive reinforcement and advance the progression of the addiction stages. The amygdala, which plays pivotal roles in emotional learning and memory, also appears to be involved in addiction-like behavior. Principal neurons in the BLA project to the NAc, and the functional role of this pathway has been initially addressed by disconnection studies. For example, selective lesion of the BLA or NAc core results in impaired acquisition of drug-seeking behavior ([@b26-molce-40-6-379]; [@b68-molce-40-6-379]). The BLA--NAc pathway was recently shown to mediate behaviors associated with positive or negative valences ([@b38-molce-40-6-379]; [@b52-molce-40-6-379]; [@b61-molce-40-6-379]). Applying optical stimulation to this pathway promotes motivated behavior, which requires D1R-expressing but not D2R-expressing MSNs ([@b61-molce-40-6-379]). [@b61-molce-40-6-379] demonstrated that intracranial self-stimulation of the amygdala projection, but not the cortical inputs, to the NAc induces positive reinforcement. The data are consistent with other studies that indicate significant alteration of the D1R-expressing MSNs after repeated drug exposure and the previous observation that the amygdala--striatal circuits are critical for selectively strengthening the innervation of D1R-expressing MSNs in the NAc ([@b43-molce-40-6-379]; [@b50-molce-40-6-379]). Furthermore, synaptic alterations in only the BLA--NAc circuit are sufficient to control locomotor sensitization ([@b47-molce-40-6-379]), CPP expression, and craving behavior through the maturation of silent synapses and recruitment of calcium-permeable AMPA receptors ([@b11-molce-40-6-379]; [@b43-molce-40-6-379]; [@b57-molce-40-6-379]). The hM4D~i~-mediated chemogenetic modulation of G~i/o~ signaling in the amygdala--striatal circuit attenuates locomotor sensitization to drug exposure, but does not affect basal locomotion ([@b47-molce-40-6-379]). Taken together, these findings suggest that the BLA--NAc circuit plays necessary and critical roles for reinforcement learning, and putatively addiction-like, behaviors. HIPPOCAMPAL--STRIATAL CIRCUIT ============================= The ventral hippocampus (vHPC) is another major source of glutamatergic inputs to the NAc, especially to the medial shell ([@b74-molce-40-6-379]). Indeed, vHPC neurons activate NAc MSNs, with stronger inputs on D1R-expressing rather than D2R-expressing MSNs. This vHPC--NAc pathway is also affected by cocaine exposure. After repeated non-contingent injections of cocaine, the bias in the amplitude of excitatory currents in D1R- and D2R-MSNs is abolished, suggesting that the vHPC--NAc pathway is capable of mediate drug-induced synaptic plasticity ([@b47-molce-40-6-379]). Indeed, lesions of the dorsal subiculum result in hyperactivity, whereas lesions of the ventral subiculum reduce locomotor responses to amphetamine and impair acquisition of cocaine self-administration ([@b13-molce-40-6-379]; [@b54-molce-40-6-379]). Interestingly, the vHPC--striatal pathway is potentiated after drug exposure ([@b9-molce-40-6-379]) and supports discrimination of drug-associated actions in the operant chamber ([@b50-molce-40-6-379]). Thus, hippocampal inputs to the NAc, especially to the shell, would be highly involved in both the psychomotor stimulant effect and information processing of the contextual values. The preponderance of evidence suggests that the hippocampus is necessary for the expression of drug addiction-like behaviors. STRIATAL DIRECT AND INDIRECT PATHWAYS ===================================== As described above, GABAergic MSNs constitute either the direct or indirect pathway based on their projection targets. The direct pathway comprises D1R-expressing MSNs that directly project to basal ganglia output nuclei, such as the substantia nigra or subthalamic nucleus. By contrast, the indirect pathway is composed of D2R-expressing MSNs that project to other basal ganglia nuclei that subsequently innervate output nuclei (e.g., the globus pallidus externa) ([@b28-molce-40-6-379]). The D1R is a G~s/a~ protein-coupled receptor whose activation results in stimulation of adenylyl cyclase, whereas the D2R is a G~i/a~ protein-coupled receptor whose activation inhibits adenylyl cyclase ([@b48-molce-40-6-379]). Chemogenetic inhibition of D1R-MSNs in the dorsal striatum suppresses locomotor sensitization, while inhibition of D2R-MSNs promotes locomotor activity after amphetamine exposure ([@b24-molce-40-6-379]). Furthermore, dorsal striatal D1R-MSNs likely mediate acquisition of reinforced behavior and place preference behavior, whereas D2R-MSNs play a sufficient role for place aversion ([@b42-molce-40-6-379]). Chemogenetic inhibition of striatal D2R-MSNs increases motivation for cocaine ([@b5-molce-40-6-379]). Expression of D1R is necessary to produce cocaine self-administration behavior ([@b15-molce-40-6-379]). By contrast, D2R is not essential for self-administration behavior ([@b14-molce-40-6-379]), but the activation of striatal D2R-MSNs rather impairs locomotor sensitization ([@b44-molce-40-6-379]). Furthermore, the ablation of striatal D2R-expressing MSNs results in increased amphetamine CPP ([@b20-molce-40-6-379]), suggesting that D2R-expressing MSNs in the NAc play an inhibitory role in addiction-like behaviors. Taken together, this evidence suggests that the expression of addiction-like behaviors is controlled by the balanced activity of D1Rs and D2Rs, which are differentially expressed in distinct subtypes of projection neurons in the striatum. However, it still remains challenging to conclusively establish differential roles for each MSN type in addiction-like behaviors. Axons from both D1R-MSNs and D2R-MSNs in the NAc innervate the ventral pallidum (VP) ([@b16-molce-40-6-379]). These pathways appear to encode the overall direction of the behavioral outputs. Normalization of cocaine-induced plasticity at NAc--VP synapses by optogenetic modulation of the direct pathway indicates that the collateral NAc--VP pathway composed of D1R-MSNs is necessary for locomotor sensitization and maintenance of motivation for cocaine seeking ([@b16-molce-40-6-379]). Interestingly and also in agreement with the optogenetic results, drug-induced (i.e., amphetamine) sensitization is blocked by G~s~-coupled receptor activation of the adenosine A2a receptor, a marker of D2R-MSNs, expressing neurons ([@b22-molce-40-6-379]). Thus, activation of D2R-MSNs seems to lead to lateral inhibition of the D1R-MSNs in the NAc to control reward-related behaviors. Exposure to cocaine suppresses this lateral inhibition, which thus promotes behavioral sensitization ([@b19-molce-40-6-379]). ADDITIONAL COMPONENTS UNDERLYING ADDICTION-LIKE BEHAVIORS ========================================================= In the progression of drug addiction, relapse is the recurrence of addiction that had advanced to recovery or remission. Stress is a major priming stimulus for triggering relapse ([@b36-molce-40-6-379]), and addictive drugs that have hedonic effects may help cope with the stressful conditions. There is ample evidence that stress increases the occurrence of relapse, but the cellular and molecular mechanisms have just begun to be addressed. For example, activation of extracellular signal-regulated kinase by brain-derived neurotrophic factor (BDNF) in the mesostriatal pathway is required for acquisition of drug-induced sensitization and CPP ([@b44-molce-40-6-379]). BDNF-mediated dopamine neuron activation is controlled by corticotropin-releasing factor (CRF; also known as corticotropin-releasing hormone), which is released under stressful conditions ([@b66-molce-40-6-379]). CRF signaling, which arises from the extended amygdala structures, including the central amygdala, may contribute to the priming of drug seeking in stressful conditions ([@b56-molce-40-6-379]). Another factor that needs to be addressed in drug addiction is the connectivity between neural ensembles that arise from the association between sensory inputs and the hedonic effect of drugs. Considering that drug-induced plasticity occurs at a small subset of activated striatal neurons ([@b41-molce-40-6-379]), neuronal connectivity would change between drug-recruited neurons and the other neuronal components, which would sculpt the acquisition and expression of drug-related memory. Additional research dedicated to this line of study will benefit further understanding of circuit-mediated addictive behavior. CONCLUSION ========== The aim of circuit-wide and circuit-specific investigations for addiction-like behavior is to elucidate addiction mechanisms and offer successful therapeutic intervention for addiction. Accumulated data indicate that the striatum is a key brain area involved in drug addiction, as striatal circuits play critical roles in setting-up of addiction-like behaviors and are critically involved in all stages of addiction progression, from initial exposure to relapse. Studies using optogenetic and chemogenetic strategies have revealed distinct neuronal circuits relevant to the progression of addiction and shared circuits with common behavioral consequences after exposure to various psychostimulants ([Fig. 2](#f2-molce-40-6-379){ref-type="fig"}). Striatal circuit-selective activation--inactivation or potentiation--depotentiation precedes the significant alteration of addiction-like behaviors, substantiating the net effect of an individual circuit on the progression of addiction. After an exposure to psychostimulant drugs, motor activity is controlled by inputs to the striatum from the vHPC and the amygdala and via the direct and indirect pathways to increase striatal dopamine levels. These pathways are also necessary for encoding components of addictive drug-related learning and memories after repeated use. Furthermore, relapse to psychostimulant drugs after abstinence largely involves the PFC, which projects to the ventral striatum, for the expression of craving or compulsive drug-seeking behaviors. Among the striatal circuits involved in the progression of addiction, activation of the IL--NAc shell and striatal D2R-MSN indirect pathways are effective for inhibiting related behavioral expression. Indeed, natural protective mechanisms of the striatal indirect pathway have been described ([@b5-molce-40-6-379]), and striatal circuit-selective restoration of synaptic transmission has been shown to normalize circuit functions and rescue animal behaviors ([@b45-molce-40-6-379]). Therefore, circuit-specific modulations provide a promising key solution for the development of effective therapeutic interventions that ameliorate (or even cure) addiction at each step of the addiction processes. This work was supported by grants from the National Research Foundation of Korea (2014051826 and NRF-2017R1 A2B2004122) to J.-H.K. {#f1-molce-40-6-379} {#f2-molce-40-6-379}

Introduction ============ Single-nucleotide mutations (SNMs), including single-nucleotide substitutions, insertions and deletions, are important biomarkers for human diseases and drug resistance.^[@cit1],[@cit2]^ Various technology platforms^[@cit3]^ have been applied to the discrimination and detection of low abundance SNMs by using synthetic nucleic acid primers and probes, for example, through polymerase chain reaction (PCR),^[@cit4],[@cit5]^ next-generation sequencing,^[@cit6],[@cit7]^ microarrays^[@cit8],[@cit9]^ and fluorescent *in situ* hybridization.^[@cit10],[@cit11]^ Crucially, the discrimination ability of all these technologies and methods relies on the specificity of Watson--Crick base pairing at some step of their workflow, which is limited by the small difference in thermodynamic stability caused by a single-base mismatch.^[@cit12]^ Many efforts have been devoted to the improvement of the discrimination ability by using denaturation approaches,^[@cit13],[@cit14]^ enzymatic methods,^[@cit15]--[@cit26]^ synthetic nucleotide analogues^[@cit27],[@cit28]^ and rationally designed probes such as molecular beacons,^[@cit29]--[@cit32]^ binary probes,^[@cit33],[@cit34]^ triple-stem probes^[@cit35],[@cit36]^ and toehold probes.^[@cit37]--[@cit42]^ However, most of these efforts have focused on the optimization of reaction condition and probe or primer design, and the discrimination ability is still seriously limited by the cross-reactivity with closely related unintended sequences. Recently, several competitive systems using the sequence-specific DNA sinks,^[@cit3],[@cit43]^ the controller DNAs,^[@cit44]^ the DNA-blocker strand^[@cit45]^ and the peptide nucleic acid clamps^[@cit46]^ were designed for SNM discrimination. These advances effectively improved the discrimination ability but suffered from complexity and required stringent condition control. As a result, it remains a major challenge to develop simple, robust and highly specific hybridization-based SNM discrimination strategies. Herein, for the first time, we propose a sequestration-assisted molecular beacon (MB) strategy for highly specific SNM discrimination in homogeneous solutions. The new SNM discrimination system consists of a target-specific MB and a series of hairpin sequestering agents (SEQs). The rationally designed hairpin SEQs can sequester the closely related unintended sequences and thus effectively eliminate the cross-reactivity. By using fluorescence measurements, we quantitatively evaluated the discrimination ability of the developed SNM discrimination method against a series of SNMs, and also investigated the superiority of the hairpin SEQ as well as the condition robustness of the developed SNM discrimination method. Moreover, we explored the feasibility of combining our SNM discrimination method with PCR amplification for the detection of *KRAS* G12D (c.35G\>A) and G12V (c.35G\>T) mutations at low abundance to demonstrate the potential application in clinical diagnosis. Results and discussion ====================== The design principle of the proposed sequestration-assisted MB strategy is illustrated in [Fig. 1](#fig1){ref-type="fig"}. The SNM discrimination system consists of a target-specific MB and a series of specific hairpin SEQs which can sequester the corresponding single-base mismatched sequences (MMs). The loop portion of the MB serves as a probe sequence that is perfectly complementary to the target sequence, and the stem of the MB is formed by two complementary 5-nt arm sequences with a fluorophore (F) and a quencher (Q) attached to the ends of the two arms respectively. The structure of the hairpin SEQs is similar to that of the MB, except that the hairpin SEQs have no fluorophore or quencher, and their loop sequences are complementary to the corresponding MMs. In the absence of the perfectly matched target sequence (PM), the 6-carboxyfluorescein (6-FAM) at the 5′ end of the MB is quenched by the black hole quenchers-1 (BHQ-1) at the 3′ end. After the addition of the PM, the MB hybridizes with the PM regardless of the SEQs, and the fluorescence signal is recovered because the unfolding of MB increases the spatial distance of 6-FAM and BHQ-1 (pathway a). In the presence of MMs only, a large excess of hairpin SEQs almost completely sequesters the MMs, so the MB still keeps the closed state (pathway b). When the PM and the MMs are present simultaneously, the MB can still be opened by the binding of PM to restore the fluorescence, and the MMs are also sequestered by the hairpin SEQs (pathway c). It is noteworthy that the sequestration-assisted MB strategy presented here (1) is developed from the classic MB system^[@cit47]^ and is quite simple and enzyme-free, (2) can effectively eliminate the cross-reactivity of the MB with MMs by using hairpin SEQs, and (3) can be applied to the discrimination of other SNMs by easily altering the loop sequences of the MB and the hairpin SEQs. {#fig1} To quantitatively evaluate the discrimination ability of the developed SNM discrimination method, we performed time-resolved fluorescence measurements to get discrimination factors (DFs) of a series of SNMs at three different positions. The inset of [Fig. 2A](#fig2){ref-type="fig"} shows the corresponding locations of these positions in the MB. The DF is defined as the ratio of the net fluorescence intensity gain obtained with the PM to that obtained with the MM under the same conditions (DF = Δ*F* ~PM~/Δ*F* ~MM~). Thus, a larger DF value is indicative of greater specificity. To systematically investigate the specificity of our SNM discrimination method, we calculated the DFs of all possible 20 SNMs (including 12 substitutions, 4 insertions and 4 deletions) at the position 7. The oligonucleotide sequences of all strands are provided in Tables S1 and S2.[†](#fn1){ref-type="fn"} As can be seen from [Fig. 2A](#fig2){ref-type="fig"}, our strategy shows excellent discrimination ability with remarkable DF values ranging from 12 to 1144 with a median of 117, which is better than that of most SNM discrimination methods reported recently (Table S3[†](#fn1){ref-type="fn"}). To demonstrate the versatility of our method, we tested two additional positions (5 and 9). For SNMs at the position 5 and 9, the DFs of six representative SNMs are in the range from 27 to 1105 ([Fig. 2A](#fig2){ref-type="fig"}), indicating that our method is reliable for the discrimination of mutations at different positions. The real-time fluorescence responses to three representative PM/MM pairs at the position 5, 7 and 9 are shown in [Fig. 2B--D](#fig2){ref-type="fig"}. The above results indicate excellent discrimination ability of our SNM discrimination method, which is attributed to the following factors: (1) the MB itself provides a competing reaction for the probe--target hybridization, which possesses good specificity, (2) the MMs are sequestered by the rationally designed hairpin SEQs, thus non-specific hybridization of MMs with the MB is effectively eliminated, and (3) the hairpin structure of the SEQ increases the sequestration specificity, leading to further improvement in discrimination ability. These experimental results clearly demonstrate the remarkable discrimination ability of our SNM discrimination method. {ref-type="fn"} The inset shows the MB sequence and three positions of the corresponding SNMs. Real-time fluorescence responses of our SNM discrimination system to PM/MM pairs of A \> T substitution at the position 5 (B) and 7 (C), and C \> G substitution at the position 9 (D). The concentrations of MB, SEQ, PM and MM are 20, 800, 20 and 20 nM, respectively. The error bars represent the standard deviation of three measurements.](c6sc03048c-f2){#fig2} According to the design rationale of the proposed discrimination strategy, the structure and concentration of the SEQ are considered as crucial factors for remarkably improving the SNM discrimination ability. To prove the superiority of the hairpin SEQ, we compared the specificity of three SNM discrimination systems with the hairpin SEQ, with the linear SEQ and without the SEQ, respectively. We performed fluorescence measurements and calculated the DFs of these SNM discrimination systems using G \> T substitution at the position 7 as a model. As depicted in [Fig. 3A](#fig3){ref-type="fig"}, the maximum DF values 131, 19 and 3.1 are obtained with the hairpin SEQ, with the linear SEQ and without the SEQ (*i.e. c* ~SEQ~ = 0), respectively. The SNM discrimination system with 800 nM hairpin SEQ achieves the highest DF and shows about 7-fold and 42-fold improvements compared to the SNM discrimination systems with the linear SEQ and without the SEQ respectively ([Fig. 3B](#fig3){ref-type="fig"}), clearly demonstrating the significant contribution of the hairpin SEQ to the discrimination ability. We also investigated the sensitivity of the proposed SNM discrimination system. The detection limits of our system and the simple MB system are 0.98 nM and 0.44 nM calculated by the 3S/N method from the corresponding linear relationships (Fig. S3[†](#fn1){ref-type="fn"}), respectively. Our method caused a slight reduction (about 2-fold) in sensitivity, due to the possible cross-reactivity of the SEQs with the PM. The results confirm that the rationally designed hairpin SEQ plays a key role in the dramatically enhanced specificity of the proposed sequestration-assisted MB strategy. {#fig3} Condition robustness and fast discrimination are key factors of SNM discrimination methods for the potential application in clinical diagnosis. We first investigated the robustness of the developed SNM discrimination method (using A \> T substitution at the position 7 as a model) by obtaining DFs in a wide range of conditions. The results are shown in [Fig. 4](#fig4){ref-type="fig"}. When the concentrations of the target or the MB are changed in the range of 2.5--100 nM, the DFs remain greater than 47 and 66 respectively ([Fig. 4A and B](#fig4){ref-type="fig"}), implying that our SNM discrimination method can maintain good specificity in a wide concentration range of the target or the MB. The remarkable DFs (≥1144) are produced in up to 50 μM of 50-nt random DNA sequences ([Fig. 4C](#fig4){ref-type="fig"}), and the DFs are greater than 140 in buffers with different concentrations of Mg^2+^ and Na^+^ (0.5--50 mM Mg^2+^ and 30--3000 mM Na^+^, [Fig. 4D](#fig4){ref-type="fig"}). Therefore, biological samples or PCR products might be analyzed directly without purification or buffer-exchange procedures. Our SNM discrimination method is also robust throughout the temperature range of 20--45 °C with the corresponding DFs no less than 72 ([Fig. 4E](#fig4){ref-type="fig"}), meaning precise temperature-control equipment is not required. We next discussed how quickly our SNM discrimination method could distinguish PM/MM pairs. For all possible 20 SNMs at the position 7, a median DF of 76 can be achieved in only 10 minutes after the initiation of the reaction (Table S4[†](#fn1){ref-type="fn"}). The experiment results demonstrate that our highly specific SNM discrimination method (1) can work robustly over a wide range of temperatures, salinities, target/MB concentrations and in the presence of high concentration of 50-nt random DNA sequences, and (2) is capable of quickly distinguishing SNMs, thus having great potential to detect disease-related SNMs in biological samples. {ref-type="fn"}](c6sc03048c-f4){#fig4} The excellent specificity makes our SNM discrimination method well-suited for the detection of low abundance mutations. To evaluate this potential, we used A \> T substitution at the position 7 as a model and measured the fluorescence responses of our SNM discrimination system to the PM at different abundances. As can be seen from [Fig. 5A](#fig5){ref-type="fig"}, the fluorescence intensity shows a gradual rise with the increasing PM percentage. An obvious fluorescence intensity increase can still be seen when only 0.1% PM is present ([Fig. 5B](#fig5){ref-type="fig"}), indicating that the PM can be successfully identified at abundance as low as 0.1%. The results well demonstrate the capability of our assay in detecting low abundance mutations. {#fig5} Mutated *KRAS* genes are associated with lung cancer, colorectal cancer, ovarian cancer and pancreatic cancer.^[@cit46],[@cit48]^ To further prove the potential application of our SNM discrimination method in clinical diagnosis, we combined this SNM discrimination method with PCR amplification to detect *KRAS* G12D (c.35G\>A) and G12V (c.35G\>T) mutations. We first performed the detection of *KRAS* G12D (c.35G\>A) mutation. The mutant-type (mutant A) and wild-type sequences were mixed at 0 : 100, 0.5 : 99.5, 1 : 99, 5 : 95, 10 : 90 and 100 : 0 ratios to total concentrations of 0.5 pg μl^--1^, and amplified by asymmetric PCR to generate single-stranded amplicons. Two synthetic oligonucleotides were added to the PCR amplicons to unwind the secondary structure, and then the amplicons were analysed by our SNM discrimination method. As shown in [Fig. 6A--C](#fig6){ref-type="fig"}, the mutant-type target can be successfully identified at abundance as low as 0.5% in the presence of wild-type strands. In contrast, the classic MB systems (without the SEQs) could hardly distinguish and detect this mutation even at 10% abundance ([Fig. 6D](#fig6){ref-type="fig"}). The results demonstrate the capability of our assay in specific and sensitive detection of low abundance SNMs. Besides the wild-type sequence, other mutant-type sequences may also interfere with the detection of the exact mutant-type of interest. So we mixed the intended mutant-type (mutant A) with same amounts of the other two unintended mutant-types (mutant T and mutant C) and 100-fold excess of wild-type. After PCR amplification, three SEQs were added to simultaneously sequester the wild-type as well as the other two unintended mutants. As can be seen from [Fig. 6I and J](#fig6){ref-type="fig"}, the mixtures of three mutant-types and the wild-type at the ratio of mutant A : mutant T : mutant C : wild-type = 0 : 1 : 1 : 100 and 1 : 1 : 1 : 100 are successfully discriminated. To demonstrate the versatility of our approach, we also tested the proposed method against *KRAS* G12V (c.35G\>T) mutation. The results are similar to that of the *KRAS* G12D (c.35G\>A) mutation (shown in [Fig. 6E--H, K and L](#fig6){ref-type="fig"}). The results indicate that our sequestration-assisted MB strategy is versatile and can be applied to the detection of low abundance SNMs in PCR amplicons with high specificity, thus holding great potential for clinical application. {#fig6} Conclusions =========== In summary, we have successfully developed a simple and robust SNM discrimination method with remarkably high specificity using the sequestration-assisted MB strategy. The crucial element of the proposed strategy is the rationally designed hairpin SEQs that can effectively sequester the closely related unintended sequences and thus dramatically improve the hybridization specificity of the MB in recognizing SNMs. Our SNM discrimination method can work rapidly and robustly over a wide range of conditions and can be easily combined with PCR amplification to detect *KRAS* G12D (c.35G\>A) and G12V (c.35G\>T) mutations at low abundance, demonstrating the capability of our assay in specific and sensitive detection of low abundance SNMs. Moreover, the proposed strategy provides a general SNM discrimination method through simply altering the loop sequences of the MB and SEQs. We anticipate that this work offers a new route to design SNM discrimination strategies for clinical application. This work was financially supported by the National Natural Science Foundation of China (21535006, 21675005 and 21605134). [^1]: ‡These authors contributed equally. [^2]: †Electronic supplementary information (ESI) available: Details in experimental section and supporting tables and figures. See DOI: [10.1039/c6sc03048c](10.1039/c6sc03048c) Click here for additional data file.

Introduction {#S0001} ============ Bipolar disorder (BP) is a mental disorder that consists of manic/hypomanic and depressive episodes or manic episodes separated by periods of a stable mood. Manic episodes include elevated or irritable mood, hyperactivity, pressure of speech, inflated self-esteem, and a decreased need for sleep.[@CIT0001] Lifetime prevalence of BP is 1.0% for BP-I, 1.1% for BP-II, and 2.4% for subthreshold BP.[@CIT0002] BP is a debilitating disorder due to its early onset, severity, and chronicity.[@CIT0003] Moreover, there is huge caregivers' burden related to patient behavior and patients' and caregivers' role dysfunction.[@CIT0004] The general public's beliefs and attitudes toward mental illness may affect the level of care and treatment those with the illness receive.[@CIT0005] The public's knowledge, beliefs, and attitudes toward BP are discussed extensively in the literature. A study in the United Kingdom explored the beliefs of lay people about the cause and treatment of BP. Brain's chemical imbalance, drug use, emotional childhood trauma, and genetics were the most highly reported causes.[@CIT0006] A study in Spain[@CIT0007] on the opinions and beliefs about schizophrenia and BP found that 72% of the Spanish population endorsed psychological treatments as an effective therapeutic choice. This study also demonstrated prevalent concerns, including social rejection and lack of information and resources. Furthermore, more than half of participants in the first national survey of knowledge, attitudes, and behaviors toward BP in France[@CIT0008] cited stressful life events, lifestyle/environment, and drugs and alcohol abuse as the most important risk factors for BP, whereas one-quarter of participants selected genetic factors. Although both biomedical and psychosocial causes of BP were endorsed over fate causes by United Kingdom residents,[@CIT0009] this may not be same in other cultures that have supernatural beliefs. In a Saudi Arabia, a misunderstanding of psychiatric disorders was demonstrated by the staff in general hospital.[@CIT0010] Poor knowledge and negative attitudes were reported by both doctors and patients in another Saudi general hospital, which negatively influenced the referral rates.[@CIT0011] A qualitative research conducted in Saudi primary healthcare centers has reported traditions, cultural norms as a main reason of stigma associated with psychiatric disorders. In addition, other significant reasons of stigma were reported, e.g. fear the reaction of psychiatric patients, lack of community awareness about psychiatric disorders and the role of psychiatrist, belief that psychiatric disorders cannot be cured, and that psychiatric disorders are hereditary. Whether patients will seek psychiatric help for BP also varies. The participants in the qualitative research have frequently mentioned seeking help from family or friends before consulting psychiatrist. On the other hand, going to "sheiks" (religious people) for Qur'an treatment was also reported.[@CIT0012] Regarding patients with BP, a study from Egypt conducted on patients with BP showed that 40.8% sought help from traditional healers before seeking psychiatric services.[@CIT0013] Other studies conducted in Saudi Arabia and Sudan have also reported patients with BP as being among visitors to traditional healers who interpret psychiatric disorders as being caused by spiritual and supernatural causes, e.g. jinn, evil eye, and witchcraft. This likely represents an important cause of delay in seeking psychiatric services, which may worsen the patients' prognosis.[@CIT0014]--[@CIT0017] Due to lack of data from Saudi Arabia, the objective of this study was to determine the public's knowledge, beliefs, and attitudes toward BP. Materials And Methods {#S0002} ===================== Study Design {#S0002-S2001} ------------ Data for this study were obtained using a cross-sectional survey during September and October, 2016. Ethical approval was obtained from the Institutional Review Board at the Faculty of Medicine at King Saud University in Riyadh, Saudi Arabia. Informed written consent was obtained from the participants after a detailed explanation of the aims of the study. Sample Population {#S0002-S2002} ----------------- This study included both male and female, who are Arabic speakers over 17 years old currently living in Riyadh, Saudi Arabia. Survey participants under the age of 18 were permitted and approved by the Institutional Review Board at the Faculty of Medicine at King Saud University. People without the capacity to consent were excluded. Recruitment {#S0002-S2003} ----------- Eighteen medical students were trained to distribute the questionnaire to the residents of all regions of Riyadh (North, South, East, West). Medical students had small booths in shopping malls, public parks, hospitals, and on the walkway streets. Participants were selected conveniently. Data Collection {#S0002-S2004} --------------- A data collection tool was constructed from the literature on assessing mental health literacy in Arab regions.[@CIT0018]--[@CIT0021] It was adapted mainly from a validated questionnaire which has been used in Saudi study on depression literacy with its parametrized answers.[@CIT0018] We chose items that assess BP awareness based on causes and treatments. We also added items that assess beliefs and attitudes toward people with BP. The questionnaire was translated into Arabic, approved by four psychiatrists, and then tested for both comprehension and readability by 20 subjects who were not included in the study. The time required to complete the survey was targeted to be around five minutes. The final questionnaire consisted of the following sections: 1) sociodemographic characteristics including age, gender, marital status, nationality, educational level, and working status; 2) BP awareness based on: a) information had two items, b) sources of information used by participants were structured from none to eight potential sources (internet, social media, television, newspapers/magazines, leaflets/posters, hospitals/clinics, pharmacist/chemist, and relatives/friends/spouses), c) knowledge about the causes of BP had nine items, and d) knowledge about the treatment of BP had ten items; and 3) beliefs and attitudes toward patients with BP. Responses for BP awareness (Sections a--d) were either "yes" or "no". Responses for beliefs and attitudes toward patients with BP were: "strongly agree," "agree," "neutral," "disagree," and "strongly disagree". The scale of BP awareness was created from the sum of responses to the 21 items in Sections a, c, d. Positive responses were considered by answering "yes" to the positive statements: 2 items in section (a), 7 items in section (c), and 3 items in section (d). On the other hand, positive responses were considered by answering "no" to incorrect statements; 2 items in section (c) and 7 items in section (d). The 21 statements are illustrated in [Table 1](#T0001){ref-type="table"}.Table 1Percentages Of Positive Responses Toward Bipolar Awareness, Including Information And Knowledge (N=416)Item NumberStatementsPositive Response (%)**Information**1Heard about Bipolar Disorder\*49.52Bipolar is a common disorder in Saudi Arabia\*57.0**Causes of Bipolar**3Neurophysiologic or neurochemical imbalance\*45.24Certain medical conditions, e.g. Cancer, Diabetes, Heart diseases, hypothyroidism \*26.05Heredity or genetic\*34.16Unhealthy lifestyle\*50.27Weakness of character, e.g. Weak personality59.98Substance abuse49.09Medications\*23.110Weak faith52.211Traumatic event in life, e.g. Death of a close one, physical injury, theft, disaster, etc.\*59.9**Treatment of Bipolar**12Psychiatric medications (e.g. antidepressants, mood stabilizers, antipsychotics, sedatives)\*47.613Talk to family or friends48.114Head bandage by traditional therapist58.715Smoking96.616Having an occasional alcoholic drink96.917Amphetamine or Hash97.418Electroconvulsive therapy under psychiatrist supervision\*12.719Using brief counselling therapies (e.g. cognitive and/or behavioral therapies)\*44.020Hitting and choking by faith healers97.821Involve in recreational activities46.4[^1] Statistical Analysis {#S0002-S2005} -------------------- Data were analyzed by using the Statistical Package for Social Studies (SPSS 22; IBM Corp., New York, NY, USA). Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables were expressed as percentages. A Student's *t*-test was used for continuous variables and the chi-square test was used for categorical variables. Because bipolar awareness score had a normal distribution, participants scoring more than or equal to the mean score of 12 points (\~\>70%) were defined arbitrary as bipolar literate.[@CIT0018] The differences in BP awareness were examined by sociodemographic characteristics, using BP awareness score ≤12 as a low awareness and BP awareness score \>12 as a high awareness, which is illustrated in [Table 2](#T0002){ref-type="table"}. Univariate and multivariate logistic regression models were used to calculate the odds ratio (OR) and its 95% confidence interval (CI). Low score (≤12) for BP awareness was used as an outcome (dependent variable) while sociodemographic variables were used as predictors (independent variables). Variables that were significant in univariate analysis were adjusted in multivariate analysis. A p-value \<0.05 was considered statistically significant.Table 2Univariate And Multivariate Logistic Regression Analysis To Predict Poor Bipolar Awareness Using Sociodemographic Characteristics (N=416)VariablesLow Awareness(Score ≤12)\*High Awareness(Score \>12)Univariate AnalysisMultivariate Analysis\*\*N(%)N(%)OR95% CIOR95% CI**Age** \<25 years109(40.1)70(48.6)1.00.50--2.17 25--44 years133(48.9)54(37.5)1.70.78--3.46 ≥45 years30(11.0)20(13.9)1.0**Gender** Male112(41.2)49(34.0)1.40.89--2.07 Female160(58.9)95(65.97)1.0**Marital Status** Married142(52.21)47(35.34)1.5\*\*\*1.01--2.311.080.56--2.06 Single130(47.79)86(64.66)1.01.0**Nationality** Non-Saudi60(22.1)13(9.0)2.6\*\*\*1.40--4.871.220.56--2.66 Saudi212(77.9)131(90.97)1.01.0**Educational level** High school or less82(30.2)23(15.97)2.0\*\*\*1.20--3.361.930.98--3.79 College and above190(69.9)121(84.0)1.01.0**Monthly income** Less than 5000 SR119(43.8)79(54.9)1.80.78--2.821.890.77--4.62 5000--10,000 SR93(34.19)16(11.1)2.2\*\*\*1.41--6.002.080.91--4.75 10,000--15,000 SR30(11.0)17(11.8)1.50.66--3.581.520.58--3.94 More than 15,000 SR30(11.0)32(22.2)1.01.0**Career status** Student68(25)75(52.1)1.01.0 Employed139(51.1)52(36.1)2.7\*\*\*1.69--1.692.44\*\*\*1.13--5.27 Unemployed65(23.9)17(11.8)3.6\*\*\*1.89--6.652.80\*\*\*1.10--7.11**Healthcare professions status** Yes35(48.6)37(51.4)1.01.0 No237(68.9)107(31.1)2.3\*\*\*1.40--3.922.15\*\*\*1.14--4.04[^2] Results {#S0003} ======= Sociodemographic Characteristics {#S0003-S2001} -------------------------------- Of the 542 questionnaires distributed, 416 were completed (response rate of 76.75%). The demographic characteristics of the surveyed population are presented in [Table 3](#T0003){ref-type="table"}.Table 3Socio-Demographic Characteristics (N=416)CharacteristicsNumber\*%\***1- Age** (Mean ± SD)\*29.5 ± 10.7**2- Gender** Male16138.7 Female25561.3**3- Marital Status**  Single21551.7 Married19045.7 Divorced61.4 Widowed51.2**4- Nationality** Saudi34382.5 Non-Saudi7317.5**5- Educational level** No formal education51.2 Elementary school education30.7 Intermediate school education174.1 High school education8119.5 Diploma degree286.7 Bachelor degree25060.1 Master degree266.3 PhD degree51.2 Other10.2**6- Career** Student13833.2 Employed19446.6 Unemployed8420.2**7- Health professional status** Yes14133.9 No27566.1**8- Monthly income** Less than 5000 SR19145.9 5000--10,000SR12329.6 10,000--15,000SR4711.3 More than 15,000 SR5513.2[^3] BP Awareness Based On Information And Knowledge {#S0003-S2002} ----------------------------------------------- [Table 1](#T0001){ref-type="table"} displays the positive responses from the 21-items scale provided by the participants on BP awareness based on information and knowledge about causes and treatments. Of the 416 participants, 50.5% had heard about BP, mainly from the internet (22.4%), social media (13%), and spouse/partner/relatives/friends (9.4%), while only 7% had heard it from clinics or hospitals. Moreover, 50% think that BP is common in Saudi Arabia. About half (45.2%) of the participants believed that neurophysiological or neurochemical imbalance is a cause of BP and 47.6% believed that psychiatric medications are a treatment for BP. In terms of BP origin, heredity or genetics was considered by 34.1% of participants and certain medical conditions were considered by 26% of them. Almost half of the participants believed that substance abuse is a cause of BP and most of them believed that smoking, having an occasional alcoholic drink, and amphetamine or hash use are not valid treatments to be used for BP (96.6%, 96.9%, and 97.4%, respectively). In addition, a traumatic life event, e.g., death of a close one, physical injury, theft, and other disaster, was considered as a cause of BP by 59.9% of the participants. Using brief counseling therapies (e.g. cognitive and/or behavioral therapies) was considered to be a treatment for BP by 44% of the participants. Less than half of the participants believed that weakness of character and a weak faith represented causes of BP, at 40.1% and 47.8%, respectively. Moreover, supernatural causes of BP were mentioned in addition to the provided scale, e.g. evil eye, magic, and jinn possessions at 19.2%, 18.8%, and 17.1%, respectively. On the other hand, prayer and faith-healing were mentioned as treatments for BP by 66.6% and 42.3% of the participants, respectively. Differences In The Awareness Of BP Based On Sociodemographic Characteristics {#S0003-S2003} ---------------------------------------------------------------------------- Using univariate analysis, all sociodemographic characteristics except age and gender were significantly associated with low awareness (BP awareness score ≤12). In multivariate analysis adjusted for the significant variables in univariate analysis including marital status, nationality, educational level, career, health professional status, and monthly income, only career status and healthcare professional status were significantly associated with low awareness ([Table 2](#T0003){ref-type="table"}). As indicated by adjusted odds ratios, students and healthcare professionals had significantly higher levels of awareness about BP ([Table 2](#T0003){ref-type="table"}). Students earned significantly higher numbers of positive responses in seven items compared to the employed or unemployed participants. Three items were from the causes of BP: "Neurophysiologic or neurochemical imbalances," "Medications," and "Weak faith" (p-value \<0.001, 0.005, and 0.013, respectively). The other four items were from the treatments of BP: "Psychiatric medications," "Having an occasional alcoholic drink," "Electroconvulsive therapy under psychiatrist supervision," and "Hitting or choking by faith healer" (p-value \<0.001, 0.049, \<0.001, and 0.001, respectively). Healthcare professionals also showed significantly more positive responses on four items compared to non-healthcare professionals. "Neurophysiologic or neurochemical imbalances" and "Medications" were endorsed by 66.7% and 33.3% of healthcare professionals as causes of BP compared to 40.7% and 20.9% of non-healthcare professionals (p-value \<0.001 and 0.023, respectively). "Psychiatric medications" and "Using brief counseling therapies" were endorsed by 62.5% and 55.6% of healthcare professionals as treatments for BP compared to 44.5% and 41.6% of non-healthcare professionals (p-value 0.005 and 0.030, respectively). In general, the level of awareness toward BP was not affected by the level of education. Nevertheless, compared with participants with lower education (high school or less), the participants with higher education (college and above) believed less that certain medical conditions may cause BP (22.3% versus 37.3%; p-value 0.003), believed more that certain medications may cause BP (25.7% versus 15.7%; p-value 0.039), and believed more that BP can be treated with psychiatric medications (50.7% versus 34.3%; p-value 0.004). ### Attitudes And Beliefs About BP {#S0003-S2003-S3001} For approaches to patients with BP, a substantial number (85.8%) believed that a person with BP can work effectively, 46.7% believed that patients with BP have to pull themselves together to get over it, and 31.8% considered it a shame to mention that someone in the family has BP. Regarding having a relationship with person with BP, 21.9% were not willing to maintain a friendship with someone with BP, 39.3% were not willing to marry a person who was previously diagnosed with BP, 19% were not willing to marry a person who has family members diagnosed with BP, and 16.8% would not consider telling their future spouse if they were diagnosed with BP ([Table 4](#T0004){ref-type="table"}).Table 4Attitudes And Beliefs About Bipolar Disorder (N=416)In Your Opinion:Agreement PercentageIf someone is suffering from bipolar, he/she has to pull him/herself together for getting over it46.7A person with bipolar disorder can work effectively85.8It is a shame to mention that someone in a family has bipolar disorder31.8You would not be willing to maintain a friendship with someone with bipolar disorder21.9You would not be willing to marry a person who was previously diagnosed with bipolar disorder39.3You would not be willing to marry a person who has family members diagnosed with bipolar disorder19You would not tell your future spouse if you were diagnosed with bipolar disorder16.8 Discussion {#S0004} ========== This study represents the first community-based survey of current public information, knowledge, beliefs, and attitudes toward BP in Saudi Arabia. This study showed that the internet and social media (35.4%) were major sources of information about BP, instead of clinics or hospitals (7%). This is not surprising as 77% of patients with BP reported using the internet to look up information about BP in an international multisite survey.[@CIT0022] Similarly, in the Arab world, there is a high interest in searching for online health information due to its fast and easy accessibility.[@CIT0023]--[@CIT0025] However, the lack of reliable and credible Arabic online health information has led to improving the quality of the Arabic health web through launching the King Abdullah Bin Abdulaziz Arabic Health Encyclopedia website by the National Guard Health Affairs (NGHA), in collaboration with the Health On the Net Foundation (HON) and the World Health Organization (WHO). This Encyclopedia has multiple articles about mental health, including BP[@CIT0026] Such certified websites need more advertisement on the internet to be used by the general population. The causes of BP cited by the Saudi population in this study, in descending order, were: traumatic life event, unhealthy lifestyle, substance abuse, neurophysiological or neurochemical imbalance, and heredity or genetics. These results are similar to those found in the United Kingdom and France.[@CIT0006],[@CIT0008],[@CIT0009] On the other hand, the highly endorsed treatments for BP by the Saudi population were: psychiatric medications and brief counseling therapies, which are also similar to the United Kingdom and Spain.[@CIT0006],[@CIT0007],[@CIT0009] However, in our study, supernatural beliefs and weak faith were endorsed by half of the participants as causes of BP. Therefore, unsurprisingly, prayer and faith-healing ‎were highly endorsed as a treatment for BP. Head bandaging by a traditional therapist was also a highly recognized treatment for BP. In this historical traditional method, a bandage is applied to the head, usually by wrap-around technique, that uses bony prominences as anchors or stays in order to treat mental illnesses.[@CIT0027] As per some traditional therapists' claims, this will prevent any air from entering between the skin and the scalp which may cause headache and other neuropsychiatric and somatic complaints.[@CIT0028] Comparable findings from a study conducted in Saudi Arabia investigating the prevalence of psychiatric disorders among visitors to faith healers reported that 12.3% of participants attributed their psychiatric illnesses to evil eye, magic (5.5%), social and financial stress (16.5%), and 49.3% reported more than one of the aforementioned reasons. However, none of the participants attributed psychiatric illness to a biological reason. In the same study, the most commonly used treatment was the Holy Quran (95.9%) and using blessed water (71.3%).[@CIT0015] In an Egyptian study, 40.8% of the patients with BP sought traditional faith healers and, of those, 62.2% did so before seeking psychiatric services.[@CIT0013] Also, a study conducted in Sudan reported that 20.7% attributed their mental illness to jinn, 19.3% to Shiatan, 28.4% to evil spirits, and 43.7% to magic.[@CIT0014] The belief in supernatural causes could be explained by poor knowledge about psychiatric conditions, projections, and defense mechanisms from the patients and their relatives to blame their problem on an external influence (supernatural causes), to relieve their guilty feelings regarding their responsibility for their illness, and to avoid the stigma associated with mental disorders and psychiatric medications[@CIT0017] As faith healers are highly appreciated and trusted by general population, we suggest to establish a mutually respectful relationship between mental health providers and faith healers, form a two-way dialogue to understand each other, in addition to licensing faith healers to formalize their practice, as well as educating them and promoting their training needs to recognize common psychiatric symptoms and refer in need patients to clinical care.[@CIT0015],[@CIT0017],[@CIT0029] These practitioners can also have a great impact on patients, so their role should not be neglected, and they should work in collaboration with the health mental system as per the WHO[@CIT0030] In our study, we found that only 12.7% believe that ECT can treat BP despite it being a safe and highly effective tool to manage BP that is severe, treatment resistant, or occurs during pregnancy.[@CIT0031] Brief educational intervention has been found to improve ECT knowledge and attitudes toward ECT.[@CIT0032] Nevertheless, ECT, but not antidepressant medication utilization, is associated with global government expenditures on mental health. In Saudi Arabia, although the overall health budget has been increasing annually, there is still no independent budget for mental health.[@CIT0033] Although the mortality rate for serious mental illnesses, including BP, was not significantly associated with recreational activities,[@CIT0034] involvement in recreational activities was highly endorsed as a treatment for BP in our study. Also, almost half of the participants in our study believe that if someone has BP, he/she has to pull him/herself together to get over it. These results hint at the tendency of underestimating the seriousness of BP and blaming the patient for having the illness. Adjusted estimates from our study showed that students and healthcare professionals had significantly higher awareness about BP, especially in terms of causes and treatments. Similar findings were reported by other studies that showed both psychiatrists and nurses had greater biopsychosocial understandings of mental illness than the lay public, likely from their training and experience[@CIT0035] Another study showed that non-physician mental health clinicians were generally knowledgeable about BP but less knowledgeable about the pharmacotherapy for treating it[@CIT0036] Also, a Saudi study found that males with lower levels of education and income were more likely to use faith-healing[@CIT0037] It calls to attention the high rate of bachelor's degrees in the sample (60%), which is comparable to the results of the demographic survey of Saudi Arabia that was done by the General Authority for Statistics in 2016, and showed that (52.7%) of Saudi population have high level of education (high school and above).[@CIT0038] The increased knowledge among students may be attributed to their frequent chatting, discussion, and file sharing on social network sites.[@CIT0039] These findings support the benefit of extending the training efforts of health clinicians (physician and non-physician)[@CIT0036] and of developing reliable information about BP for student and healthcare professionals on social networks using professional channels. Although our study reflected the positive attitudes and beliefs about BP in terms of ability to work effectively (85.8%), a recent study conducted in Saudi Arabia on stigma in mood disorders found that 66% of people with BP were unemployed mainly due to psychiatric or medical problems.[@CIT0040] In the social domain, negative attitudes in our study included (in descending order): being unwilling to marry a person who was previously diagnosed with BP, considering it a shame to mention that someone in the family has BP, being unwilling to maintain a friendship with someone with BP, to marry a person who has family members diagnosed with BP, or to tell their future spouse if they were diagnosed with BP. These negative attitudes about marriage may be explained by believing in heredity or genetic causes of BP, which was endorsed by a third of the participants. Similarly, a previous qualitative research study in Saudi Arabia revealed that the widely held belief that psychiatric disorders were either hereditary or incurable was a reason behind their stigma. Other potential reasons for the stigma held by the Saudi community that may contribute to the negative attitudes found in our study include traditions, cultural norms, upbringing, lack of public awareness, and fear of aggression and violence from psychiatric patients (Koura et al, 2012). The experience of stigma in Saudi Arabia has affected patients with BP in terms of their quality of life (36%), ability to interact with family (24%), and ability to make or keep friends (20%). Patients with BP also think that the average person is afraid of someone with a serious mental illness (48%) and they try to avoid situations that may be stigmatizing (56%) (Alateeq et al, 2018). This may lead them to stop taking their medications prior to marriage -- which is a stressful event -- resulting in relapses during their honeymoon. Also, getting married in a conservative Islamic country like Saudi Arabia, where a man will usually marry a woman without a prior personal relationship, may lead people to not choose a future wife who has a psychiatric disorder. Eventually, this may contribute to a high rate of being spouseless among patients with psychiatric illnesses in Saudi Arabia[@CIT0041] Moreover, higher levels of burden and depressed mood and lower levels of social support were associated with more perceived stigma among caregivers[@CIT0042] Therefore, it is crucial to consider the caregivers of patients with BP in addition to the patients themselves. Limitations {#S0005} =========== Although our study represents the first community-based survey of BP literacy among residents of the largest city in Saudi Arabia, we acknowledge a number of limitations. One of them is the convenient sampling of only one city in Saudi Arabia, which may limit generalizability of the study finding. Moreover, the cross-sectional design precludes making causal conclusions. Also, face validity was used as the main form of validity, which is a weak form of validity. It is subjective and cannot be quantified. Future studies should be longitudinal prospective ones, have a more representative samples, and use well-validated survey. Conclusions {#S0006} =========== This study is the first national survey in Saudi Arabia to document some of the misconceptions in public information, knowledge, beliefs, and attitudes about BP. The internet and social media were the participant's major sources of information, which highlights the need to support and improve the quality of and advertisement for Arabic online health information due to the fast and easily accessible nature of these sources. The impact of using social networking sites may also explain the high levels of awareness about BP's causes and treatments among students and healthcare professions in addition to their training and experience. Although the general population showed positive responses about BP's causes and treatments, supernatural beliefs are still predominant, which emphasizes the role of faith healers. Based on our findings, faith healers should be educated on the detection and treatment of mental diseases. When necessary, they can then refer patients to the mental health specialists and prevent any further delay in seeking help and appropriate treatment. Implementing a national community awareness program may raise the level of awareness through mass media communication strategies and schools' curriculum resources.[@CIT0043]--[@CIT0045] However, further studies are needed to investigate which potential public interventions will best improve the literacy of ‎BP and other mental illnesses. The authors would like to thank Dr. Mohammed Alangari and Dr. Abdulaziz Al-Muhanna for their great cooperation and participating in data collection. This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program. The abstract of this study was presented as a poster in the 19th Annual Conference of the International Society for Bipolar Disorders, Washington DC, USA, and in the 25th European Congress of Psychiatry (EPA 2017), Florence, Italy. Mental health literacy in bipolar disorder: A cross-sectional survey in Saudi Arabia. Alosaimi F, et al. *European Psychiatry*, Volume 41, S418. Disclosure {#S0007} ========== The authors report no conflicts of interest in this work. [^1]: **Notes:** \*Indicates positive statements, where positive responses were given for agreement. As the rest of statements were incorrect, positive responses were given for disagreement. [^2]: **Notes:** \*The outcome is low awareness (BP awareness score ≤12). \*\*Multivariate analysis is adjusted for the significant variables in univariate analysis including marital status, nationality, educational level, career, health professional status, and monthly income. \*\*\*Significant (p\<0.05). **Abbreviations:** OR, odds ratio; 95% CI, 95% confidence interval. [^3]: **Notes:** \*Number and percentage except otherwise marked; mean ± standard deviation.

Authored by a member of IFATS. Significance StatementArthroscopic microfracture (AM) and stem cell therapy have been used clinically to treat osteoarthritis (OA). This study evaluated the clinical effects of AM in the presence (treatment group) and absence (placebo group) of a stromal vascular fraction (SVF) injection in the knee for OA. The SVF was suspended in platelet‐rich plasma (PRP) before injection. Treatment efficacy differed significantly between placebo and treatment groups. All treatment group patients had significantly improved pain and arthritis index scores compared with the placebo group. These findings suggest that the SVF/PRP injection efficiently improved OA after 18 months. This study will be continuously monitored for 24 months. Introduction {#sct312040-sec-0001} ============ Osteoarthritis (OA) is a chronic progressive disease characterized by cartilage degeneration, osteophyte formation, bone reorganization, and loss of joint function [1](#sct312040-bib-0001){ref-type="ref"}. OA is the most frequent cause of disability among adults in the United States, and it occurred in \>10% of the U.S. adult population in 2009. In 2009, 905,000 knee and hip replacements were carried out in OA patients, costing approximately \$42.3 billion in total. At present, OA is mainly treated with pharmaceuticals [2](#sct312040-bib-0002){ref-type="ref"}, [3](#sct312040-bib-0003){ref-type="ref"}, hyaluronic acid [4](#sct312040-bib-0004){ref-type="ref"}, and neridronate [5](#sct312040-bib-0005){ref-type="ref"}, [6](#sct312040-bib-0006){ref-type="ref"}. However, these treatments only reduce symptoms and pain or control the inflammation process [7](#sct312040-bib-0007){ref-type="ref"} [8](#sct312040-bib-0008){ref-type="ref"} [9](#sct312040-bib-0009){ref-type="ref"}; none of these drugs actually prevents the progression of OA [10](#sct312040-bib-0010){ref-type="ref"}, [11](#sct312040-bib-0011){ref-type="ref"}. Arthroscopic microfracture (AM) has recently gained popularity as a therapy for OA [12](#sct312040-bib-0012){ref-type="ref"} [13](#sct312040-bib-0013){ref-type="ref"} [14](#sct312040-bib-0014){ref-type="ref"}, with some studies reporting significant symptom and functional improvement following the procedure [15](#sct312040-bib-0015){ref-type="ref"}. Consequently, AM is indicated as a routine treatment for OA. However, meta‐ and systematic analyses indicate that although AM initially improves OA symptoms [16](#sct312040-bib-0016){ref-type="ref"}, [17](#sct312040-bib-0017){ref-type="ref"}, this effect is only short term [16](#sct312040-bib-0016){ref-type="ref"}. In some cases, particularly among older people, AM can be harmful [16](#sct312040-bib-0016){ref-type="ref"}, [18](#sct312040-bib-0018){ref-type="ref"}, [19](#sct312040-bib-0019){ref-type="ref"}. As an alternative approach, OA has been treated using platelet‐rich plasma (PRP). PRP contains the pool of cytokines and growth factors stored in platelets [20](#sct312040-bib-0020){ref-type="ref"}. Some studies have shown that PRP improves OA symptoms [21](#sct312040-bib-0021){ref-type="ref"}, [22](#sct312040-bib-0022){ref-type="ref"}. However, this effect has not been not observed for a prolonged period [22](#sct312040-bib-0022){ref-type="ref"} [23](#sct312040-bib-0023){ref-type="ref"} [24](#sct312040-bib-0024){ref-type="ref"} [25](#sct312040-bib-0025){ref-type="ref"} [26](#sct312040-bib-0026){ref-type="ref"} [27](#sct312040-bib-0027){ref-type="ref"}. To improve the effects of PRP, previous studies have investigated the combined injection of PRP with stem cells. Mesenchymal stem cells (MSCs) in conjunction with PRP have been found to mildly improve cartilage healing, and had improved Knee Injury and Osteoarthritis Outcome Score subscores and visual analog pain scores (VAS) compared with PRP‐only therapy [28](#sct312040-bib-0028){ref-type="ref"}. Using this approach, it is hypothesized that MSCs differentiate into chondrocytes, which participate directly in cartilage repair and also contribute to immune modulation to inhibit knee joint inflammation. To date, various stem cell sources have been used to treat OA, such as bone marrow‐derived MSCs (BM‐MSCs) for autograft [29](#sct312040-bib-0029){ref-type="ref"} [30](#sct312040-bib-0030){ref-type="ref"} [31](#sct312040-bib-0031){ref-type="ref"} [32](#sct312040-bib-0032){ref-type="ref"} or allograft [33](#sct312040-bib-0033){ref-type="ref"}, adipose‐derived stem cells (ADSCs) [34](#sct312040-bib-0034){ref-type="ref"} [35](#sct312040-bib-0035){ref-type="ref"} [36](#sct312040-bib-0036){ref-type="ref"}, and peripheral blood‐derived stem cells [37](#sct312040-bib-0037){ref-type="ref"} [38](#sct312040-bib-0038){ref-type="ref"} [39](#sct312040-bib-0039){ref-type="ref"}. Other MSC sources include enriched mononuclear cells (MNCs) from bone marrow or umbilical cord blood, stromal vascular fractions (SVFs) from adipose tissue (AT) and purified MSCs obtained from culture‐expanded MNCs. In their published study, Enea et al. [40](#sct312040-bib-0040){ref-type="ref"} combined autologous bone marrow‐derived cells with microfracture to repair cartilage defects. Their results showed that single‐stage treatment of focal cartilage defects of the knee with microfracture followed by coverage with a polyglycolic acid (PGA)‐hyaluronic acid (HA) matrix augmented with autologous BMCs (PGA‐HA‐CMBMC) was safe and improved knee function. To date, no clinical studies have compared the efficacy of arthroscopic surgery with and without SVF injection in the treatment of OA. This study, therefore, aimed to evaluate the clinical effects of AM alone and in combination with SVF injection on the function and satisfaction of patients with OA. Materials and Methods {#sct312040-sec-0002} ===================== All experimental protocols were approved by the National Ethical Committee Ministry of Health, Vietnam. This study was registered at [clinicaltrials.gov](http://clinicaltrials.gov) with identifier NCT02142842. Inclusion and Exclusion Criteria {#sct312040-sec-0003} -------------------------------- All patients enrolled in this study were required to sign a consent form. Patient inclusion criteria were as follows: patients must be older than 18 years, have OA with grade 2 to 3 cartilage degeneration at the time of presentation, failed drug treatment and autologous cartilage transplantation, a Lysholm score less than 65, committed with an artheroplasty condition, and be HIV negative. A total of 30 patients were enrolled in the study: 15 patients were treated using traditional AM and 15 patients were treated with AM plus an injected mixture of SVF and PRP. The follow‐up time was 18 months for all patients. Liposuction {#sct312040-sec-0004} ----------- Patients were restricted from taking corticosteroids, aspirin, nonsteroidal anti‐inflammatory drugs and oriental herbal medications for a minimum of 1 week before liposuction. For the liposuction, patients were given spinal anesthesia with 2--3 ml (5 g/L) of bupivacaine hydrochloride. The lower abdomen was also anesthetized. Liposuction was performed using a tumescent solution (500 ml of normal saline and 0.5 ml of 1:1,000 epinephrine). We used a TriPort Harvester cannula (Tulip Medical Products, San Diego, CA, <http://www.tulipmedical.com>) and a 60‐ml BD Luer‐Lock syringe (BD Biosciences, East Rutherford, NJ, <http://www.bd.com>) to harvest 100--500 ml of adipose tissue from each patient. SVF Isolation {#sct312040-sec-0005} ------------- The SVF was isolated from the abdominal adipose tissue of each patient. Approximately 100 ml of lipoaspirate collected from each patient was divided into two 50‐ml sterile syringes. The syringes were stored in a sterile box at 2--8°C and immediately transferred to the laboratory. The SVF was isolated using an ADSC Extraction Kit (GeneWorld, Ho Chi Minh City, Vietnam, <http://geneworld.vn>) according to the manufacturer\'s instructions. Briefly, 100 ml of lipoaspirate was placed in a sterile, disposable 250‐ml conical centrifuge tube (Corning Life Sciences, Tewksbury, MA, <https://www.corning.com>) and washed twice with sterile phosphate‐buffered saline (PBS) by centrifugation at 400*g* for 5 minutes at room temperature. The adipose tissue was then digested using SuperExtract Solution (GeneWorld) containing collagenase at 37°C, for 30 minutes with agitation at 5‐minute intervals. The suspension was centrifuged again at 800*g* for 10 minutes, and the SVF was harvested as a pellet. The pellet was washed twice with PBS to remove any residual enzyme, and resuspended in PBS so that the cell quantity and viability could be measured using an automatic cell counter (NucleoCounter; Chemometec, Lillerød, Denmark, <https://chemometec.com>). Activated PRP Preparation {#sct312040-sec-0006} ------------------------- Activated PRP was derived from the peripheral blood of the same patients as the adipose tissue, using a New‐PRP Pro Kit (GeneWorld) according to the manufacturer\'s guidelines. Briefly, 20 ml of peripheral blood was collected in vacuum tubes and centrifuged at 800*g* for 10 minutes. The plasma fraction was collected and centrifuged at 1,000*g* for 5 minutes to produce a platelet pellet. Most of the plasma was then removed, leaving 3 ml of plasma for resuspension of the platelets. The inactivated PRP was then activated using activating tubes containing 100 µl of 20% CaCl~2~. Preparation of Product for Injection {#sct312040-sec-0007} ------------------------------------ The final injection product was composed of a mixture of the harvested SVF and activated PRP. Activated PRP was used to dilute the SVF to achieve a suitable dose for injection at 10^7^ SVF cells/ml. AM and SVF/PRP Injection {#sct312040-sec-0008} ------------------------ All patients in both groups received AM, which was used to confirm the degree of OA in each patient. Local chondral lesions were removed using medical instruments and an arthroscopic shaver. Microfractures were performed in accordance with the methods described by Steadman et al. [41](#sct312040-bib-0041){ref-type="ref"}. The 30 patients were grouped into a treatment group and a placebo group (*n* = 15 per group). After arthroscopic marrow stimulation by AM, the water flow was stopped and excess water was aspirated from the joint cavity. In the treatment group, the SVF and activated PRP mixture (5 ml per knee) was injected. Patients in the placebo group were injected with saline. Follow‐Up and Evaluation {#sct312040-sec-0009} ------------------------ Patients were monitored in the hospital for 1 week postinjection. During this time, all complications, including shock, infection, and inflammation, were noted. After this, patients were followed for 18 months. Western Ontario and McMaster Universities Arthritis Index (WOMAC), Lysholm, and VAS scores were assessed 1, 6, 12, and 18 months after surgery. Radiographic imaging and magnetic resonance imaging (MRI) were performed 6 and 12 months post‐treatment. In this study, we used the modified VAS scores. with 4 indicating no pain; 3, mild pain; 2, moderate pain; 1, severe pain; and 0, worst pain possible. Patients began continuous passive motion 4--5 days post‐treatment. Partial weight bearing was permitted at 2 weeks, progressing to full weight bearing 4 weeks after surgery. Isometric quadriceps and hamstring training with straight‐leg raises were advised during the non‐weight‐bearing period. Light sport activities such as swimming, cycling, or jogging on even, soft ground were permitted at 6 months. Permission to participate in unrestricted sports activity was given after 12 months. Statistical Analysis {#sct312040-sec-0010} -------------------- Results were expressed as the mean ± SD. One‐way analysis of variance and two‐tailed *t* tests were used for all statistical analyses, which were performed with GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, <https://www.graphpad.com>). *p* values \<.05 were considered statistically significant. Results {#sct312040-sec-0011} ======= Patient Characteristics {#sct312040-sec-0012} ----------------------- This study was performed from April 2013 to September 2015 at two hospitals (Van Hanh General Hospital and 115 Hospital, both in Ho Chi Minh City, Vietnam). The 30 patients who satisfied the study standard were divided into 2 groups: placebo (*n* = 15) and treatment (*n* = 15). Demographic analysis found that these groups had an equivocal age, body mass index, sex, and Kellgren‐Lawrence OA grade (Table [1](#sct312040-tbl-0001){ref-type="table-wrap"}). The Kellgren‐Lawrence grade was based on x‐rays, and was confirmed during AM ( supplemental online Fig. 1). ###### Study participant demographic characteristics -- -- Adverse Effects {#sct312040-sec-0013} --------------- No adverse events were observed during the study in either group. We identified four cases with complications not related to the AM or SVF injection; these complications included high blood pressure, chest pain, dyspnea, and urinary retention. Changes in WOMAC Scores {#sct312040-sec-0014} ----------------------- Figure [1](#sct312040-fig-0001){ref-type="fig"} shows the WOMAC score results. Pretreatment WOMAC scores were equivocal, with a small nonsignificant difference observed between the placebo and treatment group (47.27 ± 17.13 vs. 42.87 ± 16.29, respectively; *p* \> .05). At 6 and 12 months after treatment, the WOMAC scores in both groups significantly decreased compared with the pretreatment scores. In the placebo group, WOMAC scores decreased from 47.27 ± 17.13 to 23.27 ± 15.61 and 25.60 ± 19.69 at 6 and 12 months after surgery, respectively. In the treatment group, WOMAC scores decreased from 42.87 ± 16.19 to 19.27 ± 14.87 and 17.33 ± 14.91 at 6 and 12 months after surgery, respectively. At 6 and 12 months after surgery, the differences in the WOMAC scores between the treatment and placebo groups were nonsignificant (*p* \> .05). However, a slight difference was observed between the 2 groups 12 months after surgery. WOMAC scores in the treatment group gradually decreased at 6 and 12 months compared with the pretreatment scores, although the WOMAC score 12 months after surgery was slightly increased compared with the score 6 months after the procedure. {#sct312040-fig-0001} The difference in the WOMAC scores of the placebo and treatment groups became more pronounced after 18 months of monitoring. In the placebo group, the WOMAC score increased from 25.60 ± 19.69 at 12 months to 37.08 ± 21.45 at 18 months. More importantly, WOMAC scores at 18 months in the placebo group were not significantly different compared with pretreatment scores. The WOMAC scores of the treatment group decreased at 6, 12, and 18 months (19.27 ± 14.87, 17.33 ± 14.91, and 12.40 ± 13.44, respectively) after surgery compared with the pretreatment score (42.87 ± 16.29). The 18‐month WOMAC scores were also significantly different between the placebo and treatment groups (*p* \< .05; Fig. [1](#sct312040-fig-0001){ref-type="fig"}). Changes in Lysholm Scores {#sct312040-sec-0015} ------------------------- The results presented in Figure [2](#sct312040-fig-0002){ref-type="fig"} show that Lysholm scores changed in both the treatment and placebo groups, but in opposite directions. The Lysholm scores increased significantly in both groups 6 months post‐treatment compared with the pretreatment score (*p* \< .05). In the placebo group, however, the Lysholm scores were decreased dramatically 18 months after surgery to a level comparable to the pretreatment score (75.80 ± 16.05, 76.47 ± 12.44, and 65.17 ± 14.74 at 6, 12, and 18 months, respectively, compared with 64.13 ± 10.19 pretreatment). In the treatment group, the Lysholm scores gradually increased over 6, 12, and 18 months compared with pretreatment scores (80.53 ± 7.86, 82.13 ± 8.98, 84.73 ± 19.54, and 53.47 ± 14.56, respectively). At 18 months, the mean Lysholm score of the placebo and treatment groups was significantly different (*p* \< .05). {#sct312040-fig-0002} Changes in VAS Scores {#sct312040-sec-0016} --------------------- Similar to the Lysholm scores, VAS scores in both the treatment and placebo groups changed, but in opposite directions (Fig. [3](#sct312040-fig-0003){ref-type="fig"}). In the placebo group, VAS scores significantly increased after 6 months compared with those at pretreatment (2.67 ± 0.62 vs. 1.40 ± 0.51, respectively; *p* \< .05). However, the scores then decreased from 12 to 18 months (2.53 ± 0.83 and 2.08 ± 1.08, respectively). In the treatment group, VAS scores continuously increased from 1.60 ± 0.83 at pretreatment to 3.01 ± 0.59, 3.20 ± 0.68, and 3.47 ± 0.74 at 6, 12, and 18 months, respectively (*p* \< .05). {#sct312040-fig-0003} Cartilage Injury Evaluation by MRI {#sct312040-sec-0017} ---------------------------------- Based on the MRI results and the Outerbridge classification system (OS), changes in cartilage injury were recorded and are presented in Figure [4A](#sct312040-fig-0004){ref-type="fig"}. OS scores gradually increased in the placebo group from pretreatment to 6, 12, and 18 months post‐treatment (2.67 ± 1.35, 2.93 ± 1.34, and 3.20 ± 1.08, respectively). However, scores decreased in the treatment group from pretreatment to 12 months post‐treatment (3.33 ± 0.97 vs. 2.93 ± 0.88, respectively). {#sct312040-fig-0004} Although differences in OS scores were nonsignificant (*p* \> .05), the trend was clearly different between the two groups: OS scores increased in the placebo group over time but decreased in the treatment group. MRI imaging demonstrated that the cartilage layer was thicker in the treatment group 12 months after AM ( supplemental online Fig. 3). Bone Marrow Edema {#sct312040-sec-0018} ----------------- Bone marrow edema (BME) was also recorded based on the MRI results. The results presented in Figure [4B](#sct312040-fig-0004){ref-type="fig"} and supplemental online Figure 2 show that BME was considerably deceased 12 months after surgery in the treatment group, although it was moderately increased in the placebo group. In the treatment group, BME gradually decreased from pretreatment to 6 and 12 months post‐treatment (2.40 ± 0.63, 1.86 ± 0.64, and 1.33 ± 0.62, respectively), with a significant difference at 12 months (*p* \< .05). In the placebo group, BME increased moderately at 6 to 12 months post‐treatment compared with pretreatment measurements (1.87 ± 0.74 at pretreatment vs. 2.00 ± 0.53; 2.13 ± 0.64 at 6 to 12 months post‐treatment, respectively). Correlating OA Stage With Treatment Efficacy {#sct312040-sec-0019} -------------------------------------------- Although the number of patients included in this study was low, we were able to evaluate the relative efficacy of AM plus SVP/PRP treatment between patients with stage 2 (*n* = 4) and stage 3 (*n* = 11) OA. The results presented in Figure [5A](#sct312040-fig-0005){ref-type="fig"} and [5B](#sct312040-fig-0005){ref-type="fig"} shows that the SVF/PRP injection affected patients with stage 2 and 3 OA differently with respect to both WOMAC and Lysholm scores, with significant differences observed at 18 months post‐treatment. Although the WOMAC and Lysholm scores were significantly improved in both stage 2 and 3 OA groups at 18 months post‐treatment compared with pretreatment, only in stage 2 OA patients were both WOMAC and Lysholm scores significantly improved at 18 months compared with 12 months post‐treatment (*p* \< .05). {#sct312040-fig-0005} When we separately compared the stage 2 and stage 3 treatment groups with the placebo group, the differences became clearer (Fig. [6](#sct312040-fig-0006){ref-type="fig"}). Compared with the stage 2 OA members of the placebo group, the stage 2 treatment group had significantly improved WOMAC and Lysholm scores. Compared with the stage 3 OA placebo group, the stage 3 treatment group was improved but to a lesser extent. Patients in the stage 2 treatment group continuously improved in both their WOMAC and Lysholm scores at 12 and 18 months post‐treatment, whereas the improvement rate was slower in the stage 3 OA group. {#sct312040-fig-0006} Changes in Knee Joint Function {#sct312040-sec-0020} ------------------------------ The knee joint function of treated patients was significantly improved at 18 months post‐treatment, and their joint motion amplitude (JMA) increased from 116.2 ± 27.1 at pretreatment to 138.8 ± 12.0 at 18 months post‐treatment. JMA also increased in the placebo group from 120.6 ± 24.3 pretreatment to 133.3 ± 17.9 at 18 months post‐treatment but to a lesser extent than in the treatment group. Discussion {#sct312040-sec-0021} ========== AM is the conventional method to treat cartilage degeneration, including OA lesions. However, the benefits of AM are gradually lost in the 18 months following treatment. This study aimed to combine the AM approach with an injection of SVF and PRP to improve treatment efficacy. Autologous ADSCs and autologous PRP from the peripheral blood were used in this study. Although previous studies used allogeneic‐derived MSCs to effectively improve OA, we used an autologous source to minimize the side effects relating to host factors, specifically inflammation. Both SVF and ADSCs (the purified form of SVF) have been used clinically in the treatment of conditions such as multiple sclerosis [42](#sct312040-bib-0042){ref-type="ref"}, femoral head necrosis [43](#sct312040-bib-0043){ref-type="ref"}, [44](#sct312040-bib-0044){ref-type="ref"}, chronic myocardial ischemia [45](#sct312040-bib-0045){ref-type="ref"}, critical limb ischemia, progressive supranuclear palsy [46](#sct312040-bib-0046){ref-type="ref"}, and acute respiratory distress syndrome [47](#sct312040-bib-0047){ref-type="ref"}. Our results indicate that AM with a combined SVF/PRP injection significantly improved and prolonged the treatment efficacy of AM for OA. At 6 months post‐treatment, the WOMAC, Lysholm, and VAS scores were significantly improved compared with pretreatment scores. These scores were further and significantly improved at 12 and 18 months post‐treatment in the SVF/PRP group. Some of the patients obtained scores similar to that of healthy individuals. The WOMAC is a widely used, proprietary set of standardized questionnaires used by health professionals to evaluate the condition of patients with OA of the knee and hip, including pain, stiffness, and joint function. Higher WOMAC scores correspond with a higher level of pain, stiffness, and functional limitation. In the treatment group, the mean WOMAC score was 12.40 ± 13.44 at 18 months after surgery. The WOMAC Index is sensitive to change and, therefore, is considered a suitable scale to assess OA. In addition to the WOMAC Index, the Lysholm scale is one of the most commonly used scoring systems for measuring OA. It was first published in 1982 and comprises 8 questions designed to evaluate joint instability in younger patients. This scale measures disability and focuses on the patient\'s perception of their ability to perform activities of daily living, as well as various intensities of physical activity [48](#sct312040-bib-0048){ref-type="ref"}. According to this scale, a score of 84--90 is considered a good result. The average Lysholm score of patients in the AM plus SVF/PRP group 18 months after treatment was 84.73 ± 19.54. Supporting the change seen in the WOMAC and Lysholm scores, the VAS scale scores also showed clear improvements in the treatment group. The VAS is a psychometric response scale that can be used in questionnaires. It is a measurement approach for subjective characteristics or attitudes that cannot be directly measured. The VAS scale for pain is divided into 4 points: 4 (no pain), 3 (mild pain), 2 (moderate pain), and 0 (severe pain). The WOMAC, Lysholm, and VAS scores demonstrated that at 18 months post‐treatment, all patients in the treatment group had significantly improved pain, movement, and capacity for physical activity. Some patients' scores appeared similar to those of healthy individuals. AM resulted in significantly reduced pain and improved knee function 6 months after the procedure, and these persisted for up to 12 months. However, by 18 months post‐AM, the symptoms of OA in the majority of patients reverted back to pretreatment levels. These results support those of several published studies. Thorlund et al. [16](#sct312040-bib-0016){ref-type="ref"} reviewed 1,789 reports of AM used in degenerative knees. They found that AM had a small, inconsequential benefit in the management of OA, was effective for a limited time, and any benefits were absent 1 to 2 years after surgery. Furthermore, in patients with moderate to severe OA of the knee, Risberg [18](#sct312040-bib-0018){ref-type="ref"} showed that the addition of arthroscopy to a regimen of physiotherapy and medication did not improve the physical function, pain, or health‐related quality of life of patients with OA. Our results showed that SVF in combination with PRP significantly improved the outcomes of AM for OA of the knee. SVF and PRP not only maintained and prolonged the effects of AM, but also increased overall treatment efficacy. All WOMAC, Lysholm, and VAS scores were noticeably improved compared with AM alone at 6 and 12 months post‐treatment. From the MRI results, we showed that OS scores and BME were significantly improved at 12 months post‐treatment. Whereas OS scores and BME improved after AM in the placebo group, both of these indicators were decreased in the treatment group. In particular, BME was significantly decreased at 12 months post‐treatment. OS classification is a grading system for joint cartilage breakdown: grade 0 represents normal joint cartilage; grade 1 represents cartilage with softening and swelling; grade 2 represents a partial‐thickness defect with fissures on the surface that do not reach the subchondral bone or exceed 1.5 cm in diameter; grade 3 represents fissuring to the level of subchondral bone in an area with a diameter more than 1.5 cm; and grade 4 represents exposed subchondral bone. Our results showed that the OS scores decreased from 3.33 ± 0.97 pretreatment to 2.93 ± 0.88 at 12 months post‐treatment in the treatment group. These results showed that the cartilage layer was thicker 12 months after the knee was injected with SVF and PRP, a finding congruent with our previously published study [36](#sct312040-bib-0036){ref-type="ref"}. Other studies have shown that SVF in combination with PRP stimulates cartilage regeneration, with a thicker cartilage layer observed using post‐treatment MRI evaluation [34](#sct312040-bib-0034){ref-type="ref"}, [44](#sct312040-bib-0044){ref-type="ref"}. We have shown in a mouse model that SVF and PRP can stimulate knee cartilage regeneration [49](#sct312040-bib-0049){ref-type="ref"}. The impact of a SVF/PRP injection in our study was also similar to effects noted in canine [50](#sct312040-bib-0050){ref-type="ref"} [51](#sct312040-bib-0051){ref-type="ref"} [52](#sct312040-bib-0052){ref-type="ref"}, rabbit [53](#sct312040-bib-0053){ref-type="ref"}, [54](#sct312040-bib-0054){ref-type="ref"}, horse [55](#sct312040-bib-0055){ref-type="ref"}, rat [56](#sct312040-bib-0056){ref-type="ref"}, and goat [57](#sct312040-bib-0057){ref-type="ref"} models. Cartilage regeneration in these models was attributed to neocartilage triggered by SVF and PRP. In a rabbit model, Dragoo et al. [58](#sct312040-bib-0058){ref-type="ref"} showed that autologous ADSCs were able to re‐establish the joint surface in rabbits. They found neocartilage was present in 100% of treated rabbits (12 of 12), whereas only 8% of control rabbits (1 of 12) had neocartilage. The mechanisms of action of SVF and ADSCs have been investigated in previous studies. In 2003, Gimble and Guilak [57](#sct312040-bib-0057){ref-type="ref"} showed that injected ADSCs were able to protect and heal injured cartilage. Other benefits of ADSCs have been reported for cartilage regeneration, including anti‐inflammatory properties [59](#sct312040-bib-0059){ref-type="ref"}, [60](#sct312040-bib-0060){ref-type="ref"} and immune modulation. ADSCs can produce and secrete cytokines and growth factors that can trigger chondrogenesis, including transforming growth factor‐β (TGF‐β), bone morphogenic protein 2 (BMP‐2), BMP‐4, BMP‐7, insulin‐like growth factor 1, and fibroblast growth factor 2 (FGF‐2). ADSCs also produce cytokines that modulate the recipient immune system, including TGF‐β, hepatocyte growth factor, nitric oxide, indolamine‐2,3‐dioxygenase, TNF‐α [61](#sct312040-bib-0061){ref-type="ref"} and interferon‐γ [62](#sct312040-bib-0062){ref-type="ref"}, [63](#sct312040-bib-0063){ref-type="ref"}. In vitro, cultured ADSCs suppress the host\'s immune response and the T‐cell proliferation as effectively as do BM‐MSCs [61](#sct312040-bib-0061){ref-type="ref"}, [64](#sct312040-bib-0064){ref-type="ref"}. Further studies have demonstrated that ADSCs actually stimulate a lesser proliferative response than do allogeneic PBMCs, but a similar response to BM‐MSCs [65](#sct312040-bib-0065){ref-type="ref"} [66](#sct312040-bib-0066){ref-type="ref"} [67](#sct312040-bib-0067){ref-type="ref"}. These findings suggest that ADSCs can replace BM‐MSCs in the field of regenerative medicine [61](#sct312040-bib-0061){ref-type="ref"}. The anti‐inflammatory roles of ADSCs and PRP were also confirmed in our study by the obvious improvement of BME in the treatment group. BME is a condition characterized by the accumulation of excessive fluid in bone marrow‐related structures. BME is a predictor for the progression of knee OA in the compartment ipsilateral to the bone marrow lesion [68](#sct312040-bib-0068){ref-type="ref"}. BME was significantly reduced and the cartilage layer thickness was increased in the SVF/PRP‐treatment group, indicating that OA was significantly improved. The increased BME observed in the placebo group may have been related to the progression of OA and inflammation after AM. Cartilage regeneration in OA knees following AM and the combined SVF/PRP injection was likely because of the combination of SVF and PRP. However, SVF is likely to be the main contributor to this healing response. PRP has been used to treat knee OA in previous studies [69](#sct312040-bib-0069){ref-type="ref"} [70](#sct312040-bib-0070){ref-type="ref"} [71](#sct312040-bib-0071){ref-type="ref"}, but almost all of these studies showed that PRP significantly reduced short‐term pain without concurrent cartilage regeneration [21](#sct312040-bib-0021){ref-type="ref"}, [69](#sct312040-bib-0069){ref-type="ref"}, [71](#sct312040-bib-0071){ref-type="ref"}, [72](#sct312040-bib-0072){ref-type="ref"}. In combination with ADSCs, PRP can improve chondrogenesis in vitro and in vivo [73](#sct312040-bib-0073){ref-type="ref"}. The components of PRP play important roles in stimulating grafted and endogenous cell growth and differentiation. PRP contains at least six known growth factors, including: platelet‐derived growth factor, which promotes blood vessel growth and cell division; TGF‐β, which promotes cell mitosis and bone metabolism; vascular endothelial growth factor, which promotes blood vessel formation; epidermal growth factor, which promotes cell growth and differentiation, angiogenesis, and collagen formation; FGF‐2, which promotes cell differentiation and angiogenesis; and IGF, which is a regulator of all of the body\'s cell types [74](#sct312040-bib-0074){ref-type="ref"} [75](#sct312040-bib-0075){ref-type="ref"} [76](#sct312040-bib-0076){ref-type="ref"}. We also observed that the regeneration response of cartilage to injected SVF/PRP was different between patients with grade 2 and 3 OA. Both WOMAC and Lysholm scores showed that the recovery of patients with grade 2 OA was faster than that of those with grade 3 disease. In particular, the improvement of WOMAC and Lysholm scores in patients with OA grade 2 were significant at 18 months compared with 12 months post‐treatment. This demonstrated that OA grade 2 was treated with higher efficacy than OA grade 3 following SVF/PRP injection. Although this study was limited with respect to the sample size of patients with either grade 2 or 3 OA, these results are similar to other treatment options for OA, such as HA and PRP injections [24](#sct312040-bib-0024){ref-type="ref"}, [25](#sct312040-bib-0025){ref-type="ref"}. Finally, JMA was compared between treated and placebo group patients. JMA was clearly increased in the treatment group compared with the placebo group, which agrees with both our subjective and radiographic analyses. More importantly, almost all patients in the treatment group exhibited a JMA similar to healthy individuals. The mean JMA was 138.8 ± 12 at 18 months post‐treatment. The mean JMA of healthy individuals has been reported to be 140.0 (range, 113.9--166.4) [77](#sct312040-bib-0077){ref-type="ref"}. We believe that our study is the first to evaluate AM with and without SVF for OA treatment with an 18‐month follow‐up time. Although Freitag et al. [78](#sct312040-bib-0078){ref-type="ref"} recently performed a similar study to ours, their follow‐up time was only 12 months. Conclusion {#sct312040-sec-0022} ========== This study showed that AM with SVF/PRP injection was effective for knee OA and had better long‐term outcomes than AM alone. Our preliminary analysis also showed that grade 2 knee OA was improved to a greater extent than grade 3 disease following AM with SVF injection. AM with SVF injection significantly improved WOMAC, Lysholm, and VAS scores over the entire 18‐month study period. MRI findings showed that the regenerated cartilage layer of patients treated with AM and SVF was thicker at 12 and 18 months after the procedure. Furthermore, the JMA of SVF/PRP‐treatment patients 18 months after surgery was significantly improved and comparable with that of healthy individuals. No adverse effects were recorded in any treated patients. From these findings, we conclude that AM with SVF/PRP injection may be a suitable treatment for grade 2 and 3 OA of the knee. Author Contributions {#sct312040-sec-0023} ==================== P.D.N.: conception and design, administrative support, provision of study material or patients; T.D.‐X.T., H.T.‐N.N., and H.T.V.: provision of study material or patients, collection and/or assembly of data; P.T.‐B.L.: conception and design, administrative support, provision of study material or patients, collection and/or assembly of data; N.L.‐C.P. and N.B.V.: provision of study material or patients; N.K.P.: conception and design, data analysis and interpretation; P.V.P.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript. Disclosure of Potential Conflicts of Interest {#sct312040-sec-0024} ============================================= The authors indicated no potential conflicts of interest. Supporting information ====================== ###### Supporting Information ###### Click here for additional data file. This study was funded in part by GeneWord Ltd.

1. Introduction {#sec0005} =============== In 1785 William Withering observed that digitalis glycoside beyond its diuretic effects also \"has a power over the motion of the heart with a degree yet unobserved in any other medicine\" \[[@bib0005]\]. In subsequent years, it was recognised that digitalis could also stabilise a rapid and irregular heart-beat \[[@bib0010]\] and some hundred years later, Fothergill reported that digitalis also enhanced ventricular contraction \[[@bib0015]\]. In the 1980s Hamlyn and colleagues reported the presence of endogenous sodium potassium pump inhibitors, termed cardiotonic steroids (CTS), in patients with essential hypertension \[[@bib0020]\] and healthy controls \[[@bib0025], [@bib0030], [@bib0035]\]. Despite almost four decades of intense research into the role of endogenous CTS in physiology and disease unanswered questions, heightened discussion and disagreement dominate the research field. Even the mere existence of endogenous, circulating CTS is questioned! Further disagreements and questions relate to their structure, biological target, interaction with digoxin, mechanism of action and function in health and disease. Digoxin, an archetypal CTS, is still used for treatment of patients with atrial fibrillation and heart failure. Research into the role of endogenous CTS continues and the clinical use of digoxin, while reduced, is unlikely to come to a halt. Aim of this review is to highlight the current state-of-the-art of the endogenous CTS in health and disease and discuss some of the key questions that remain to be addressed. 2. Sodium potassium pump is the primary target for CTS {#sec0010} ====================================================== Sodium potassium pump (NKA) is ubiquitous and as such plays a key role in many physiological processes in the body. NKA uses the free energy of hydrolysis of ATP to exchange three intracellular Na^+^ ions for two extracellular K^+^ ions, thus setting the electrochemical gradient for both Na^+^ and K^+^ across the cell membrane. NKA is therefore vital for maintaining the resting potential and Na^+^ and K^+^ gradients in almost every eukaryotic cell. These gradients ensure basic cellular homeostasis such as regulation of cell volume, essential ionic, glucose and amino acid transport processes. In excitable cells NKA activity restores the Na^+^ and K^+^ gradients following depolarisation and in the kidney its activity provides the driving force for Na^+^ reabsorption essential to control extracellular volume and blood pressure. Among the many Na^+^-dependent transmembrane transport processes in muscle cells, the activity of the NKA drives Na^+^/Ca^2+^ exchanger (NCX) and thus regulates the concentration of Ca^2+^ in both the cytosol and the sarcoplasmic reticulum (SR). Furthermore, recent evidence points towards a crucial role for the NKA in neurological function \[[@bib0040],[@bib0045]\]. The work of Skou \[[@bib0050]\] and others on the NKA structure and function over many years identified that CTS bind to the conserved binding site and thereby inhibit NKA activity. Commonly referred to as the "ouabain binding site", CTS binding site resides within the catalytic domain of the NKA α subunit \[[@bib0055]\], see [Fig. 1](#fig0005){ref-type="fig"}. It is highly conserved across the species, suggesting an important physiological function. Mutations of the ouabain binding site, leading to CTS desensitisation, have been identified in monarch butterflies and leaf beatles that feed on plants rich in digitalis \[[@bib0060],[@bib0065]\]. Similar mutations are also observed in toads, presumably developed as adaptational protection to high concentrations of CTS in their skin \[[@bib0070]\]. Remarkably, mice and rats also contain a CTS insensitive α-1 isoform \[[@bib0075],[@bib0080]\]. It is unclear as to the benefits of this adaptational mutation and further studies are warranted and could shed light on the physiological and pathophysiological roles of CTS.Fig. 1**Structure of the NKA and the chemical structures of its commonly described ligands ouabain, marinobufagenin and digoxin.** NKA is made up of the α and β subunits and the accessory FXYD protein. Conserved CTS binding site is found within the α subunit. Three commonly studied CTS are shown on the right. Ouabain and marinobufagenin have been detected in circulation in patients and healthy controls. Ouabain and digoxin have a five-membered lactone ring, whereas marinobufagenin contains a six-membered lactone ring.Fig. 1 NKA is composed of a catalytic α subunit, regulatory β subunit and an accessory FXYD protein \[[@bib0085],[@bib0090]\]. β subunit stabilises the NKA and helps the complex traffic through the secretory pathway to the plasma membrane \[[@bib0095],[@bib0100]\]. In the heart, FXYD protein named phospholemman modulates NKA activity in response to kinase/phosphatase mediated stimuli \[[@bib0090],[@bib0105], [@bib0110], [@bib0115], [@bib0120]\]. NKA functional unit is made up of α and β macromolecular complex with four α (α1, α2, α3 and α4), three β isoforms (β1, β2 and β3) \[[@bib0125],[@bib0130]\] and a FXYD protein, allowing a formation of a number of different NKA isozymes with distinct transport and pharmacological properties \[[@bib0135]\]. Whether multiple isozyme combinations exist within the same heart, whether they demarcate distinct chambers or even distinct topologies within a single cardiomyocyte, is not known and warrants investigation. It has been clearly demonstrated that CTS differentially affect alpha subunits, yet little information exists as to the biological relevance of these (see Pavlovic review \[[@bib0140]\]). Preliminary work shows that in the heart and kidneys α1 is the dominant isoform regulating bulk Na^+^ whereas α2 plays a more prominent role in smooth muscle contraction and has been shown to significantly contribute to regulation of T-tubular Na^+^ and thus modulation of cardiac contraction, via its indirect effects on the Na^+^/Ca^2+^ exchanger (NCX) \[[@bib0090],[@bib0145]\]. Digoxin and ouabain have all been shown to reduce heart rate \[[@bib0150]\], and this is likely to be the primary mechanism for beneficial effects observed in patients with atrial fibrillation and heart failure. When compared to ivabradine, another rate lowering drug, digoxin performed as well in risk reduction for worsening heart failure \[[@bib0155]\]. NKA activity has been demonstrated to play an important role in the brain. Mutations in the brain α1 and α3 NKA isoforms affecting the NKA activity have been identified and linked with the neuronal defects \[[@bib0040],[@bib0045]\]. Whether CTS-mediated regulation of the brain NKA plays a significant functional role in neuronal control remains to be determined.Relevant questions:What is the relevance of different NKA isoforms in different tissues?Are the NKA isoforms differentially expressed in different chambers of the heart?Why is the CTS binding site not conserved in mice and rats?What evolutionary advantage does desensitisation of the CTS binding site confer to mice and rats? 3. Structure and biosynthesis of the endogenous CTS {#sec0015} =================================================== Whilst the existence of an endogenous, circulating, natriuretic factor, targeting the NKA may have been suggested as early as 1960s, Hamlyn and colleagues were the first to demonstrate and identify the presence of a ouabain-like compound in human plasma \[[@bib0030],[@bib0035]\]. Initial mass spectrometry studies showed the compound to be similar to ouabain, with adrenals showing 500-fold higher concentrations, possibly indicating the origins \[[@bib0025]\]. Development of an immunoassay for detection of endogenous ouabain \[[@bib0160]\] allowed multiple studies in serum of experimental animals and humans. Subnanomolar concentrations of ouabain were detected in the blood of patients with essential hypertension \[[@bib0165], [@bib0170], [@bib0175]\], heart failure \[[@bib0180],[@bib0185]\], acute kidney injury \[[@bib0190], [@bib0195], [@bib0200]\], and end stage renal failure \[[@bib0205]\]. Some evidence exists for the presence of digoxin in human urine \[[@bib0210]\] and of deglycosylated congeners of digoxin in mammals \[[@bib0215],[@bib0220]\]. Marinobufagenin was isolated and identified in urine of patients with myocardial infarction \[[@bib0225]\] and serum of patients with terminal renal failure \[[@bib0230]\]. Structurally related telocinobufagin, the reduced form of marinobufagenin, was identified as a constituent of human serum in patients with terminal renal failure \[[@bib0230]\], at higher concentration than that of marinobufagenin and ouabain. It is likely that other bufadienolides may also be present in circulation. Indeed there are reports of proscillaridin A- \[[@bib0235]\] and bufalin-like \[[@bib0240]\] immunoreactive substances detected in human serum. Some of the studies have relied on the use of antibody-based ELISA methods for detection of the endogenous CTS whereas others have used high-resolution mass spectrometry and NMR, though often without detailed validation. Baecher et al. have recently developed a validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for detection and quantification of ouabain, with a lower detection limit of 1.7 pmol/L \[[@bib0245]\]. Controversially, they found no ouabain in the plasma of 5 control and 25 heart failure patients and thus concluded that endogenous ouabain detected previously is an artifact. Disqualifying 30 years of work with a single study in 30 patients, though well executed, seems premature. Unsurprisingly, this precipitated a flurry of robust exchanges between the interested parties \[[@bib0250], [@bib0255], [@bib0260]\] with strong views being expressed on both sides. In their review \[[@bib0265]\], Hamlyn and Blaustein rightly argue for possible presence of several isomers of ouabain, and considering detection of several structurally related CTS, e.g. marinobufagenin, telocinobufagin, deglycosylatyed digoxin congeners, it is possible that several CTS species exist in circulation. No attempts have been made thus far to develop validated LC-MS/MS methods for detection and quantification of multiple CTS in plasma or serum. With the recognition that steroids do not exist nor function in isolation of others, development of methods for detection of CTS panel is necessary in order to move the field forward. Biosynthesis pathways for the CTS are unclear, though existing evidence suggests that CTS are synthesised from cholesterol in the adrenal glands \[[@bib0270],[@bib0275]\] and possibly hypothalamus \[[@bib0280]\]. Indeed, missense polymorphism (rs2254524 V642 L) in lanosterol synthase, an enzyme involved in cholesterol biosynthesis, were shown to affect ouabain biosynthesis \[[@bib0285]\]. Can this pathway be targeted pharmacologically requires investigation. The trigger for biosynthesis initiation seems complex, with serum concentrations of ouabain and marinobufagenin increasing in response to volume expansion \[[@bib0290]\], adrenocorticotropic hormone, angiotensin II, vasopressin, and phenylephrine \[[@bib0270],[@bib0295]\], adding another layer of complexity.Relevant questions:Which CTS are present in circulation, if any?What is the effect of multiple different CTS on cardiac, smooth muscle, neuronal and kidney function? Do they act synergistically or is there antagonism?How do endogenous CTS get synthesised and can these biosynthetic pathways be targeted? 4. CTS mechanism of action and interaction with digoxin {#sec0020} ======================================================= Textbook model describes CTS-mediated inhibition of the cardiac NKA, thereby, raising intracellular Na^+^ and Ca^2+^ (via NCX), see [Fig. 2](#fig0010){ref-type="fig"}. The effects of acutely increased Ca^2+^ on contractility \[[@bib0300]\] are well described and this is postulated to be the inotropic mechanism providing improved cardiac output in heart failure patients. Digoxin, and digitoxin also reduce heart rate, presumably by parasympathomimetic effects, and as such are used for rate control in patients with atrial fibrillation. Of course, it may be that bradycardic effects are the primary mechanism for improved morbidity in patients with heart failure. It is unclear, whether the effects on heart rate are mediated directly on the vagus, sino-atrial node, on cardiac conduction, or possibly on all of them. Levels required to achieve these effects in patients are around 1 nM. Considering that circulating concentrations of CTS are in the range of 0.05-0.7 nM \[[@bib0020],[@bib0025],[@bib0165],[@bib0225],[@bib0230],[@bib0305], [@bib0310], [@bib0315], [@bib0320], [@bib0325], [@bib0330], [@bib0335], [@bib0340]\], it is questioned whether inotropic and bradycardic effects are likely to be observed. Alternative theory that does not involve modulation of intracellular Na^+^ and Ca^2+^ concentrations was presented \[[@bib0345]\]. This hypothesis proposes that the NKA acts as a receptor for the CTS and thus plays a signaling role \[[@bib0350]\], regulating early response genes associated with cell growth \[[@bib0355]\]. In this model, Lie and Xie suggest that a fraction of inactive NKA subunits are localised in the caveolae and are physically associated with other key signaling proteins such as EGFR and Src \[[@bib0360],[@bib0365]\] see [Fig. 2](#fig0010){ref-type="fig"}. Binding of the CTS leads to activation of hypertrophic and fibrotic signaling cascades via NKA-Src-ERK \[[@bib0355]\]. Evidence also exists for the involvement of other signaling molecules, phospholipase C, TRP proteins, PI(3)K, and PKC \[[@bib0345],[@bib0365], [@bib0370], [@bib0375], [@bib0380], [@bib0385]\] and that the CTS induce the endocytosis of the CTS-NKA-Src-EGFR complex \[[@bib0370], [@bib0375], [@bib0380]\]. There is disagreement whether the activation of signaling cascades via CTS indeed occurs independently of NKA inhibition and the accompanying increases in intracellular Na^+^ and Ca^2+^ \[[@bib0390],[@bib0395]\]. There is certainly substantive evidence that acute CTS inotropic effects and activation of NKA-Src-ERK signaling cascades depend on the presence of functional NCX and intracellular sodium and calcium increases \[[@bib0300],[@bib0400],[@bib0405]\].Fig. 2**Schematic diagram of ionic versus signaling pathways for cardiotonic steroid effects.** In the classic pathway (shown on the left), CTS binds and inhibits the NKA, which in turn is accompanied by changes in cytosolic \[Na^+^\]. The increase in cytosolic \[Na^+^\] then reduces NCX activity and induces an increase in cytosolic \[Ca^2+^\]. High cytosolic \[Ca^2+^\] mediates muscle contraction (inotropy) or activates a variety of signaling pathways. It is unclear whether the effects of CTS on heart rate (bradycardia) are also mediated via the ionic pathway. The signaling pathway (shown on the right hand side) occurs in the caveolar domain and involves the physical interaction with Src. When the CTS binds the NKA, SRC gets activated inducing further activation of mitogen-activated protein kinase (ERK) through activation of its mitogen-activated protein kinase kinase (MEK).Fig. 2 Presence of endogenous CTS raises an important therapeutic query, if endogenous CTS exist in patients treated with digoxin or digitoxin, how do these affect outcomes? It is possible that administration of digoxin to patients with already raised endogenous CTS can be detrimental. Link between mortality and elevated serum levels of CTS in heart failure patients has been clearly established. Heart failure patients treated with therapeutic doses of digoxin, where serum levels of digoxin are \>1.2 ng/ml (as measured by ELISA with poor specificity), are associated with 11.8 % increase in mortality \[[@bib0410]\]. Thus, differing levels of endogenous cardiotonic steroids may partly explain the varied success of digoxin in this patient population and also the susceptibility to adverse drug reactions and overt toxicity. Surprisingly, experiments in rat arteries suggest potential antagonism of ouabain and digoxin \[[@bib0415]\], and digoxin was shown to oppose ouabain-induced hypertension \[[@bib0420]\]. This may explain the beneficial effects of digoxin therapy in heart failure patients \[[@bib0425]\], however, whether antagonism between digoxin and ouabain, and other CTS exists in the heart and what the therapeutic implications of these findings are, remains to be investigated.Relevant questions:Are the clinical benefits of CTS in heart failure patients primarily mediated via heart rate reduction?Do endogenous CTS levels affect response to digoxin therapy in patients with heart failure and atrial fibrillation?Does the antagonism between digoxin and endogenous CTS exist in the heart? 5. Endogenous CTS in physiology and pathophysiology {#sec0025} =================================================== CTS were shown to be involved in cardiac contraction, heart rate modulation, blood pressure regulation and natriuresis. Chronic exposure to CTS can lead to remodelling of the heart, kidneys and arterial walls but also cardiac fibrosis and arrhythmias ([Fig. 3](#fig0015){ref-type="fig"}). Their secretion seems to be driven by kidney dysfunction and sodium and volume status and is likely involved in a number of diseases from uremic cardiomyopathy, preeclampsia, hypertension, congestive heart failure, myocardial ischemia-induced arrhythmias and diabetes mellitus.Fig. 3**Graphical representation of the proposed biosynthesis and function of endogenous cardiotonic steroids.** Synthesis of endogenous cardiotonic steroids is thought to occur in the adrenal cortex from cholesterol. Acute effects of endogenous cardiotonic steroids are mediated via inhibition of the NKA. Isoform specific effect in each tissue is shown in red. Acute and chronic effects of elevated cardiotonic steroids levels on the specific organs are shown in black boxes. Adapted from Schoner and Scheiner-Bobis \[[@bib0535]\].Fig. 3 Regulation of sodium content and thus blood volume is proposed as the primary physiological role of endogenous CTS. The basis of this natriuretic control is thought to be dependent on the direct inhibition of the α1 NKA isoform in renal tubular cells \[[@bib0430]\]. The most convincing evidence in support of this are the studies by Periyasami and colleagues where marinobufagenin antibodies given to salt loaded rats lowered urinary excretion and increased NKA activity \[[@bib0435]\]. Given, high similarity in chemical structures between different CTS, antibodies raised against individual CTS tend to have poor specificity \[[@bib0440]\] and thus marinobufagenin may not necessarily be the primary modulator. Indeed, increases in both endogenous ouabain and marinobufagenin are observed in states of volume expansion and volume expansion mediated hypertensive syndromes that are related to water and salt accumulation \[[@bib0310],[@bib0430],[@bib0445]\]. Beyond the expected inhibition of the NKA, administration of ouabain or marinobufagenin also resulted in translocation of the NKA to intracellular compartments via clathrin-dependent endocytosis \[[@bib0380]\]. This translocation required PI(3)K activation, the plasmalemmal pump to be in the context of caveola, and signaling through the Src-EGFR pathway \[[@bib0375]\]. It is therefore suggested that salt loading leads to increases in ouabain and marinobufagenin in the proximal tubules, both of which act to decrease Na^+^ through NKA inhibition and/or activation of a signaling mechanism mediated by NKA-Src-EGFR cascades. This decrease in renal Na^+^ reabsorption is expected to lead to an increase in urinary Na^+^ excretion. In studies of healthy individuals, salt loading for up to 6 days lead to a transient elevation in plasma ouabain and sustained increase in marinobufagenin, followed by an increase in urine Na^+^ excretion \[[@bib0290],[@bib0450]\]. CTS are also implicated in control of blood pressure and infusion of ouabain or marinobufagenin, at concentrations comparable with *in vivo* plasma levels, lead to an increase in blood pressure \[[@bib0330],[@bib0455], [@bib0460], [@bib0465]\]. Indeed, increased levels of endogenous ouabain were detected in patients with essential hypertension \[[@bib0165],[@bib0170]\] and preeclampsia \[[@bib0470], [@bib0475], [@bib0480]\]. Hypertension induced by salt loading, ouabain infusion or preeclampsia was reduced by immune-neutralisation with anti-CTS antibodies \[[@bib0485],[@bib0490]\] or CTS antagonists rostafuroxin \[[@bib0495]\] and resibufogenin \[[@bib0500]\]. It should be noted however that rostafuroxin did not reduce blood pressure in hypertensive patients enrolled in Ouabain and Adducin for Specific Intervention on Sodium in Hypertension (OASIS-HT) phase-2 Trial \[[@bib0505]\]. Whilst the evidence for the role of endogenous CTS in natriuresis and blood pressure homeostasis is mounting it is unclear which endogenous CTS are involved and whether the acute and chronic responses are differentially modulated.Relevant questions:Do endogenous CTS directly modulate natriuresis and blood pressure or are they simply by products of these homeostatic mechanisms?Are there differences in CTS biosynthesis between the acute and chronic responses to salt loading?Which CTS are involved in physiological volume and blood pressure control? Endogenous CTS levels were also shown to be increased in animal models and patients with acute kidney injury \[[@bib0200]\], chronic kidney disease \[[@bib0330]\], end stage renal disease \[[@bib0205],[@bib0230]\], myocardial infarction \[[@bib0510]\] and heart failure \[[@bib0180],[@bib0515],[@bib0520]\]. Most of the studies demonstrate increases in ouabain and marinobufagenin from circa 0.3 nM in healthy individuals to circa 0.7--1.0 nM in patients. The evidence in favor of the hypothesis that endogenous CTS drive the disease progression rather than simply act as biomarkers of pathology is growing. In healthy adult rats administration of marinobufagenin for 4 weeks led to increases in blood pressure, diastolic dysfunction and ventricular hypertrophy \[[@bib0330],[@bib0525]\]. Remarkably, active and passive immunisation against marinobufagenin reversed most of these alterations \[[@bib0330],[@bib0525]\]. Independent studies by Ferrandi et al. showed that infusion of 0.7 nM of ouabain for 18 weeks in healthy adult rats induced hypertension and left ventricular hypertrophy and could be reversed by ouabain antagonist PST2238 \[[@bib0460]\]. These studies demonstrate that chronic exposure to pathophysiological concentrations in healthy animals can lead to some of the cardiovascular changes observed in patients, however, utilisation of animal models with a preserved NKA sensitivity to CTS is required to provide further mechanistic insights. Whether immunisation for CTS or intravenous administration of antibodies raised against CTS (e.g. in patients on renal replacement therapy) is beneficial to these patient populations is an easily testable and attractive proposition.Relevant questions:Which CTS are synthesised in patients with renal and heart failure?Is the cardiovascular disease progression mediated by chronic elevation of CTS?Does quenching of endogenous CTS by immunisation or administration of antibodies against CTS represent a viable therapeutic option for patients with chronically high CTS levels? 6. Conclusion {#sec0030} ============= Administration of CTS was conclusively shown to modulate contraction, blood pressure regulation, natriuresis and chronically can contribute to remodelling of the heart, kidneys and arterial walls. Although endogenous CTS have been detected in blood of patients with kidney and cardiovascular dysfunction as early as 1981, the chemical identities of CTS and mechanistic insight into the cardiovascular physiology and pathogenesis of these molecules remains unclear. Antibody-based assays cannot distinguish different CTS due to their high structural similarity. Furthermore there is a clear need for human studies as murine NKA shows a 1000 fold reduced sensitivity to CTS \[[@bib0530]\]. A clear barrier to further expansion of knowledge is our lack of tools to accurately detect and measure the CTS present in circulation and thus identify patients with a positive CTS status. Understanding how these endogenous molecules interact with digoxin is also important if we are to come up with novel stratified therapies, better tailored for treatment of patients with heart failure and atrial fibrillation. These studies must be carried out in either human tissue or animal models with preserved NKA sensitivity to CTS, rather than mice and rats, where NKA sensitivity is reduced. Sources of funding {#sec0035} ================== DP is supported by the British Heart Foundation (PG/17/55/33087, RG/17/15/33106, FS/19/16/34169, FS/19/12/34204) and Wellcome Trust (109604/Z/15/Z). CRediT authorship contribution statement {#sec00005} ======================================== **Davor Pavlovic:** Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Validation, Visualization, Writing - original draft, Writing - review & editing. Declaration of Competing Interest ================================= None.

{#sp1 .308} {#sp2 .309}

Haida Z, Hakiman M. A comprehensive review on the determination of enzymatic assay and nonenzymatic antioxidant activities. Food Sci Nutr. 2019;7:1555--1563. 10.1002/fsn3.1012 1. INTRODUCTION {#fsn31012-sec-0001} =============== It is a fact that human exposure to vast chronic ailments has never been higher than this present age. Free radicals are being generated at high quantities, particularly at metabolic and contact processes. Tissues and cells are damaged by oxidation as macromolecules (fatty acids, nucleic acids, and proteins) deteriorate (Gupta et al., [2014](#fsn31012-bib-0019){ref-type="ref"}). Basically, the reaction that takes place between free radicals and electrons derived from other molecules harms the human body due to its negative effect on the enzyme system (Bjelakovic, Nikolova, Gluud, Simonetti, & Gluud, [2007](#fsn31012-bib-0009){ref-type="ref"}). Hence, the complex systems of enzymatic and nonenzymatic antioxidants have the capability to address the deteriorating impacts of oxidants and free radicals that may lead to infirmity. An antioxidant is a type of compound that stabilizes, scavenges, and suppresses the generation of oxidants and free radicals. Therefore, consumption of antioxidants based on natural resources (greens, fruits, and herbs) may help to shield one from oxidants and free radicals without side effects. All enzymatic and nonenzymatic antioxidant assays described in the review are based on UV--vis spectrophotometry method. Apart from spectrophotometry method for analysis of antioxidants, other methods such as electrochemical and chromatography methods have also been used. The electrochemical methods include cyclic voltammetry, superwave voltammetry, differential pulse voltammetry, coulometry, potentiometry, amperometry, and biamperometry. The chromatography methods include thin layer chromatography (TLC), liquid chromatography--mass spectrophotometry (LC‐MS), high‐performance liquid chromatography (HPLC), and gas chromatography. However, among all the methods mentioned above, the spectrophotometry method has been the most commonly used method because it is the easiest to handle and inexpensive to run when compared to the electrochemical and chromatography methods. Spectrophotometry method can be performed by using a cuvette or a microplate reader. Analysis of antioxidant by using cuvette is usually conducted for small number of samples. The method is time consuming as only one sample is allowed to be read at one time which takes about 1--2 min. This method also requires high amount of reaction mixture due to large volume of cuvette (approximately 1.5--2 ml). In contrast, the use of a microplate is more convenient and time saving. It allows reading of 96 samples at one time. Presently, a microplate reader of spectrophotometer has been upgraded to allow reading of 384 samples at one time. By using a microplate reader, the volume of samples needed is significantly low (approximately 100--200 µl). Hence, the amount of reaction mixture needed to prepare is low, thus saving the use of chemicals. As such, a number of methods have been proposed to quantify antioxidant content in many samples. With that, this paper presents a review pertaining to the common techniques employed to analyze both enzymatic and nonenzymatic antioxidants. 2. FREE RADICALS, REACTIVE OXYGEN SPECIES (ROS), AND REACTIVE NITROGEN SPECIES (RNS) {#fsn31012-sec-0002} ==================================================================================== Free radicals are claimed to be harmful to humans because its unpaired electron(s) extracts electron(s) from other molecules in the body to gain stability, hence damaging DNA, proteins, and lipids. These free radicals, which can be found as nitrogen derived (RNS) or oxygen derived (ROS), have rather high reactivity and short life. Components that are present as free radicals in ROS are O~2~ (superoxide), HO^•^ (hydroxyl), HO~2~ (hydroperoxyl), ROO^•^ (peroxyl), and RO^•^ (alkoxyl), while those nonradicals refer to H~2~O~2~(hydrogen peroxide), HClO (hypochlorous acid), O~3~ (ozone), and ^1^O~2~ (singlet oxygen). Meanwhile, NO^•^ (nitric oxide), NO~2~ (nitrogen dioxide), N~2~O~3~ (dinitrogen trioxide), and ONOO^−^ (peroxynitrite) are the free radicals derived from RNS (Ali et al., [2008](#fsn31012-bib-0004){ref-type="ref"}; Evans & Halliwell, [1999](#fsn31012-bib-0016){ref-type="ref"}). The major source of ROS is the environment that is filled with car exhaust fume, cigarette smoke, ozone, and low‐wave electromagnetic and ultraviolet (UV) radiation. Meanwhile, some sources of the endogenous ROS are amino acids auto‐oxidation, respiratory burst by phagocytes, ischemia reperfusion injury, and mitochondrial electron transport chain. Hydroxyl radical (HO^•^), which is generated from H~2~O~2~ and O~2~ via Harber--Weiss reaction, refers to a damaging ROS with 10^−5^ s half‐life (Beauchamp & Fridovich, [1970](#fsn31012-bib-0007){ref-type="ref"}). The H~2~O~2~ component, although with poor reactivity and relatively stable properties, can cross cell membranes easily and arrack various sites after conversion to HO. Besides, H~2~O~2~ produces free radicals with the existence of transition metal ions. One essential RNS‐based free radical refers to nitric oxide (NO) as this component is one of the 10 smallest molecules that are naturally found in 30‐Da molecular weight. The processes of redox, substitution, addition, and chain termination reaction are closely associated to NO. According to Stamler ([1994](#fsn31012-bib-0036){ref-type="ref"}), the primary targets of NO are proteins that contain metals, intracellular thiol, and low molecular weight thiols (glutathione). The peroxynitrite (ONOO^−^) is also an essential antioxidant with a range of targets. The mechanism of ONOO^−^ is as follows: peroxidation of lipid, break of DNA strand, nitration of tyrosine, and, lastly, death of cell. Upon reaction with NO, ONOO^−^ produces NO~2~ and causes nitrosative stress, while generating N~2~O~2~ with more NO (Koppenol, Moreno, Pryor, Ischiropoulos, & Beckman, [1992](#fsn31012-bib-0022){ref-type="ref"}). Harmful oxidative stress, which can lead to chronic disease, is produced when RNS and ROS concentrations exceed the amount of antioxidants in the human body. 3. ANTIOXIDANTS {#fsn31012-sec-0003} =============== Oxidative stress can be addressed by antioxidants by delaying or preventing the oxidative chain reaction promulgation. Plant‐based antioxidants scavenge free radicals to inhibit tissue/cell damages, hence minimizing risk of chronic diseases (Soobrattee, Bahorun, Neergheen, Googoolye, & Aruoma, [2008](#fsn31012-bib-0035){ref-type="ref"}). Besides, antioxidants donate their electrons in order to neutralize both exogenous and endogenous free radicals (Jacob, [1995](#fsn31012-bib-0021){ref-type="ref"}). Antioxidant components are composed of lipid‐soluble (hydrophobic) and water‐soluble (hydrophilic) substances. Plant‐based antioxidants are mostly hydrophilic, in which some instances are phenolics, flavonoids, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), as well as uric, lipoic, benzoic, and ascorbic acids. On the other hand, hydrophobic antioxidants are linked to biological membrane functional, which are found in carotenoids, tocopherols, vitamin K, ubiquinone, and phospholipids. Additionally, antioxidants are categorized depending on defense lines: preventive antioxidant as the initial line of defense, radical scavenging antioxidant in the second line of defense, and, lastly, repair and de‐novo enzymes as the third line of defense, as further elaborated: The first line of defense hinders the production of free radicals that leads to oxidative stress via enzymes, such as SOD, CAT, GPx, glutathione reductase, and several types of minerals, for example, selenium (Se), manganese (Mn), copper (Cu), and iron (Fe).The second line of defense inhibits the production of damaged species, apart from making the free radicals less harmful that further reduces damages caused by oxidative reaction. Some excellent scavengers of free radicals are vitamins E and C, flavonoids, and uric acid.The third line of defense serves to repair damaged DNAs, proteins, peroxides, and oxidized lipids, besides inhibiting the propagation of chain reaction in peroxyl lipid radical. The classification of antioxidants depends on their catalytic action type, either enzymatic or nonenzymatic antioxidants. The enzymatic antioxidants possess certain cofactors and tend to be highly specific for substrate reactive species. Some instances of enzymatic antioxidants refer to SOD, CAT, and GPx. Meanwhile, those nonenzymatic antioxidants differ from the former as they do not possess any specific substrate and, hence, could nullify the negative effects of both RNS and ROS. 4. ENZYMATIC ANTIOXIDANT ASSAY {#fsn31012-sec-0004} ============================== 4.1. Superoxide dismutase assay {#fsn31012-sec-0005} ------------------------------- The enzyme extract was prepared by homogenizing fresh samples (200 mg) in 5 ml of 100 mM potassium phosphate buffer (pH 7.8) that contained 0.1% (v/v) Triton X‐100, 2% (w/v) polyvinyl pyrrolidone (PVP), and 0.1 mM EDTA. Next, the extract was filtered and centrifuged at 22,000 × *g* for 10 min at temperature 4°C. The resultant supernatant was collected and dialyzed by using cellophane membrane tubing for 240 min against cold extraction buffer. The remaining extract was used for enzyme assay. The final volume of incubation mixture is 3 ml that contains 50 mM of potassium phosphate buffer (pH 7.8), 45 µM of methionine, 20 µM of potassium cyanide, 84 µM of nitroblue tetrazolium (NBT), and 5.3 mM of riboflavin. The amount of homogenate added to the mixture is kept under one unit of enzyme so as to ensure high accuracy. The mixture is incubated at 25°C with the presence of 15 W fluorescent lamps in an aluminum foil‐lined box. After 10 min of exposure to light, the reduction in NBT is measured at absorbance of 600 nm. Absence of enzyme is indicated by the highest reduction. One unit of enzyme activity is defined as the amount of enzyme that leads to 50% inhibition of NBT reduction (Misra & Fridovich, [1972](#fsn31012-bib-0027){ref-type="ref"}). 4.2. Catalase assay {#fsn31012-sec-0006} ------------------- Extraction of catalase assay was prepared by homogenizing fresh samples (200 mg) in 5 ml of 50 mM Tris‐NaOH at pH 8.0 that contained 0.5% (v/v) Triton X‐100, 2% (w/v) PVP, and 0.5 mM EDTA. The homogenate was centrifuged for 10 min at 4°C at 22,000 × *g*, and the resultant supernatant was dialyzed prior to enzyme assay. Catalase assay can be conducted by adhering to the method suggested by Aebi ([1984](#fsn31012-bib-0001){ref-type="ref"}). One milliliter of reaction mixture containing 50 mM of potassium phosphate buffer (pH 7.0) and 250 µl of enzyme extract is initiated by adding 60 mM of hydrogen peroxide. The absorbance is measured by using a spectrophotometer at an absorbance rate of 240 nm for 3 min. The H~2~O~2~ decomposition is calculated by using extinction coefficient of 39.4 mM^−1^ cm^−1^. One unit of activity is equivalent to 1 mM of H~2~O~2~ degraded per minute and is expressed as unit per milligram of protein. 4.3. Peroxidase assay {#fsn31012-sec-0007} --------------------- Two grams of samples had been homogenized with 10 ml of 0.1 M phosphate buffer (pH 6.0). The extract was filtered through cheesecloth and centrifuged for 30 min at 12,000 × *g*. The resultant supernatant was collected and heated at 65°C for 3 min to inactivate any catalase present in the extract. Peroxidase assay is prepared by performing the guaiacol oxidation method as depicted by Britton and Mehley ([1955](#fsn31012-bib-0011){ref-type="ref"}). The final reaction mixture (3 ml) in the test tube consists of 10 mM of potassium phosphate buffer (pH 7.0), 8 mM of guaiacol, and 100 µl of enzyme extract, in which 2.75 mM of hydrogen peroxide is added to initiate the reaction. Increment in absorbance measured at 470 nm within 30 min indicates the formation of tetraguaiacol. The change in absorbance per min and specific activity as enzyme units per mg soluble protein with extinction coefficient 6.39 mM^−1^ cm^−1^ refers to a unit of peroxidase activity. The enzyme activity is expressed as unit per milligram of protein. 4.4. Ascorbate peroxidase assay {#fsn31012-sec-0008} ------------------------------- The enzyme assay was extracted from 200 mg of samples. Next, the samples were homogenized in 50 mM potassium phosphate buffer (5 ml, pH 7.8) that contained 1 mM ascorbic acid, 1 mM phenylmethane sulfonyl fluoride (PMSF), and 1% PVP. The reaction mixture was centrifuged at 22,000 × *g* at 4°C for 10 min. The resultant supernatant was collected and dialyzed prior to enzyme assay. The reaction mixture that contains 100 mM of Tris‐acetate buffer (pH 7.0), 2 mM of ascorbic acid, and enzyme extract is added with 2 mM of hydrogen peroxide to initiate the reaction. The decrease in absorbance rate is measured by using a spectrophotometer at 290 nm for 100 s. The extinction coefficient 2.8 mM^−1^ cm^−1^ is used to calculate the reaction. The specific enzyme activity is expressed as unit per milligram of protein (Ali, Hahn, & Paek, [2005](#fsn31012-bib-0003){ref-type="ref"}). 4.5. Ascorbate oxidase assay {#fsn31012-sec-0009} ---------------------------- The plant sample tissue was extracted in 20 mM potassium phosphate (pH 7.4), 1.5% PVPP, and 0.5 mM PMSF. After that, the mixture was homogenized with Polytron, incubated on ice for 20 min, and vortexed for every 2‐min interval. Next, the mixture was centrifuged at 15,000 × *g* at 4°C for 15 min. The resultant supernatant was collected and dialyzed prior to enzyme assay. The final reaction mixture consists of 1.0 ml that is comprised of 20 mM of potassium phosphate buffer (pH 7.0) and 2.5 of mM ascorbic acid. The 10 µl of enzyme extract is added to initiate the reaction. Due to ascorbate oxidation, the decrease in absorbance is monitored for 3 min at an absorbance rate of 265 nm and calculated by using extinction coefficient, 14 mM^−1^ cm^−1^ (Diallinas et al., [1997](#fsn31012-bib-0014){ref-type="ref"}). 4.6. Guaiacol peroxidase assay {#fsn31012-sec-0010} ------------------------------ The enzyme extract for determination of guaiacol peroxidase assay was performed by homogenizing 200 mg of fresh samples in 5 ml of cold 50 mM sodium phosphate buffer at pH 7.0. Next, the dialyzed enzyme extract was used for assay after being centrifuged at 22,000 × *g* for 10 min. The assay mixture (5 ml) contained 2 mM H~2~O~2~, 9 mM guaiacol, 40 mM sodium phosphate (pH 6.1), and 50 µl enzyme. The increment in absorbance was measured at 420 nm and calculated by using extinction coefficient of 26.6 mM^−1^ cm^−1^ for 2 min with 30‐s interval. The outcomes are expressed as unit per milligram of protein (Egley, Paul, Vaughn, & Duke, [1983](#fsn31012-bib-0015){ref-type="ref"}). 4.7. Glutathione reductase assay {#fsn31012-sec-0011} -------------------------------- Glutathione reductase assay was carried out by adhering to the method depicted by Schaedle and Bassham ([1977](#fsn31012-bib-0033){ref-type="ref"}). The enzyme extract was prepared prior to enzyme assay. Briefly, 200 mg of fresh samples was homogenized by using chilled mortar and pestle in 5 ml of 50 mM Tris‐HCl buffer at pH 7.6. The resultant supernatant was collected after being centrifuged at 22,000 × *g* for 4 min and dialyzed prior to enzyme assay. The final reaction mixture (1 ml) was composed of 200 µl enzyme extract, 50 mM Tris‐HCl buffer (pH 7.6), 1 mM glutathione disulfide (GSSG), 0.15 mM NADPH, and 3 mM MgCl~2~. A decrease in NADPH absorbance was observed at 340 nm. The specific activity of enzyme is expressed as unit per milligram of protein. 5. NONENZYMATIC ANTIOXIDANTS ASSAY {#fsn31012-sec-0012} ================================== 5.1. Total polyphenol content {#fsn31012-sec-0013} ----------------------------- Polyphenols are polyhydroxylated phytochemical that are synthesized by plants and have many benefits to the human health. Polyphenols have abilities to trap and to scavenge free radicals by donating hydrogen ion to stabilize the free radicals. In addition, polyphenols can regulate nitric oxide, induce apoptosis, inhibit cell proliferation and angiogenesis, and prevent high blood pressure, apart from possessing anti‐aging, anti‐bacterial, and anti‐tumor properties. The two major subclasses of polyphenols are phenolic acids and flavonoids (Arts & Hollman, [2005](#fsn31012-bib-0006){ref-type="ref"}; Yazaki, Sasaki, & Tsurumaru, [2009](#fsn31012-bib-0039){ref-type="ref"}). The total polyphenols content in the plant can be determined by employing the method suggested by Marinova, Ribarova, and Atanassova ([2005](#fsn31012-bib-0025){ref-type="ref"}). A total of 10 µl of sample extract are mixed with 2.5 ml of 10‐fold of diluted Folin--Ciocalteu reagent. After 5 min, 2.5 ml of 7% sodium carbonate is added and the mixture is incubated at room temperature. After an hour of incubation, the absorbance of reaction mixture is measured at 725 nm. The total polyphenol content is expressed as milligram gallic acid equivalents per gram of samples. 5.2. Total phenolic acids {#fsn31012-sec-0014} ------------------------- Phenolic acids are hydroxylated that derive from benzoic and cinnamic acids. Hydrobenzoic acid is mainly present in the form of glucosides in foods, while hydrocinnamic acid, such as *p*‐coumaric, caffeic acid, and ferrulic acid, is mostly found in food as simple esters (Mattila & Kumpulainen, [2002](#fsn31012-bib-0026){ref-type="ref"}). Total phenolic acids can be determined by adhering to the method proposed by Singleton and Rossi ([1965](#fsn31012-bib-0034){ref-type="ref"}), which is known as the Folin--Ciocalteu phenol reagent technique. Briefly, 1 ml of sample extract is added into a test tube that contains 9 ml of distilled water. Then, 1 ml of Folin--Ciocalteu phenol reagent is added to it and the mixture is mixed thoroughly via vortex. After 5 min, 10 ml of 7% sodium carbonate is added. Next, 4 ml of distilled water is added and the mixture is adjusted to 25 ml of final volume. The reaction mixture is incubated for 90 min at room temperature, and the absorbance is measured at 750 nm. The total phenolic acids are expressed as milligram of gallic acid equivalents per gram of samples. 5.3. Total flavonoids {#fsn31012-sec-0015} --------------------- Flavonoids are widely found in plants, and they consist of a large group of polyphenolic compounds that can be characterized by benzo‐y‐pyrone structure. Flavonoids are low molecular phenolics, and they can be divided into several subclasses, such as flavones, flavanones, isoflavones, anthocyanins, flavanols, and flavonols. The most crucial function of flavonoids is their antioxidant activity. Flavonoids can scavenge various oxidizing species, including superoxide anion, hydroxyl or peroxyl radicals by quenching the singlet oxygen. Flavonoids can also act as an inhibitor in oxidation of low density lipoprotein (LDL), anti‐bacterial, and anti‐fungal, besides preventing malaria (Harborne & Williams, [2000](#fsn31012-bib-0020){ref-type="ref"}; Subramanian, Stacey, & Yu, [2007](#fsn31012-bib-0037){ref-type="ref"}). The content of total flavonoids can be determined based on the method depicted by Marinova et al. ([2005](#fsn31012-bib-0025){ref-type="ref"}) by employing the aluminum chloride colorimetric technique. Briefly, 1 ml of sample extract is added into a test tube containing 4 ml of distilled water. After that, 0.3 ml of 5% sodium nitrite is added. After 5 min, 0.3 ml of 10% aluminum chloride is added into the mixture. At the sixth min, 2 ml of 1 M sodium hydroxide is added. Next, the mixture is adjusted to 10 ml by adding 2.4 ml of distilled water and mixed thoroughly with a vortex machine. The absorbance of the reaction mixture is measured at 510 nm. The total flavonoid content of the extract is expressed as milligram rutin equivalents per gram of samples. 5.4. Total ascorbic acid {#fsn31012-sec-0016} ------------------------ Ascorbic acid or vitamin C is an organic acid that is the most crucial vitamin for human nutrition. Ascorbic acid is synthesized in mitochondria and transported to other cell through proton--electron chemical gradient or through facilitated diffusion. It also has the ability to scavenge many types of free radicals. The main sources of ascorbic acid are fruits and vegetables. Ascorbic acid can fight against chronic diseases, such as cardiovascular diseases and certain types of cancer (Aro & Ohad, [2003](#fsn31012-bib-0005){ref-type="ref"}; Borland et al., [2006](#fsn31012-bib-0010){ref-type="ref"}; Gil, Tomás‐Barberán, Hess‐Pierce, & Kader, [2002](#fsn31012-bib-0017){ref-type="ref"}). The total ascorbic acid can be determined by using 1% phosphate citrate buffer (Davies & Masten, [1991](#fsn31012-bib-0013){ref-type="ref"}). Briefly, fresh plant sample is extracted by using chilled pestle and mortar with the addition of 1% phosphate citrate buffer (pH 3.5). Next, the sample is homogenated and centrifuged at 14,000 x *g* for 10 min at 4°C. The supernatant is collected, and 1.72 mM of 2,6‐dichloroindophenol (2,6‐DCPIP) is added. The absorbance is immediately measured after mixing at 518 nm by using a spectrophotometer. 5.5. Anthocyanin content {#fsn31012-sec-0017} ------------------------ Anthocyanins are chemicals that have a single aromatic structure known as cyaniding, which refers to a widely distributed pigment group found in the plant kingdom. The colors range from red to blue of the visible spectrum due to the water‐soluble pigments. The colors of these substances are varied due to addition or removal of hydroxyl groups or because of methylation or glycosylation (Harborne & Williams, [2000](#fsn31012-bib-0020){ref-type="ref"}; Sakakibara, Honda, Nakagawa, Ashida, & Kanazawa, [2003](#fsn31012-bib-0032){ref-type="ref"}). Determination of anthocyanin content can be carried out based on the technique proposed by Bharti and Khurana ([2003](#fsn31012-bib-0008){ref-type="ref"}). Briefly, fresh leaves are added into 10 ml of acidic methanol (1% v/v HCl) and the mixture is incubated overnight. To partition anthocyanin from chlorophyll, 10 ml of chloroform and 9 ml of double deionized water are added. The test tube is shaken gently, and the mixture is allowed to settle. The absorbance of the reaction mixture is measured at 505 nm. 5.6. DPPH scavenging activity {#fsn31012-sec-0018} ----------------------------- Molecule 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) is classified as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole that prevents it from dimerize. When a solution of DPPH is mixed, it donates hydrogen atom that reduces its form and loses its violet color. The DPPH radical scavenging activity can be carried out by preparing 1 ml of sample extract in a test tube. Next, 2 ml of 1 mM of methanolic DPPH is added. The solution is mixed thoroughly and incubated for 30 min at 37°C. The blank sample is prepared without adding any standard or sample extract. The change in absorbance is measured at 515 nm, and the percentage of inhibition is calculated (Alhakmani, Kumar, & Khan, [2013](#fsn31012-bib-0002){ref-type="ref"}).$$\text{Inhibition}\left( \% \right) = \left\lbrack {\left( {\text{A}_{515}\,\text{Control} - \text{A}_{515}\,\text{Sample}} \right)/\text{A}_{515}\,\text{Control}} \right\rbrack \times 100$$ 5.7. Ferric reducing‐antioxidant power (FRAP) assay {#fsn31012-sec-0019} --------------------------------------------------- The FRAP assay measures the reduction of ferric iron and 2,3,5‐triphenyl‐1,3,4‐triaza‐azoniacyclopenta‐1,4‐diene chloride to blue ferrous complex by antioxidants under acidic condition (pH 3.6). The FRAP unit is the reduction of one mole of Fe (lll) to Fe (ll). The FRAP assay can be prepared based on the method described by Wong, Leong, and Koh ([2006](#fsn31012-bib-0038){ref-type="ref"}). Briefly, 200 µl of sample extract is added with 3 ml of FRAP reagent that is prepared with a mixture of 300 mM of sodium acetate buffer (pH 3.6), 10 mM of 2,4,6‐tri(2pyridyl)‐s‐triazine (TPTZ) solution, and 20 mM of FeCl.6H~2~O at the ratio 10:1:1. The reaction mixture is incubated for 30 min at 37°C. Increment in absorbance is measured at 593 nm, and the percentage of inhibition (antioxidant) is calculated.$$\text{Inhibition}\left( \text{antioxidant} \right)\left( \% \right) = \left\lbrack {\left( {\text{A}_{593}\,\text{Sample} - \text{A}_{593}\,\text{Control}} \right)/\text{A}_{593}\,\text{Sample}} \right\rbrack\, \times 100$$ 5.8. Hydrogen peroxide (H~2~O~2~) scavenging activity {#fsn31012-sec-0020} ----------------------------------------------------- Hydrogen peroxide is found naturally at low concentration levels in air, water, human body, plants, foods, and microorganism. H~2~O~2~may enter the human body by inhalation of vapor or mist and through eye or skin contact. Hydroxyl radicals (OH^•^) are the by‐products of H~2~O~2~decomposed into oxygen and water that initiate lipid peroxidation and damages to DNA. In order to determine the scavenging activity of hydrogen peroxide, 40 mM of hydrogen peroxide solution is prepared in 50 mM phosphate buffer (pH 7.4) and the absorbance is measured at 230 nm. Next, 1 ml of sample extract or standard is added with 2 ml of hydrogen peroxide solution. After 10 min, the absorbance is measured against blank solution. The blank solution is prepared with phosphate buffer without adding hydrogen peroxide. Then, the percentage of hydrogen peroxide scavenge can be calculated (Nabavi, Ebrahimzadeh, Nabavi, Hamidinia, & Bekhradnia, [2008](#fsn31012-bib-0028){ref-type="ref"}).$$\text{H}_{2}\text{O}_{2}{\mspace{6mu}\text{scavenge}}\left( \% \right) = \left\lbrack {\left( {\text{A}_{230}\,\text{Control} - \text{A}_{230}\,\text{Sample}} \right)/\text{A}_{230}\,\text{Control}} \right\rbrack \times 100$$ 5.9. Nitric oxide scavenging activity {#fsn31012-sec-0021} ------------------------------------- NO^•^ is generated in biological tissues by specific NO synthase that metabolizes arginine to citrulline via five‐electron oxidative reaction. At a physiological pH of 7.2, the sodium nitroprusside compound is decomposed into aqueous solution and generates NO^•^. Stable products (nitrate and nitrite) are produced when NO^•^reacts with oxygen under aerobic condition, which can be determined by using the Griess reagent. The scavenging activity of nitric oxide can be determined based on the method proposed by Marcocci, Maguire, Droylefaix, and Packer ([1994](#fsn31012-bib-0024){ref-type="ref"}). Briefly, 2 ml of 10 mM of sodium nitroprusside is prepared in 0.5 ml of phosphate buffer saline (pH 7.4). Next, 0.5 ml of sample extract is added and incubated at 25°C. After 150 min of incubation, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H~3~PO~4~, and 0.1% naphthylethylenediamine dihydrochloride) is added to 0.5 ml of incubated solution. The reaction mixture is re‐incubated for 30 min at room temperature. The rate of absorbance is measured at 546 nm, and the inhibition percentage is calculated.$$\text{NO\ inhibition} = \left\lbrack {\left( {\text{A}_{546}\,\text{Control} - \text{A}_{546}\,\text{Sample}} \right)/\text{A}_{546}\,\text{Control}} \right\rbrack \times 100$$ 5.10. Superoxide radical scavenging activity {#fsn31012-sec-0022} -------------------------------------------- Although the superoxide anion is a weak oxidant, it can ultimately produce powerful and dangerous hydroxyl radicals and singlet oxygen, which contributes to oxidative stress. This assay can be prepared based on the method developed by Robak and Gryglewski ([1988](#fsn31012-bib-0031){ref-type="ref"}). The final mixture of 16 mM Tris‐HCl buffer at pH 8.0 (3 ml) is comprised of 0.3 mM of NBT (0.5 ml), 0.936 mM of NADH solution (0.5 ml), and 1 ml of sample extract. The mixture reaction is initiated by adding 0.5 ml of 0.12 mM phenazine methosulfate (PMS), and the mixture is incubated at 25°C for 5 min. The absorbance is measured at 560 nm against blank sample, and the percentage of inhibition is calculated.$$\text{Inhibition}\left( \% \right) = \left( {1 - \text{A}_{560}\,\text{Sample}/\text{A}_{560}\,\text{Control}} \right) \times 100$$ 5.11. Hydroxyl radical scavenging activity {#fsn31012-sec-0023} ------------------------------------------ The hydroxyl refers to a dangerous free radical that belongs to ROS. Hydroxyl radical reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids, which can inflict damages to the human cell. The ability displayed by hydroxyl radicals to scavenge can be determined by adhering to the method described by Kunchandy and Rao ([1990](#fsn31012-bib-0023){ref-type="ref"}). The final mixture (1.0 ml) contains 28 mM of 2‐deoxy‐Dribose (100 µl) in 20 mM of potassium phosphate buffer (pH 7.4), 500 µl of sample extract, 1.04 mM of EDTA (200 µl) and 200 µM of FeCl~3~ (1:1 v/v), 1.0 mM of H~2~O~2~ (100 µl), and 1.0 mM of ascorbic acid (100 µl). Next, the mixture is incubated for an hour at 37°C. After that, 1.0 ml of 2.8% trichloroacetic acid and 1.0 ml of 1% thiobarbituric acid are added and the mixture is re‐incubated for 10 min at 100°C. The solution mixture is cooled on ice, and the absorbance rate is measured at 532 nm. After that, the extract is substituted with distilled water as blank, while the sample blank contains the sample solution, but without deoxyribose. Lastly, the inhibition percentage is calculated.$$\text{Inhibition}\left( \% \right) = \left\lbrack {\text{A}_{532}\,\text{Blank} - \left( {\text{A}_{532}\,\text{Extract} - \text{A}_{532}\,\text{Sample\ blank}} \right)/\text{A}_{532}\,\text{Blank}} \right\rbrack \times 100$$ 5.12. Phosphomolybdate assay (total antioxidant capacity) {#fsn31012-sec-0024} --------------------------------------------------------- The total antioxidant capacity assay can be used to determine the capacity of antioxidants through the formation of phosphomolybdenum complex. The assay is based on the reduction of Mo (Vl) to Mo (V) by using sample analyte, which subsequently produces a green phosphate Mo (V) complex at acidic condition. Briefly, 0.1 ml of the sample extract is added to 0.3 ml of reagent solution that contains 0.6 M of sulfuric acid, 28 mM of sodium phosphate, and 4 mM of ammonium molybdate. The test tube is covered and incubated at 95°C for 90 min. After that, the mixture is cooled at room temperature and the absorbance is measured at 695 nm. The blank solution that functions as control contains both the reagent solution and the solvent. The total antioxidant capacity is calculated (Priya, Rajaram, & Suresh‐kumar, [2012](#fsn31012-bib-0030){ref-type="ref"}).$$\text{Total\ antioxidant\ capacity}\left( \% \right) = \left\lbrack {\left( {\text{A}_{695}\,\text{Control} - \text{A}_{695}\,\text{Sample}} \right)/\text{A}_{695}\,\text{Control}} \right\rbrack \times 100$$ 5.13. Reducing power {#fsn31012-sec-0025} -------------------- The principle of this method is based on the increment of absorbance that indicates an increase in the antioxidant activity. Compounds with reducing power ability are electron donors and, hence, possess the ability to reduce the oxidized intermediate process of lipid peroxidation. Briefly, the reaction mixture contains 2.5 ml of sample extract, 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6), and 2.5 ml of 1% potassium ferric cyanide. Next, the reaction mixture is incubated at 50°C. After 20 min of incubation, 2.5 ml of 10% (w/v) trichloroacetic acid and the mixture is centrifuged at 1,000 x *g*for 10 min. Then, 2.5 ml of the upper layer in the supernatant is collected and mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride. The absorbance of reaction mixture is measured at 700 nm against blank, which consists of all reagents without the sample extract (Gulcin, Tel, & Kirecci, [2008](#fsn31012-bib-0018){ref-type="ref"}). 5.14. Metal ion chelating activity {#fsn31012-sec-0026} ---------------------------------- Formation of chelates with Fe^2+^ ~,~ferrozine can generate a complex that is red in color. Nevertheless, the fading of red shade in ferrozine‐Fe^2+^ complexes is only restricted in the presence of other chelating agents. The measurement of the reduced color determines the chelating activity that competes with ferrozine for ferrous ion. Briefly, 800 µl of sample extract is added to 2 mM of ferrous chloride (100 µl). In order to initiate the reaction, 5 mM of ferrozine (400 µl) is added and the mixture is incubated for 10 min at room temperature. The absorbance of the mixture is measured at 562 nm against the blank sample. Citric acid or EDTA can serve as control, whereas distilled water can function as blank. The capacity to chelate ferrous ion is calculated (Prieto, Pineda, & Aguilar, [1999](#fsn31012-bib-0029){ref-type="ref"}).$$\text{Chelation}\left( \% \right) = \left\lbrack {1 - \left( {\text{A}_{562}\,\text{Sample}/\text{A}_{562}\,\text{Control}} \right)} \right\rbrack \times 100$$ 5.15. β‐carotene/linoleic acid bleaching {#fsn31012-sec-0027} ---------------------------------------- β‐carotene bleaching is one of the fastest methods that can be employed to screen antioxidants based on the principle that linoleic acid, which is an unsaturated fatty acid, gets oxidized by ROS that is produced by oxygenated water. The produced resultant can initiate the oxidation of β‐carotene, thus leading to discoloration. In brief, β‐carotene (0.2 mg/ml) is dissolved in chloroform and added into a round bottom flask, along with 20 µl of linoleic acid and 200 µl of Tween 20. A volume of 200 µl of the sample extract, either control or standard, is added. The mixture is evaporated at room temperature to dry under vacuum. Next, 50 ml of distilled water is added and the mixture is shaken to form a liposome solution. The initial absorbance (Abs^0^) of the mixture is measured at 470 nm. After that, the remaining solution is placed in water bath at 50°C for 120 min and the absorbance is recorded at 470 nm (Abs^120^). As for the control solution, 80% of methanol excluding sample extract can be used. The percentage of antioxidant activity is calculated (Dastmalchi, Dorman, Laakso, & Hiltunen, [2007](#fsn31012-bib-0012){ref-type="ref"}).$$\text{Antioxidant}\left( \% \right) = \left\lbrack {1 - \left( {\text{Abs}^{0}\,\text{Sample} - \text{Abs}^{120}\,\text{Sample}} \right)/\left( {\text{Abs}^{0}\,\text{Control} - \text{Abs}^{120}\,\text{Sample}} \right)} \right\rbrack \times 100$$ 6. PRECAUTIONS, REPEATABILITY, AND LIMIT OF DETECTION {#fsn31012-sec-0028} ===================================================== Performing an in vitro antioxidant assay requires several precautions in order to reduce error. Both the enzymatic and nonenzymatic in vitro antioxidant assays need to be conducted in a dark room. An antioxidant is a sensitive compound that easily deteriorates when exposed to light. Alternative to using a dark room, preparation of samples also can be performed by covering the flasks with aluminum foil or by using amber glass bottles. Enzymatic antioxidant assay needs to be performed in a cold room (approximately 4°C) due to higher rate of fluctuations. In an in vitro antioxidant assay, the reagents used need to be freshly prepared as the reactivity of the reagents decreases with time. Preferably, extraction of samples needs to be prepared fresh prior to carrying out the assay. Although samples can be stored in a freezer (at −4 to −80°C) for several days, antioxidant activity is generally lower as compared to freshly prepared samples. Repeat of in vitro antioxidant assay is usually conducted to increase precision and yield more accurate results. In order to establish repeatability, an in vitro antioxidant assay needs to be conducted in the same place, using the same procedures and instruments. Environmental conditions such as room temperature and light intensity need to be adjusted to same. An in vitro antioxidant assay for each sample needs to be repeated for at least three times, and an average reading is calculated. With the average of readings, a standard deviation and a standard error can be obtained. Significance of results is determined by conducting statistical analysis. Comparison between samples of antioxidant activity can be analyzed by calculating IC~50~ values. IC~50~ is defined as the concentration of sample required to exhibit 50% of free radical inhibition. The lower the amount of sample required to inhibit 50% of free radicals, the greater the antioxidant activity of the sample. Limit of detection (LOD) is defined as the lowest amount or concentration of test samples that can be detected from zero. Prior to calculating the LOD, a calibration curve of sample needs to be plotted and linear equation be obtained. The limit of detection is calculated by using the formula (Yilmaz, Sadikoglu, Saglikoglu, Yagmur, & Askin, [2008](#fsn31012-bib-0040){ref-type="ref"}):$$\text{Limit\ of\ detection} = 3\, s/m$$where *s* is the standard deviation and *m*is the slope. 7. CONCLUSION {#fsn31012-sec-0029} ============= At present, researches pertaining to antioxidant compounds from plant sources have attracted attention across the globe due to its therapeutically and pharmacologically potent properties with low or no side effects to human. Besides, the increasing uses of chemicals in food productions, pollution, smoking, and synthetic medicine appear to increase the chances of free radicals based diseases. Plants are the biggest source of antioxidants, and they are sufficient to be used as medicine to fight against harmful diseases. This review article looked into enzymatic and nonenzymatic assays to evaluate antioxidants. Hence, this review article serves as a comprehensive reference for those keen in studies pertaining to antioxidants. CONFLICT OF INTEREST {#fsn31012-sec-0031} ==================== The authors declare no conflict of interest. ETHICAL STATEMENT {#fsn31012-sec-0032} ================= This study does not involve any human and animal testing. The authors would like to thank Dr Thavamaran Kanesan, who helped in English editing of this article.

All data files are available at <https://dataverse.nl/dataset.xhtml?persistentId=hdl:10411/PTQG6C>. Introduction {#sec005} ============ Breast cancer (BC) is the most frequently diagnosed malignant tumor among women in both high-income countries (HICs) and low and middle-income countries (LMICs) \[[@pone.0200966.ref001]\]. The incidence of BC in LMICs is lower than for HICs, but mortality rates in LMICs are higher than in HICs because of advanced-staged diagnosis and inadequate access to care \[[@pone.0200966.ref002]\]. The mortality rates have been decreasing in many HICs since around 1990 due to early detection and improved treatment \[[@pone.0200966.ref002]\]. In Indonesia, BC continues to be the most common malignancy in women with an incidence rate of 40.3 percent and a mortality rate of 16.6 percent per 100,000 people \[[@pone.0200966.ref003]\]. The diagnostic process of BC and its treatments are often associated with negative effects that can lead to lower quality of life (QOL) \[[@pone.0200966.ref004], [@pone.0200966.ref005]\]. Consequently, the current intervention of BC should not only focus on illness control but also to maintain and improve QOL of women with BC. Throughout the process of hospital care, from diagnosis to treatment, the BC examinations and treatments affect the physical, psychological and social aspects of the life of a woman, which can significantly reduce her QOL, increase psychological distress \[[@pone.0200966.ref006]\], and uncertainty \[[@pone.0200966.ref007]\], negatively affect her body image and sexuality \[[@pone.0200966.ref008]\], illness perception \[[@pone.0200966.ref009]\], and increase unmet health needs \[[@pone.0200966.ref010]\]. Therefore, the information about QOL is crucial at every stage of the BC trajectory. However, most investigations explore various issues after definitive diagnosis, e.g., QOL in women with BC during treatment \[[@pone.0200966.ref009], [@pone.0200966.ref011], [@pone.0200966.ref012]\] and QOL in BC survivors \[[@pone.0200966.ref013]--[@pone.0200966.ref015]\]. To our knowledge, no previous investigation was conducted among the women before the definitive diagnosis in Indonesian women. This stage is essential because some psychosocial problems might already occur when women find abnormalities in their breasts. One of the issues may be the uncertainty about having or not having the disease and the future treatment process. The diagnosis of BC may have many consequences, not only related to life expectancy but also to QOL. Some women with BC in Indonesia have a belief that BC is an incurable and deadly disease \[[@pone.0200966.ref016]\]. On the other hand, despite having discovered the symptoms of BC, some women may assume that they have no severe health problems. Given the interrelation between demographic characteristics, QOL and health status in general \[[@pone.0200966.ref017]--[@pone.0200966.ref020]\], we also evaluated which demographic attributes might contribute to a lower QOL and health status in the women with BC symptoms. No studies have yet been published that compared QOL and health status between undiagnosed women with BC symptoms and women in the general population in Indonesia. Therefore, the aims of this study were: (1) to assess QOL and health status of Indonesian women with BC symptoms before receiving a definitive diagnosis; (2) to compare QOL and health status between women with BC symptoms and Indonesian women in general; (3) to evaluate the association of socio-demographic factors with QOL and health status of Indonesian women with BC symptoms before the definitive diagnosis. The current study that investigated QoL and health status for women with BC symptoms before definite diagnosis may serve as a bridge for future studies to explore whether psychosocial concerns such as impaired body image and unmet needs should be taken into account for women with BC symptoms before definitive diagnosis. Methods {#sec006} ======= Participants {#sec007} ------------ This study consisted of two groups of participants: 1) Indonesian women with BC symptoms who already consulted the hospital but yet without a definitive diagnosis (BC Symptoms Group) and (2) Indonesian women from the general population (General Population Group). ### BC symptoms group {#sec008} Participants were recruited from four district hospitals in West Java, Indonesia. They were new patients (outpatients) who visit the hospital with breast symptoms, which make them suspects of having BC, but yet without a definitive diagnosis. The following inclusion criteria were used: women with age 18 years and above, an adequate command of the Indonesian language (Bahasa Indonesia) and no major psychiatric disorder. The last criterion was determined by checking the medical record on a consultation history/record with the Psychiatric Department. Patients who have been seen by a psychiatrist were excluded from the study. ### General population group {#sec009} The data of the comparison group (which will be referred to as \"general population\" in this manuscript) were women selected from a larger study focused upon the Indonesian general population, in which several questionnaires were tested in a face-to-face setting at the home/office of the interviewer or at the homes of the subjects \[[@pone.0200966.ref021]\].This study implemented a multi-stage stratified quota sampling method to ensure representativeness with the Indonesian general population, which resulted in 1054 participants being interviewed. Only the female participants with the age of 18 years and above from the aforementioned study were included in the analysis of the present study. Instruments {#sec010} ----------- A standard socio-demographic questionnaire was used to collect participants\' background data on residence, age, education level and income level. QOL was measured using the Indonesian version of the WHOQOL-BREF, with a four weeks-time retrospection. WHOQOL-BREF has been utilized in several investigations in the BC populations in Asian countries \[[@pone.0200966.ref022]\], including Indonesia \[[@pone.0200966.ref009]\]. This instrument is a self-report questionnaire that consists of 26 items. The internal consistency of the WHOQOL BREF's domains in the present sample were 0.70, 0.78, 0.57, and 0.75 for physical, psychological, social, and environmental domain respectively. Two items measure QOL and health satisfaction in general. Twenty-four items measure four broad domains: (i) physical health (7 items), e.g., \"Do you have enough energy for everyday life?", (ii) psychological health (6 items), e.g., \"How much do you enjoy life?", (iii) social relationships (3 items), e.g., \"How satisfied are you with your personal relationships?" and (iv) environmental (8 items), e.g., \"How satisfied are you with the conditions of your living place?" Each item is rated using a 5-point Likert scale with varied wording on each scale depending on the item (for example 1 = very dissatisfied to 5 = very satisfied) \[[@pone.0200966.ref023]\]. The internal consistency of the WHOQOL BREF domains in the present sample were 0.70, 0.78, 0.57, and 0.75 for physical, psychological, social, and environmental domain respectively. The health status of the participants was measured by the EQ-5D-5L \[[@pone.0200966.ref024]\]. This instrument has been used in several BC patient populations around the world \[[@pone.0200966.ref025]\]. EQ-5D-5L is a generic health-related QOL instrument based on a descriptive system that defines health in terms of five dimensions: mobility (MO), self-care (SC), usual activities (UA), pain/discomfort (PD), and anxiety/depression (AD). Each dimension has five levels: (1) no problems, (2) slight problems, (3) moderate problems, (4) severe problems, and (5) extreme problems/unable. Therefore, the EQ-5D-5L instrument describes 3125 (5^5^) unique health states. A 1-digit number expresses the level selected for that specific dimension. A specific health state then consisted of a combination of a 5-digit number for the five dimensions. For example, state '11111' indicates 'no problems on any of the five dimensions', while state '34512' indicates 'moderate problems in walking about, severe problems washing or dressing, extreme problems doing usual activities, no pain or discomfort, and slight anxiety or depression'. This descriptive system is followed by a self-rating of overall health status on a visual analogue scale (EQ-VAS) ranging from 0 (\"the worst health you can imagine\") to 100 (\"the best health you can imagine\"). EQ-5D-5L has been proven as a valid and reliable questionnaire to be used in Indonesia \[[@pone.0200966.ref026]\]. Data collection procedures {#sec011} -------------------------- ### BC symptoms group {#sec012} The study was approved by the Health Research Ethics Committee of Dr. Hasan Sadikin General Hospital Bandung. Participants who agreed to participate by means of oral or written consent were asked to complete the following instruments in the hospital: (1) the socio-demographic and medical history form, (2) the WHOQOL-BREF, and (3) the EQ-5D-5L. If they had difficulties in completing the instruments, the interviewers helped them by reading the items out loud and asking the participants to indicate the answers. ### General population group {#sec013} The study was approved by the Health Research Ethics Committee, Faculty of Medicine, Universitas Padjadjaran, Indonesia \[[@pone.0200966.ref021]\]. A representative sample from the Indonesian general population was recruited using multi-stage stratified quota sampling. The interviewers explained the objectives of the study, followed by filling in the informed consent when the participants agreed to participate. Three instruments completed by the participants were: (1) the socio-demographic form: age, sex, income, and education, (2) the WHOQOL-BREF, (3) the EQ-5D-5L. The participants were helped by the interviewers whenever they had problems completing the questionnaires. Data analysis {#sec014} ------------- Demographic characteristics were summarized using descriptive statistics, including percentages for categorical data, and means and standard deviations for continuous data. The self-reported health problems obtained from the EQ-5D-5L were presented in percentages of each level of each dimension that was answered positively and then compared between the groups with the Chi-square test. Each participant's EQ-5D-5L responses then were transformed to a single index score based on the preference of the Indonesian general population, a so-called 'value set'\[[@pone.0200966.ref021]\]. For instance, the health state of '11111' corresponds to an EQ-5D-5L index score of 1.00, and '22211', which means 'slight problems in mobility, self-care and usual activities and no problems in pain/discomfort and anxiety/depression' leads to a value of 0.69. Mean and standard deviation were calculated for the EQ-VAS, EQ-5D-5L index score and each domain of the WHOQOL-BREF. For the comparison of the QOL between the two groups, we applied an independent t-test if the data were normally distributed or the Wilcoxon rank-sum test if not normally distributed. Normality was tested using the Shapiro-Wilk test and visual inspection of the histograms. For determining the magnitude of the differences, we calculated the effect size using Cohen\'s d, and we applied the criteria from Cohen for the interpretation: 0.2--0.5 = small, 0.5--0.8 = medium, \>0.8 = large difference \[[@pone.0200966.ref027]\]. We also applied multivariate analysis of covariance (MANCOVA) with the QOL (WHOQOL-BREF: physical, psychological, social, and environment domain scores) and health status (EQ-5D-5L: EQ-VAS and index score) scores as outcomes and a variable 'group' (BC symptoms group vs. general population group) as the predictor in the MANCOVA. Further multiple linear regression analysis was carried out to evaluate whether and if so which socio-demographic variables were significantly affect the QOL and health status scores in the BC symptom group only. Beforehand, a Spearman correlation analysis was done to check whether any significant correlation(s) between each sociodemographic variable with other demographic variables and the outcomes (QOL and health status). We found that ethnicity and religion had no significant correlation to any other variables, therefore they were excluded from the analysis. The following sociodemographic variables: residence (urban/rural), age, level of education (basic: primary school and below/middle: high school/high: all others), and monthly income (below 2 million IDR/2-4 million IDR/above 4 million IDR) were included in the multiple linear regression analysis. Statistical analyses were performed using SPSS-IBM version 21; p-values \< .05 were considered statistically significant. Results {#sec015} ======= Characteristics of participants {#sec016} ------------------------------- The demographic characteristics of women with BC symptoms and the general population are presented in [Table 1](#pone.0200966.t001){ref-type="table"}. The proportion of age and residence subgroups were similar between the two groups. The BC symptom group had a significantly higher level of education and monthly income than the general population group. 10.1371/journal.pone.0200966.t001 ###### Demographic characteristics of the Breast Cancer (BC) symptoms sample (N = 132) and the general population sample (N = 471). {#pone.0200966.t001g} ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Characteristic Level BC Symptom General Population[^a^](#t001fn001){ref-type="table-fn"} Pearson's χ^2^ (df) P-value ----------------------------------------------------------- -------------- ------------ ---------------------------------------------------------- --------------------- --------- ------------- --------- Age 18--30 years 48 (36.4) 157 (33.3) 0.4263 (2) 0.808 31--50 years 58 (43.9) 218 (46.3) \> 50 years 26 (19.7) 96 (20.4)   Residence Rural 74 (56.1) 217 (46.1) 4.1196 (1) 0.042 Urban 58 (43.9) 254 (53.9)   Level of Education[^b^](#t001fn002){ref-type="table-fn"}\ Basic 22 (16.7) 172 (36.5) 18.9003 (2) \<0.001 Middle 79 (59.8) 222 (47.1) High 31 (23.5) 77 (16.3) Income/month[^c^](#t001fn003){ref-type="table-fn"}\ Low 60 (45.5) 403 (85.6) 93.2667 (2) \<0.001 Middle 54 (40.9) 49 (10.4) High 18 (13.6) 19 (4.0) ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ^a^based on study Purba, et al., 2017 ^b^Basic: primary school and below, Middle: high school, High: all others ^c^Low:2 million IDR, Middle: 2--4 million IDR, High: above 4 million IDR QoL and health status in BC symptoms group and general population group {#sec017} ----------------------------------------------------------------------- The mean scores of the QOL domains: measured by the WHOQOL-BREF and health status: measured by the EQ-5D-5L are summarized in [Table 2](#pone.0200966.t002){ref-type="table"}. Concerning the QOL, the BC symptoms group showed significantly lower scores (less favorable) on physical and psychological domains than the general population. The effect sizes for these differences were considered as small. For health status, the EQ-VAS and index score of women with BC symptoms were significantly lower than the general population with medium effect sizes for both scores. 10.1371/journal.pone.0200966.t002 ###### Comparison of means of quality of life (QOL) and health status of the Breast Cancer (BC) symptoms and the general population sample. {#pone.0200966.t002g} BC symptom General population t-statistic[^a^](#t002fn001){ref-type="table-fn"} P-value Effect Size[^b^](#t002fn002){ref-type="table-fn"} ---------------------- ----------------- ------------ -------------------- --------------------------------------------------- --------- --------------------------------------------------- --------- ----- Quality of life Physical health 63.2 13.9 67.9 11.3 4.004 \<0.001 0.4 Psychological health 62.5 15.7 65.2 12.4 2.042 0.042 0.2 Social relations 62.8 14.4 62.0 13.5 -0.609 0.543 -0.1 Environment 59.3 12.6 58.4 12.9 -0.729 0.467 -0.1 Health status EQ-VAS 69.1 20.0 78.6 14.7 6.010 \<0.001 0.6 Index score 0.8 0.2 0.9 0.1 9.066 \<0.001 0.9 ^a^all degress of freedom (df) = 601 ^b^Effect size based on Cohen\'s d [Table 3](#pone.0200966.t003){ref-type="table"} presents the comparison of the EQ-5D-5L self-reported health status of the BC symptom and the general population samples. The proportions of responses for each severity level of problems were significantly different for all dimensions between the two samples. It can be seen that the percentage of the BC symptoms group which reported no problems in four dimensions: self-care, usual activity, pain/discomfort and anxiety/depression was lower than that of the general population. In addition, no participants from the general population group reported the worst level of problems in any dimensions, while 1.5%, 3.8%, and 6.8% of the BC symptoms group indicated unable/severe problems in usual activity, pain/discomfort, and anxiety/depression, respectively. 10.1371/journal.pone.0200966.t003 ###### Comparison of proportions of the EQ-5D-5L self-reported health status of the Breast Cancer (BC) symptoms sample with the general population sample. {#pone.0200966.t003g} Level of problems Mobility Self-Care Usual Activity Pain/Discomfort Anxiety/Depression ------------------- ---------- ----------- ------------------ ----------------- -------------------- ------------------ ------ ------ -------------------- ------ ------ -------------------- ------ ------ --------------------- No 90.9 89.0 11.95 (3); 0.008 92.4 98.0 13.20 (3); 0.004 78.0 86.7 20.88 (4); \<0.001 27.3 57.3 62.95 (4); \<0.001 18.2 63.3 147.19 (4); \<0.001 Slight 3.8 9.2 3.8 1.5 13.6 11.5 51.5 38.5 47.7 29.8 Moderate 3.0 1.5 3.0 0.2 5.3 1.7 13.6 3.1 15.9 6.5 Severe 2.3 0.2 0.8 0.2 1.5 0.0 3.8 1.2 11.4 0.4 Unable/ Extreme 0.0 0.0 0.0 0.0 1.5 0.0 3.8 0.0 6.8 0.0 BC = breast cancer symptom; GP = general population For the MANCOVA analysis, we included the educational level and monthly income as covariates because only these two characteristics differed significantly between the two groups. The results still showed significant overall differences in the QOL between women with BC symptoms and the general population (Wilks' lambda of 0.85; p-value\<0.001). A MANCOVA conducted on the health status yielded similar results (Wilks' lambda of 0.82; p-value\<0.001). Further multiple linear regression analysis conducted only in the BC symptoms group showed that participants who lived in a rural area demonstrated higher social domain scores than they who did not live in a rural area. Concerning education, participants with the lowest educational levels (i.e. primary school and below) demonstrated lower scores (less favorable) on physical, social, and environmental QOL domains than participants who had college/university level of education, while only physical health scores were significantly different between the middle and the lowest levels of education. Participants in the lowest monthly income group demonstrated less favorable scores in physical, psychological, and environment QOL domains than participants in the highest monthly income group, while only the social domain score was significantly different between the middle and lowest-income groups. With respect to health status (EQ-5D-5L), participants who had the lowest educational level demonstrated significantly lower EQ-5D-5L index scores than participants who achieved higher educational level. We found that age had no significant association with QOL and health status of women with BC symptoms. Details can be seen in [Table 4](#pone.0200966.t004){ref-type="table"}. 10.1371/journal.pone.0200966.t004 ###### Coefficients (B-values) from multiple linear regression analysis for quality of life and health status in women with Breast Cancer (BC) symptoms. {#pone.0200966.t004g} Predictors Physical QOL Psychological QOL Social QOL Environmental QOL EQ-VAS Index score ------------------------------------------------- -------------- ------------------- ------------ ------------------- -------- ------------- ------ ----- ------- ------ ----- ------- ------- ----- ------- ------ ----- ------- Group[^a^](#t004fn001){ref-type="table-fn"}   BC -6.7 1.3 0.000 -4.8 1.4 0.001 -1.8 1.5 0.205 -2.0 1.3 0.141 -11.2 1.7 0.000 -0.2 0.0 0.000 Residence[^b^](#t004fn002){ref-type="table-fn"}   Urban 0.0 0.0 0.389 0.0 0.0 0.970 -0.1 0.0 0.139 0.0 0.0 0.695 -0.1 0.1 0.084 0.0 0.0 0.001 Age -1.1 1.0 0.268 -2.2 1.1 0.041 -2.7 1.1 0.017 -1.3 1.0 0.198 -3.0 1.3 0.025 0.0 0.0 0.206 Education[^c^](#t004fn003){ref-type="table-fn"}   Middle 1.8 1.1 0.109 2.1 1.2 0.091 2.4 1.3 0.065 2.5 1.2 0.034 0.4 1.5 0.777 0.0 0.0 0.051   High 2.6 1.6 0.098 4.7 1.8 0.008 5.4 1.8 0.003 6.6 1.7 0.000 4.0 2.1 0.059 0.0 0.0 0.097 Income[^d^](#t004fn004){ref-type="table-fn"}   Middle 2.5 1.5 0.084 2.0 1.6 0.223 3.8 1.7 0.022 3.6 1.5 0.020 1.9 2.0 0.345 0.0 0.0 0.027   High 7.5 2.2 0.001 7.0 2.4 0.004 5.8 2.5 0.021 9.2 2.3 0.000 5.3 3.0 0.074 0.1 0.0 0.044 Constant 67.8 1.8 0.000 64.2 2.0 0.000 63.2 2.1 0.000 56.7 1.9 0.000 82.3 2.5 0.000 0.9 0.0 0.000 ^a^General population is the reference ^b^Rural is the reference group ^c^Basic education level: primary school and below is the reference group ^d^Low monthly income is the reference group Discussion {#sec018} ========== To our knowledge, this is the first study that compared the QOL between Indonesian women with BC symptoms before the definitive diagnosis and Indonesian women in general. We found that the QOL of Indonesian women with BC symptoms was significantly lower than in the general Indonesian population, especially in the physical and psychological domain. They also reported more problems across all dimensions, namely mobility, self-care, usual activity, pain/discomfort and anxiety/depression. These findings were also maintained after correction for demographic differences. In addition, we found that education and monthly income were positively associated with the QOL and health status among the women with BC symptoms. Previous studies among patients with BC reported that pain/discomfort and anxiety/depression are the most common symptoms reported. This might be associated with lower level of HRQOL among patients across different states of BC: after primary BC, during recurrence, and metastases \[[@pone.0200966.ref028]--[@pone.0200966.ref030]\]. Our study extended these previous results by adding that in the phase of pre-definite diagnosis, the similar results occurred: a higher percentage of reported problems in pain/discomfort and anxiety/depression. Note that because of the EQ-5D wording in the two aforementioned dimensions, we don't know yet whether the women felt pain or discomfort and anxiety or depression. Further explorations might be needed to investigate whether participants have problems on only one or both conditions, e.g. pain or discomfort. Concerning the group of women with BC symptoms, we found that higher levels of education and income were associated with more favorable physical, social, and environmental dimensions of QOL compared to those with lower levels of education. This finding is consistent with previous studies in other populations which demonstrated that both income \[[@pone.0200966.ref015], [@pone.0200966.ref031], [@pone.0200966.ref032]\] and education \[[@pone.0200966.ref033]\] level have a significant impact on QOL: the lower, the worse. It may be hypothesized that higher socio-economic and educational level of patients may lead to better access to information and health services; as a result, these individuals may have fewer problems and feel less uncertain. Certain limitations of the current study should be considered. First, the sample of women with BC symptoms was obtained from only one area in Indonesia, West Java, that might not be a representative for the whole Indonesian archipelago. Second, the participants of the present study were women with BC symptoms who visited the hospital. It could be argued that these women were anxious enough about the symptoms they observed to enable them to visit the hospital, compared to the women who did not visit the hospital although they probably observed some BC symptoms. This might have biased the results. Third, the comparison group consisting of adult women from the general population, was not screened for the presence of any diseases. Therefore, it is possible that this group included a few participants with BC symptoms, a BC diagnosis, or BC survivors. This might have influenced the results, leading to an underestimation of the actual differences between the groups. Fourth, the choice of generic instruments: WHQOL-BREF and EQ-5D-5L, might be not sensitive enough to measure the QOL and health status of the patients compared to disease-specific instruments such as the European Organization for Research and Treatment of Cancer QOL Questionnaire-C30 (EORTC QLQ-C30) \[[@pone.0200966.ref034]\] and the Functional Assessment of Cancer Therapy-General (FACT-G) \[[@pone.0200966.ref035]\]. However, since the aim of the present study was to compare QOL of women with BC symptoms to women from the general population, which are less likely to have any BC symptoms, generic QOL instruments were considered as the best tool to serve this aim. Nevertheless, there are significant differences between the groups, which indicate worse QOL and health status in the group with BC symptoms. Fifth, although the EQ-5D-5L had been validated and used in breast cancer patients' population in different countries across the world \[[@pone.0200966.ref025]\], this was not the case for Indonesia. So, it is not known for certain that the psychometric properties are supported accurately in the context of this study. Future research might investigate other factors that may contribute to the QOL of women with BC symptoms, such as social support or physical activities, and find and evaluate effective ways to promote and improve their QOL. Studies might evaluate strategies carried out by healthcare providers and professionals, e.g., physicians, nurses, psychologists, or community health workers to increase their compliance and reduce physical and psychological problems, such as provision of individualized information, symptom management, counseling, or psychosocial interventions. Conclusions {#sec019} =========== Our study showed that Indonesian women with BC symptoms before the definitive diagnosis reported lower physical and psychological QOL and more pain/discomfort and anxiety/depression compared to the Indonesian women in general. Awareness and support for them from the medical field might improve these aspects of QOL. The authors thanks Hery Susanto for his support in data analysis. BC : Breast cancer EQ-5D-5L : European Quality of Life-5 Dimensions-5 Levels HICs : High-income countries LMICs : Low-middle income countries QOL : Quality of life WHOQOL-BREF : World health organization Quality of life BREF [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

The COVID-19 pandemic has overwhelmed medical facilities worldwide and caused a shortage of typically disposable personal protective equipment (PPE), forcing medical workers to reuse or work without proper PPE.[@c1] Researchers have explored decontamination procedures that might allow PPE to be reused safely,[@c3] and medical workers have begun implementing these procedures, including decontaminating disposable masks with ultraviolet (UV) irradiation.[@c5] However, UV decontamination faces several drawbacks, including an inability to kill viruses trapped within crevices that are not illuminated and a lack of availability at clinics in low-income areas and in most peoples\' homes.[@c6] Alternative methods of decontamination, namely, steam sterilization, alcohol washing, and bleach washing, are useful for glassware and other durable materials, but have been reported to degrade single-use PPE.[@c4] On the other hand, dry heat decontamination can be performed almost anywhere (including home ovens and rice cookers) and inactivates viruses within crevices without damaging the delicate PPE.[@c7] However, dry heat decontamination guidelines for SARS-CoV-2 remain limited to a few experimental measurements constrained to specific temperatures that do not apply to all heating devices.[@c11] Meanwhile, virus transmission has been linked to variations in outdoor climate, where colder atmospheric temperatures lead to longer virus lifetimes outside of hosts. This effect has been reported for influenza,[@c12] the common cold,[@c14] SARS-CoV-2,[@c11] SARS-CoV-1,[@c16] and MERS-CoV.[@c18] Even at a local scale, a recent resurgence of COVID-19 cases in a seafood market was linked to low temperatures.[@c20] Epidemiologists would benefit from knowledge of the lifespan of SARS-CoV-2 as a continuous function of the atmospheric temperature to accurately model the spread of COVID-19. Furthermore, understanding the temperature-dictated inactivation time could help predict the resurgence of cases as colder weather returns to the Northern Hemisphere, following a similar trend to that of the seasonal flu.[@c21] We introduce an analytical model based on the rate law for a first-order reaction and the Arrhenius equation that enables prediction of the thermal inactivation rate and lifetime of coronaviruses, including SARS-CoV-2, as a function of temperature. These viruses are treated as macromolecules undergoing thermal denaturation; we confirm that coronaviruses undergo thermal denaturation because their inactivation behavior follows the Meyer--Neldel rule.[@c22] The time required to achieve the desired log-scale reduction in viable virions (e.g., by a factor of 10^3^ as typically used for viral decontamination[@c23]) was used to generate dry heat decontamination guidelines for SARS-CoV-2 relevant to temperature ranges accessible in commonly available heating devices. The model also predicts the lifetime of human coronaviruses as a continuous function of temperature in various climates, which will assist epidemiologists in understanding the regionally dependent lifetime of the SARS-CoV-2 virus, as well as the potential of a COVID-19 resurgence in autumn and winter. Reports in the literature provide abundant data to construct a predictive analytical model capturing the thermal effects on virus inactivation. We specifically focused on the inactivation of coronaviruses, a group of enveloped viruses often responsible for respiratory or gastrointestinal diseases in mammals and birds.[@c27] We compiled hundreds of data points for the inactivation of five coronaviruses, with subdivisions based on (i) strains of each virus, (ii) environmental pH levels, and (iii) relative humidity (RH) conditions, resulting in 14 datasets \[[Fig. 1(a)](#f1){ref-type="fig"}\]. These viruses include (i) Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1 and SARS-CoV-2);[@c11] (ii) Middle East Respiratory Syndrome Coronavirus (MERS-CoV);[@c18] (iii) Transmissible Gastroenteritis Virus (TGEV);[@c31] (iv) Mouse Hepatitis Virus (MHV);[@c32] and (v) Porcine Epidemic Diarrhea Virus (PEDV).[@c34] {ref-type="disp-formula"}\], where the inserted chart presents the R^2^ values for the linear fits. The resulting activation energy, E~a~, and frequency factor, ln(A), were back-calculated from each linear fit according to Eq. [(2)](#d2){ref-type="disp-formula"} and plotted (b); the linear correlation between ln(A) and E~a~ indicates protein denaturation.[@c22]](APPLAB-000117-060601_1-g001){#f1} The rate law describes the inactivation behavior of microbes.[@c35] Non-first-order rate laws have been applied to the inactivation of some microbes,[@c36] particularly bacteria with heterogeneous populations,[@c39] but the inactivation of most viruses---including the coronaviruses considered in our analysis---follows a first-order reaction, with viable virions as reactants and inactivated virions as products \[Eq. [(1)](#d1){ref-type="disp-formula"}\]: $$\left\lbrack C \right\rbrack = \left\lbrack C_{0} \right\rbrack e^{- kt}.$$The majority of primary experimental data for the inactivation of viruses is reported in plots of the log of concentration, ln(\[C\]), as a function of time, *t*, with \[C~0~\] being the initial concentration of viable virions. We applied a linear regression to each set of primary data to determine the rate constant, *k*, for the inactivation of a virus at a given temperature, *T*, determined by calculating the slope, *k* = $-$Δln(\[C\])/Δ*t*. Each of these pairs of (*k*, *T*) yields one data point in [Fig. 1(a)](#f1){ref-type="fig"}, with details in the [supplementary material](https://doi.org/10.1063/5.0020782#suppl), Figs. S1--S28. Virus inactivation occurs due to thermal denaturation of the proteins that comprise each virion. The temperature dependence of the thermal denaturation process is captured by the Arrhenius equation,[@c40] which yields a linear relationship between ln(*k*) and 1/*T* \[Eq. [(2)](#d2){ref-type="disp-formula"}\]: $$\ln\left( k \right) = - \frac{E_{a}}{RT} + \ln\left( A \right),$$where *R* is the gas constant, *E~a~* is the activation energy associated with the inactivation of the virus (i.e., the energy barrier to be overcome for protein denaturation), and *A* is the frequency factor. In [Fig. 1(a)](#f1){ref-type="fig"}, ln(*k*) and 1/*T* are plotted according to the Arrhenius equation \[Eq. [(2)](#d2){ref-type="disp-formula"}\]. The activation energy, *E~a~*, and the natural log of the frequency factor, ln(*A*), can be obtained by equating --*E*~a~/*R* and ln(*A*) from Eq. [(2)](#d2){ref-type="disp-formula"} with the slopes and intercepts from the linear fits in [Fig. 1(a)](#f1){ref-type="fig"}, respectively, and are plotted in [Fig. 1(b)](#f1){ref-type="fig"}. The linear correlation between ln(*A*) and *E~a~* indicates that coronaviruses undergo a thermal denaturation process following the Meyer--Neldel rule,[@c22] supporting our hypothesis that they are primarily inactivated by the thermally driven degradation of proteins. In fact, the linear regression calculated in this work, \[ln(*A*) = 0.394*E*~a~ − 5.63\], is nearly identical to those calculated in two prior studies on the denaturation of tissues and cells, which report \[ln(*A*) = 0.380*E*~a~ − 5.27\][@c22] and \[ln(*A*) = 0.383*E*~a~ − 5.95\].[@c41] The degree of inactivation of a pathogen is defined by the ratio of the concentration (amount) of a pathogen to its initial concentration, \[C\]/\[C~0~\], often in terms of orders of magnitude; an *n*-log inactivation refers to a reduction in the concentration of 10 raised to the *n*th power (\[C\]/\[C~0~\] = 10^−*n*^). Equations [(1)](#d1){ref-type="disp-formula"} and [(2)](#d2){ref-type="disp-formula"} combine to yield an analytical model used in determining the time required to achieve an *n*-log reduction in a pathogen \[Eq. [(3)](#d3){ref-type="disp-formula"}\]: $$t_{n - \mathit{\log}} = - \frac{1}{A}e^{(\frac{E_{a}}{RT})}{\ln\left( 10^{- n} \right)}.$$The U.S. Food and Drug Administration recommends a 3-log (99.9%) reduction in the number of virions for the decontamination of non-enveloped viruses (i.e., \[C\]/\[C~0~\] = 10^−3^).[@c23] Since non-enveloped viruses are shown to be more resilient to elevated environmental temperatures than their enveloped counterparts (including coronaviruses),[@c44] we refer to the time required to achieve a 3-log reduction as the coronavirus *lifetime*, indicative of a conservative prediction for both decontamination time and viable lifetime outside a host. The time required to achieve an *n*-log reduction is directly proportional to the *n* value; therefore, a more conservative decontamination time could be obtained by inserting a different value of *n* into Eq. [(3)](#d3){ref-type="disp-formula"}, which would change the *n-*log reduction predictions of lifetime by a multiplicative factor of *n~desired~*/*n~current~* (e.g., in this work, *n~current~* = 3; therefore, a 6-log reduction would require doubling of the lifetimes predicted in this work). [Figure 2](#f2){ref-type="fig"} reports the virus lifetimes generated from Eq. [(3)](#d3){ref-type="disp-formula"} as a function of temperatures ranging from room temperature to temperatures achievable using common heating devices. In [Fig. 2(a)](#f2){ref-type="fig"}, all five types of coronaviruses are plotted to show the variation across different environmental temperatures. The plot in [Fig. 2(b)](#f2){ref-type="fig"} shows similar data, with the exception of data from Casanova, *et al.*,[@c16] due to the possible experimental error in the primary data (see the [supplementary material](https://doi.org/10.1063/5.0020782#suppl), Sec. S3), and with the lifetime axis scaled linearly to highlight the exponential dependence of lifetime on temperature. The human coronaviruses SARS-CoV-2 and SARS-CoV-1 exhibit a similar trend in thermal degradation, in agreement with recent work.[@c30] We observed that SARS-CoV-2 has a slightly longer mean lifetime than SARS-CoV-1 outside a host, potentially contributing to its relatively high reproduction number, *R*~0~. However, based on uncertainty analysis, [Fig. 3](#f3){ref-type="fig"} indicates that the prediction intervals (PIs) of SARS-CoV-1 and SARS-CoV-2 overlap, suggesting that additional data would be needed to definitively support the conclusion that SARS-CoV-2 has a longer lifetime. The prediction interval is used to estimate the variation in coronavirus lifetimes predicted by the analytical model. The prediction interval can account for uncertainties corresponding to different virus strains due to genetic mutations, as well as variations in experimental conditions, such as RH and fomites, and a conservative estimate of the maximum lifetime of a coronavirus given this uncertainty can be determined with different levels of confidence (90%, 95%, and 97.5% prediction intervals are shown in [Fig. 3](#f3){ref-type="fig"}). The details of statistical uncertainty for all of the viruses are included in the [supplementary material](https://doi.org/10.1063/5.0020782#suppl), Table S3. The average lifetime for the human coronaviruses SARS-CoV-2 and SARS-CoV-1 is shown in [Table I](#t1){ref-type="table"}. The temperature values displayed in the table illustrate both (i) common environmental temperatures and (ii) temperatures appropriate for thermal decontamination. {#f2} {#f3} ###### The average lifetimes for SARS-CoV-2 and SARS-CoV-1 across a range of environmental and decontamination temperatures. The upper limit of a 95% prediction interval based on statistical analysis of the data is included in parentheses as a conservative estimate of the maximum lifetime across different mutations and environmental conditions. The mean lifetimes of all human coronaviruses considered in this work were greater than one month at temperatures below 10 °C. Temperature SARS-CoV-2 lifetime, *t*~3-log~ SARS-CoV-1 lifetime, *t*~3-log~ ---------------------------- ---------------------- --------------------------------- --------------------------------- Environmental temperatures 10 °C \>1 month 29.8 d *(\> 1 month)* 15 °C 15.5 d *(\>1 month)* 10.4 d *(\> 1 month)* 20 °C 5.9 d *(\>1 month)* 3.8 d *(\> 1 month)* 25 °C 2.3 d *(25.5 d)* 1.4 d *(25.4 d)* 30 °C 22.5 h *(10.0 d)* 13.1 h *(8.26 d)* 35 °C 9.4 h *(4.2 d)* 5.2 h *(2.9 d)* 40 °C 4.0 h *(1.8 d)* 2.1 h *(1.1 d)* Decontamination 60 °C 10.5 min *(2.3 h)* 4.8 min *(1.1 h)* 70 °C 2.5 min *(38.6 min)* 1.1 min *(18.4 min)* 80 °C \<1 min *(11.9 min)* \<1 min *(6.1 min)* 90 °C \<1 min *(4.0 min)* \<1 min *(2.3 min)* We estimated the regional lifetime of SARS-CoV-2 based on climate temperatures in the United States. We used temperatures averaged over January to March, 2020, corresponding to the onset of the COVID-19 pandemic \[[Fig. 4(a)](#f4){ref-type="fig"}\], and July to September, 2019, as a rough prediction of SARS-CoV-2 lifetimes in summer 2020 \[[Fig. 4(b)](#f4){ref-type="fig"}\]. Summer weather in the Northern Hemisphere will reduce SARS-CoV-2 outdoor-lifetime significantly, potentially slowing the transmission of COVID-19. The predictions in [Fig. 3](#f3){ref-type="fig"} are based on a constant temperature profile and do not account for daily temperature fluctuations, which may result in shorter lifetimes than predicted due to the exponential dependence of the reaction rate on temperature. Additional environmental effects, like UV from sunlight, may further reduce inactivation time; with these limitations in mind, [Fig. 4](#f4){ref-type="fig"} represents a conservative prediction of SARS-CoV-2 lifetime across the United States, and lifetimes greater than one month are not reported. {#f4} We tested the predictive ability of the thermodynamic model presented here by comparing the results to experimental data that had not been used to train the model. SARS-CoV-1 was reported to require 5 days at room temperature to achieve a 5-log reduction;[@c46] our model predicts an inactivation time of 4.2 days under the same conditions. In another report, SARS-CoV-1 was heated to 56 °C and required only 6 min to achieve a 6-log reduction;[@c29] our model predicts a time of 17 min. A third report claimed that SARS-CoV-1 required 30 min to achieve an approximately 6-log reduction at 60 °C;[@c47] our model predicts a time of 10 min. A more recent report shows that both SARS-CoV-2 and SARS-CoV-1 require 72 h for a 3-log reduction on plastic surfaces maintained around 23 °C; our model predicts lifetimes of 80 and 50 h, respectively, in good agreement with the reported data.[@c30] All of these reported lifetimes were within the uncertainty bounds of the model predictions. Considering the similarity in inactivation behavior for SARS-CoV-1 and SARS-CoV-2,[@c30] validation with SARS-CoV-1 suggests that this model will be a useful tool to estimate the lifetime of SARS-CoV-2. The model is limited to temperature-based predictive ability and does not consider relative humidity or the fomite (i.e., the surface material on which a virion rests), both of which appear to affect inactivation times.[@c11] Variations in lifetime at a given temperature due to these environmental factors can be interpreted as catalytic effects;[@c49] incorporating a corresponding adjustment to the activation energy might enable additional predictive capabilities. Another limitation of this model is its reliance on a limited set of primary data, which may contain experimental error (all primary data are reproduced in the [supplementary material](https://doi.org/10.1063/5.0020782#suppl)); statistical prediction uncertainties are described in the [supplementary material](https://doi.org/10.1063/5.0020782#suppl), Sec. S5. In addition, this model assumes that the enthalpy and entropy of the inactivation reaction are constant as temperature changes. This assumption is typically valid for macromolecules such as proteins.[@c22] Some reports suggest that multiple inactivation reaction pathways can occur near room temperature, but these reports are limited in scope and do not agree with each other, and further work would need to be done before considering or implementing such effects.[@c31] Finally, the extrapolation of our model to higher temperatures outside the range of the primary data (e.g., above 100 °C) may be unfounded if alternate inactivation reaction pathways become available at these elevated temperatures. Fortunately, the results in [Table I](#t1){ref-type="table"} indicate that dry heat decontamination is feasible for inactivation of all types of coronaviruses, including SARS-CoV-2. The most common material used in surgical masks and N95 respirators is non-woven polypropylene,[@c50] which can be decontaminated with dry heat below its melting point (156--168 °C).[@c52] Cui and colleagues show that thermal cycling (75 °C, 30 min heating, applied over 20 cycles) does not degrade the filtration efficiency of N95-level facemasks,[@c9] and Lin *et al.* report no significant degradation in the effectiveness of surgical masks after heating to 160 °C for 3 min.[@c7] Therefore, we expect that dry heat decontamination is an effective decontamination method, while also feasible within relatively short times (conservatively, less than 40 min at 70 ${^\circ}C$; [Table I](#t1){ref-type="table"}) and achievable by the majority of people with access to home ovens, rice cookers, or similar inexpensive heating devices. In summary, this work provides guidelines to medical professionals and the general public for the effective, safe thermal decontamination of PPE. In addition, the sensitivity of coronaviruses to environmental temperature variations, shown in [Table I](#t1){ref-type="table"} and [Fig. 4](#f4){ref-type="fig"}, indicates that the thermal inactivation of SARS-CoV-2 must be considered in epidemiological studies predicting its global spread and, potentially, seasonal recurrence; our model can be incorporated in these studies due to its ability to predict virus lifetime as a continuous function of environmental temperature. Finally, the modeling framework presented here offers a fundamental understanding of virus thermal inactivation that can help fight the COVID-19 pandemic, as well as future outbreaks of other coronaviruses. See the [supplementary material](https://doi.org/10.1063/5.0020782#suppl) for primary datasets for each virus studied in this work,[@c54] tables of activation energy and frequency factor calculated from the data, temperature data in the United States corresponding to winter and summer, and details on the statistical analysis and uncertainty in predictions.[@c57] AUTHORS\' CONTRIBUTIONS {#s2} ======================= T.F.Y. and D.J.P. compiled and analyzed the data and developed the analytical model. All authors contributed to the interpretation of results and writing and editing the manuscript. D.J.P. guided the work. Z.L. and R.A.S. contributed equally to this work. We gratefully acknowledge helpful discussions with Dr. Dimithree Kahanda. This work was supported by the National Science Foundation under Grant No. CBET-2030023. DATA AVAILABILITY ================= The data that support the findings of this study are available within the article and its [supplementary material](https://doi.org/10.1063/5.0020782#suppl).

Background ========== *Streptococcus mutans* is considered the primary causative agent of dental caries, and when transiently introduced into the bloodstream following daily dental hygienic practices such as toothbrushing and flossing, this bacterium can also cause potentially lethal infective endocarditis (IE) \[[@B1]-[@B4]\]. In both infectious scenarios, the virulence of *S. mutans* depends upon its ability to form biofilms and to withstand extreme changes in environmental conditions, including fluctuations in oxygenation, shear stress, as well as nutrient source and availability. For example, in the oral cavity, *S. mutans* must be able to rapidly alter its expression of transporters and metabolic enzymes to catabolize a variety of host-derived dietary carbohydrates. Internalized carbohydrates are metabolized through the glycolytic pathway, resulting in the accumulation of acidic end-products in the environment, which favors the growth of *S. mutans* and other acid-tolerant cariogenic species. Repeated cycles of acidification can lead to a net demineralization of tooth enamel and the development of caries. Sucrose, a common dietary sweetener, can also be utilized by *S. mutans* for the production of extracellular polysaccharides \[[@B5]-[@B8]\] that facilitate bacterial adhesion and biofilm formation. Aeration has also been found to have a profound effect on carbohydrate metabolism and biofilm formation by *S. mutans*\[[@B9]-[@B11]\]. It is therefore not surprising that there is overlap in the genetic regulatory circuits responsive to carbohydrate metabolism, aeration/oxidative stress resistance and control of biofilm formation in *S. mutans*, which include CcpA \[[@B12]-[@B14]\], Rex \[[@B15]\], and Frp \[[@B16]\]. More recently, an emerging trend in the study of bacterial biofilms has been a focus on the contribution of bacterial cell death and autolysis to biofilm adherence, maturation, and dispersal. It has been demonstrated in a wide variety of bacteria that death and lysis of a subpopulation of cells can facilitate biofilm formation due to the release of DNA into the extracellular environment (eDNA) \[[@B17]-[@B22]\]. Likewise, cell death and lysis have been implicated in dispersal of cells from a mature biofilm \[[@B23]-[@B25]\]. In *Staphylococcus aureus*, the Cid/Lrg system has been shown to be involved in the regulation of cell death, autolysis, and biofilm formation \[[@B17],[@B21],[@B26]-[@B28]\]. Characterization of *S. aureus cid* and *lrg* mutants has revealed that these operons have opposing effects on cell death and murein hydrolase activity \[[@B27],[@B29]\]. These observations, combined with the fact that LrgA and CidA share structural features with the bacteriophage lambda family of holin proteins \[[@B29]\], have led to the hypothesis that CidA and LrgA control cell death and lysis in a manner analogous to effector and inhibitor holins, respectively \[[@B26],[@B30]\]. Bacteriophage holins are small membrane proteins that oligomerize in the cell membrane, acting as "molecular clocks" that regulate the timing and lysis of the host cell during lytic infection \[[@B31]\]. For example, the lambda S holin regulates cell death and lysis by the formation of large lipid-excluding "rafts" that promote cytosolic leakage as well as access of the phage-encoded endolysin (murein hydrolase) to the cell wall \[[@B32]-[@B34]\]. *S. aureus* CidA and LrgA have recently been shown to oligomerize into high-molecular-mass complexes in a cysteine disulfide bond-dependent manner, a biochemical feature also shared with holin proteins \[[@B35]\]. Although the molecular details of how Cid and Lrg function to control cell death and lysis have not yet been completely elucidated, the fact that *cid* and *lrg* homologues have been identified in a wide variety of bacterial and archeal genomes supports a fundamental and conserved role for this system in cell physiology \[[@B30],[@B36]\]. In previous work it was determined that expression of potential *cidAB* and *lrgAB* homologues in *S. mutans* is highly responsive to carbohydrate availability \[[@B12],[@B37]\] and oxygenation \[[@B11]\]. Given the potential importance of these genes to biofilm development in *S. mutans*, we previously characterized a panel of *S. mutans cid* and *lrg* isogenic mutants and found that a subset of these genes did indeed influence biofilm formation, production of glucosyltransferases (enzymes that synthesize extracellular glucan polymers that contribute to biofilm adhesion), and oxidative stress tolerance \[[@B37]\]. In this study it was also found that, as demonstrated previously in *S. aureus*\[[@B38],[@B39]\], the *S. mutans* LytST two-component system was required for activation of *lrgAB* expression, but not *cidAB* expression \[[@B37]\]. Genes homologous to *lytST* appear to be present in most Gram-positive organisms that contain *lrgAB*\[[@B30]\] and these genes are often linked to one another, inferring an important role for this two-component system in fine-tuning *lrgAB* expression in response to external environmental signals. Therefore in this study, we sought to determine if LytST is involved in regulation of *lrgAB* expression in response to glucose and oxygenation in *S. mutans*, and to elaborate on the contribution of LytST to cellular homeostasis and global control of gene expression. Results ======= Effects of oxygenation and glucose metabolism on *S. mutans lrg* and *cid* expression ------------------------------------------------------------------------------------- The LytST two-component regulatory system has been shown to positively regulate *lrgAB* expression in a wide variety of bacteria, including various staphylococcal \[[@B38]-[@B40]\] and *Bacillus* species \[[@B41],[@B42]\], as well as in *S. mutans*\[[@B37]\]. The conserved nature of this regulation in Gram-positive bacteria, combined with the known effects of LytST and LrgAB on cell death/lysis \[[@B29],[@B38],[@B39],[@B43]\], biofilm development \[[@B21],[@B37],[@B38]\], and oxidative stress resistance \[[@B37]\], suggests that LytST and LrgAB are central regulators of physiologic homeostasis. However, little is known about the environmental and/or cellular cues to which LytS responds. In *S. aureus* and *B. anthracis*, it has been shown that *lrgAB* expression is responsive to disruption of cell membrane potential in a LytST-dependent manner \[[@B41],[@B44]\]. However, we were unable to determine whether this regulation also occurs in *S. mutans*, as treatment with membrane-potential disrupting agents (gramicidin, carbonyl cyanide m-chlorophenylhydrazone) did not have a measurable effect on membrane potential, as assessed by staining with DIOC~2~ (3) (data not shown). In previous studies, it was shown that oxygen and glucose metabolism have a pronounced effect on *lrg* and *cid* expression in *S. mutans*, but the specific role of LytS, if any, in this regulation was not addressed \[[@B11],[@B37]\]. Therefore, *S. mutans* UA159 and its isogenic *lytS* mutant were grown under aerobic and low-oxygen conditions to exponential (EP) and stationary (SP) growth phases in media containing 11 mM or 45 mM glucose. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed on RNA isolated from cultures at each time point to assess changes in *lrg* expression (Figure [1](#F1){ref-type="fig"}). In UA159, stationary phase *lrgAB* expression was upregulated 365-fold relative to exponential phase when grown under 11 mM glucose and low-oxygen conditions (Figure [1](#F1){ref-type="fig"}A)*.* Although mutation of *lytS* resulted in a severe loss of stationary phase *lrgAB* induction in cells grown in 11 mM glucose, *lrgAB* expression was not completely abolished. When grown under aerobic conditions and 11 mM glucose, stationary phase *lrgAB* expression was upregulated 2500-fold relative to exponential phase in the wild-type strain (Figure [1](#F1){ref-type="fig"}A), confirming previously-published observations that aerobic growth promotes *lrgAB* expression \[[@B11]\]. However, stationary-phase *lrgAB* expression was still induced 216-fold in the *lytS* mutant during aerobic growth, suggesting that (1) other as-yet-unknown regulators also contribute to the positive control of *lrgAB* expression during aerated growth, and (2) LytST is a predominant regulator of *lrgAB* expression during low oxygen growth, compared to aerobic growth. Under low-oxygen and aerated cultures, stationary phase induction of *lrgAB* expression was dramatically reduced when grown in 45 mM glucose, and similar levels of expression were observed in the wild-type and *lytS* mutant (Figure [1](#F1){ref-type="fig"}B), suggesting that growth in high levels of glucose abrogates oxygen-dependent regulation of *lrgAB* by LytST. Consistent with previously-published data \[[@B37]\], LytS did not appear to have a measurable effect on *cidAB* expression under any of the growth conditions tested here (data not shown). In summary, LytST-dependent regulation of *lrgAB* expression is much more pronounced during low-oxygen growth and at low glucose levels. ![**LytS-dependent expression of*lrgAB*in*S****.****mutans*.**Overnight cultures were diluted in THYE, containing either 11 mM (**A**) or 45 mM glucose (**B**) to an OD~600~ = 0.02 and grown at 37°C as static cultures at 5% CO~2~ ("low-O~2~") or as aerobic shaking cultures at 250 RPM ("aerobic"). RNA was harvested at exponential (EP) and stationary phase (SP). Reverse-transcription, real-time PCR reactions, and determination of copy number were performed as described previously using *lrgA* and 16S-specific primers \[[@B37],[@B77]\]. Fold-change expression of *lrgAB* and 16S under each growth condition was calculated by dividing the gene copy number of each test sample by the average gene copy number of UA159 EP. Data was then normalized by dividing each *lrgAB* fold-change value by its corresponding 16S fold-change expression value. Data represent the average of 3 biological replicates. Dark grey bars represent UA159 and light grey bars represent *lytS* mutant. Error Bars represent the standard error (SEM).](1471-2180-12-187-1){#F1} Microarray analysis of the LytS regulon --------------------------------------- Based on the transcriptional data presented above, the effects of LytST regulation on *lrgAB* expression are most evident while *S. mutans* is growing under conditions of low-oxygen (5% CO~2~) with a lower concentration of glucose. To begin to explore how LytST impacts critical phenotypes of *S. mutans*, RNA expression profiles in UA159 and the *lytS* mutant were compared using an RNA microarray approach. RNA was isolated from early exponential and late exponential growth phases from static planktonic cultures grown in BHI (containing 11 mM total glucose) at 37°C in a 5% CO~2~ atmosphere (Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). At early exponential growth phase, loss of LytS affected the expression of 40 genes (12 upregulated and 28 downregulated; *P \<* 0.005; Additional file [1](#S1){ref-type="supplementary-material"}: Table S1). Most of the upregulated genes in early exponential phase displayed only a modest increase in expression and included genes involved in DNA repair, purine/pyrimidine metabolism, competence, and a number of unassigned and hypothetical ORFs. RNA transcripts that were strongly down-regulated greater than 10-fold in cells lacking LytS during early exponential growth included those annotated as bacitracin/surfactin/gramicidin synthesis proteins, transport and binding proteins, and LrgAB. In contrast, loss of LytS affected the expression of a much larger number of genes in late exponential phase (136 genes total), with 79 upregulated transcripts and 57 downregulated transcripts (*P* \< 0.001; Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). Aside from dramatically decreased *lrgAB* expression, affected genes included those involved in amino acid and co-factor biosynthesis, carbohydrate and fatty acid metabolism, stress adaptation, toxin production, DNA repair/recombination, protein synthesis, transcriptional regulation, and competence, as well as multiple hypothetical and/or unassigned ORFs (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2 and Figure [2](#F2){ref-type="fig"}). A subset of genes was differentially expressed as a function of the loss of LytS in both early exponential and late exponential growth phases (Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). These included many genes encoded by the *S. mutans* genomic island TnSMu2 \[[@B45]\] (SMU.1335c, 1339-1342, 1344c-1346, 1354c, 1360c, 1363c, 1366c), *ssbA*, *comYB*, and *lrgAB*. Given that these genes were regulated by LytS in both growth phases examined, it is possible that they are under the direct control of LytST. To validate the microarray data, qRT-PCR was performed on late exponential phase wild-type and *lytS* mutant RNA to assess expression of 14 of the affected genes. As shown in Table [1](#T1){ref-type="table"}, the expression ratios (*lytS* mutant/wild-type) for each gene obtained by real-time PCR were similar to the microarray results. Interestingly, expression ratios of these genes were all close to 1.0 when comparing expression between the wild-type strain and a *lrgAB* mutant (Table [1](#T1){ref-type="table"}), indicating that the differential expression patterns observed in the *lytS* mutant were not a consequence of down-regulated *lrgAB* expression. {#F2} ###### Real-time PCR validation of RNA microarray results   **Microarray** **Real-time pcr** ------------ ----------------- ------------------- -------- -------- --- (SMU.1985) *comYA (comYB)* 22.9927 6.8449 0.8163   SMU.1967 *ssbA* 5.5803 4.1076 0.8791   (SMU.1515) *vicR (vicX)* 2.6764 1.7647 1.0267   SMU.924 *tpx* 2.4148 3.6168 1.058   SMU.1739 *fabF* 2.2443 2.0333 1.084   SMU.1666 *livG* 2.1183 3.4331 1.009   SMU.80 *hrcA* 0.4953 0.6107 1.0204   SMU.1424 *pdhD* 0.4769 0.4031 1.2004   SMU.580 *xseA* 0.29849 0.5409 1.1398   SMU.1600 *celB* 0.2186 0.2825 1.2979   SMU.113 *pfk* 0.1597 0.176 1.3578   SMU.82 *dnaK* 0.1523 0.2652 0.9907   SMU.1344 *fabD* 0.0223 0.012 1.0637   SMU.1341 *grs* 0.0008 0.0121 1.1027   Results are expressed in fold-change (mutant/wild-type). Investigation of the effect of LytST and LrgAB on competence ------------------------------------------------------------ In analyzing the microarray data in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2, it appeared that the gene most highly upregulated in response to loss of LytS in both phases of growth was *comYB* (SMU.1985), a homologue of the *B. subtilis comGB* gene that encodes part of an ABC transporter essential for DNA binding-uptake during competence in *S. mutans*\[[@B46]\]. Interestingly, a *comYB* mutant of *S. mutans* was shown to be unaffected in competence signaling, but showed reduced biofilm formation, which was thought to be a function of its inability to bind biofilm matrix eDNA \[[@B47]\]. Since the *lytS* mutant displayed an increase in *comYB* expression (Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2), we hypothesized that this strain may display alterations in its ability to form biofilm and/or its transformability during genetic competence. However, the *lytS* mutant did not display any appreciable difference in its ability to form static biofilm in the presence of glucose or sucrose (data not shown), and likewise, did not display a difference in its ability to uptake plasmid DNA in a quantitative competence assay, relative to the wild-type strain (Figure [3](#F3){ref-type="fig"}). Since *lrgAB* expression is so strongly regulated by LytST, the ability of isogenic *lrgA*, *lrgB*, and *lrgAB* mutants to uptake plasmid DNA via competence was also assessed (Figure [3](#F3){ref-type="fig"}). Of all the mutants tested, the *lrgA* mutant was the most severely impaired in its ability to uptake plasmid DNA relative to the parental strain, displaying a 26- and 24-fold decrease in transformation efficiency in the presence and absence of competence-stimulating peptide (CSP), respectively (Figure [3](#F3){ref-type="fig"}), suggesting that LrgA is somehow involved in genetic transformation in a CSP-independent manner. This finding has particular significance considering that LrgAB has been linked to regulation of cell death and lysis in *S. aureus*\[[@B21],[@B29]\] and *S. mutans*\[[@B37]\], and these physiological processes are also extremely important during natural competence. It is interesting to note that, similar to the competence results described here, the *lrgA* mutant was previously shown to display decreased glucose-dependent biofilm formation and decreased glucosyltransferase production, whereas the *lrgB* and *lrgAB* mutants behaved in a manner similar to the parental strain \[[@B37]\]. These phenotypic patterns suggest that the presence of LrgB alone, rather than the lack of LrgA, may be responsible for the biofilm and competence phenotypes observed in the *lrgA* mutant. ![**Transformation efficiencies of UA159 and isogenic*lytS*and*lrg*mutants.** To compare the ability of UA159 and its isogenic *lytS*, *lrgA*, *lrgB*, and *lrgAB* mutants to take up exogenously-added plasmid DNA, a quantitative competence assay was performed on n = 4-6 biological replicates of each strain as described in Methods \[[@B83]\]. Plasmid pAT28 \[encoding spectinomycin resistance; \[[@B84]\] was used to assess transformation efficiency in UA159, *lytS*, *lrgB*, and *lrgAB* mutants. Because the *lrgA* mutant was generated with a spectinomycin-resistance cassette \[[@B37]\], plasmid pORi23 \[encoding erythromycin resistance; \[[@B85]\]\] was used to assess transformation efficiency in UA159 and *lrgA* mutant. Transformation efficiencies (Y axis) in the presence (grey bars) and absence (white bars) of CSP are expressed as the percentage of transformants (CFU/ml on BHI + selective antibiotic) among total viable cells (CFU/ml on BHI). Error bars represent SEM. Brackets with *P* values denote statistically-significant differences between two samples (Mann--Whitney Rank Sum Test).](1471-2180-12-187-3){#F3} Effect of LytST on oxidative stress tolerance --------------------------------------------- Previously, our investigations disclosed a strong link between oxidative stress tolerance and the Cid/Lrg system \[[@B37]\], a role for these genes that had not been described in other organisms. Specifically, we found that *lrgAB*, *lrgB*, *cidAB*, and *cidB* mutants exhibited reduced growth in the presence of paraquat, and growth of *lrgAB*, *cidAB*, and *cidB* mutants on BHI agar plates in aerobic conditions was almost completely inhibited \[[@B37]\]. It is therefore interesting to note that in the *lytS* microarray results (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2), genes encoding antioxidant and DNA repair/recombination enzymes were significantly upregulated in the *lytS* mutant in late exponential phase. These included *yghU* and *tpx*, encoding the putative anti-oxidant enzymes glutathione S-transferase and thiol peroxidase, respectively, as well as *recJ*, which encodes a single-stranded DNA exonuclease protein that facilitates DNA repair in response to oxidative stress \[[@B48]-[@B51]\]. To further investigate the effect of *lytS* and *lrgAB* on oxidative stress tolerance, wild-type, *lytS*, and *lrgAB* mutants were grown as planktonic static BHI cultures in aerobic atmosphere and in the presence and absence of H~2~O~2~ (Figure [4](#F4){ref-type="fig"}). When challenged with H~2~O~2~, UA159 experienced an increased lag phase of growth, and the overall OD of the culture was 10-25% less than the untreated culture until 20 h growth. Under these assay conditions, the *lrgAB* mutant displayed a dramatic growth defect in both the presence and absence of H~2~O~2~. It is interesting to note that this aerobic growth defect was also previously observed when the *lrgAB* mutant was grown in aerobic atmosphere on BHI agar plates \[[@B37]\]. The *lytS* mutant displayed an increased lag in growth relative to UA159 when cultured in the presence of H~2~O~2~, but OD values were comparable to the wild-type strain by 16 h growth. These results suggest that the LytST regulon impacts the ability of cells to grow under conditions of oxidative stress. {#F4} The cell-permeable fluorescent dye CM-H~2~DCFDA (Invitrogen Molecular Probes) was also used to assess intracellular ROS in UA159 and the *lytS* mutant (Figure [5](#F5){ref-type="fig"}). This fluorescent compound is oxidized in the presence of H~2~O~2~ and other reactive oxygen species (ROS) and is considered a general indicator of intracellular oxidative stress \[[@B52],[@B53]\]. This analysis revealed that stationary-phase cultures of the wild-type and *lytS* mutant strains had similar "endogenous" intracellular levels of ROS (Figure [5](#F5){ref-type="fig"}, light grey bars). When stationary-phase cells from each strain were loaded with CM-H~2~DCFDA and then challenged with 5 mM H~2~O~2~ (Figure [5](#F5){ref-type="fig"}, dark grey bars), a greater increase in fluorescence was observed in the *lytS* mutant relative to UA159 (*P* = 0.009, Mann--Whitney Rank Sum Test), suggesting that loss of LytS has an impact on the ability of the cells to detoxify H~2~O~2~ and/or other intracellular ROS. {#F5} Discussion ========== The transcriptome analyses presented in this study have revealed that the LytST two-component system has a widespread effect on gene expression in *S. mutans*. A much higher number of transcripts were affected by the *lytS* mutation in late exponential phase and the magnitude of changes in expression was greater (n = 136 genes, Additional file [2](#S2){ref-type="supplementary-material"}: Table S2) relative to early-exponential phase (n = 40 genes, Additional file [1](#S1){ref-type="supplementary-material"}: Table S1), where most genes exhibited only a modest (1-2 fold) change in expression. These differences in gene expression patterns are unlikely to be an indirect function of altered *lrgAB* expression in the *lytS* mutant, as expression of *lytS*-regulated genes was unaltered in an *lrgAB* mutant relative to the wild-type strain (Table [1](#T1){ref-type="table"}). Taken together, these observations suggest that LytST exerts control over its transcriptome in a growth-phase dependent manner, and to our knowledge, this is the first study that has compared the scope of LytST regulation at different phases of growth. Interestingly, RNA microarray studies of *lyt* mutants have also been performed in *S. aureus*\[[@B38]\], *S. epidermidis*\[[@B40]\], and *B. subtilis*\[[@B42]\]. As we have observed here in *S. mutans*, a global effect of LytST on gene expression was also noted in *S. aureus* and *S. epidermidis*\[[@B38],[@B40]\]. In *S. aureus*, LytST appeared to exert primarily positive effects on gene expression in exponential phase when aerobic cultures were grown in media containing excess (35 mM) glucose, as only 7 genes were found to be upregulated in the *lytS* mutant \[[@B38]\]. In *S. epidermidis*, a large number of genes were up- or down-regulated as a function of the presence of LytST during exponential phase during aerobic growth in medium containing 12 mM glucose \[[@B40]\]. In contrast, mutation of *lytS* only appeared to affect the expression of *lytST* itself and genes encoding *lrgAB* and *cidAB* homologues in *B. subtilis*\[[@B42]\]. However, due to the differences in growth conditions used (glucose levels and/or culture aeration) and the differing metabolic pathways present in these organisms, it is difficult to establish direct correlations between these studies and the *S. mutans* microarray results presented here. As demonstrated previously \[[@B37]\], expression of *lrgAB* was also shown to be tightly controlled by the LytST two-component system in *S. mutans* in this study. Specifically, we have found that LytST-dependent expression of *lrgAB* is regulated in part by glucose metabolism and oxygen in *S. mutans* (Figure [1](#F1){ref-type="fig"}). Furthermore, control of *lrgAB* expression by LytST appears to be highly growth-phase dependent: *lrgAB* expression in the *lytS* mutant exhibited only a modest decrease in expression in early exponential phase (0.49 relative to UA159, Additional file [1](#S1){ref-type="supplementary-material"}: Table S1), whereas *lrgAB* expression was down-regulated some 200-fold in the *lytS* mutant at late exponential phase (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). Alternatively, it is possible that control of *lrgAB* expression by LytST is related to higher glucose availability during early exponential phase. Although detailed mechanistic studies have not yet been performed, there is mounting evidence that these proteins are critical for oxidative stress resistance in *S. mutans*: (1) *lrgAB* expression is highly regulated by oxygen (\[[@B11]\] and this study); (2) a *lrgAB* mutant was defective in aerobic growth on BHI agar plates \[[@B37]\]; (3) a *lrgAB* mutant displayed a decreased growth rate in the presence of paraquat (a superoxide-generating agent) relative to the wild-type strain \[[@B37]\]; and (4) a *lrgAB* mutant displayed a strong growth defect during static planktonic aerobic growth in BHI in the presence and absence of H~2~O~2~ challenge (this study). Interestingly, a link between LrgAB and oxidative stress was also demonstrated in *S. aureus*, where *lytSR* and *lrgAB* expression were upregulated 2-5 fold in response to azurophilic granule proteins, H~2~O~2~, and hypochlorite \[[@B54]\]. In agreement with a role for LrgAB in oxidative stress resistance, several LytST-regulated genes identified in this study have also been implicated in bacterial oxidative stress responses. Upregulated potential oxidative stress genes include *yghU*, a putative anti-oxidant enzyme \[[@B50]\], *tpx*, a predicted thiol peroxidase \[[@B55]\], and *recJ*, a single-stranded DNA exonuclease protein that facilitates DNA repair in response to oxidative stress \[[@B51]\]. Conversely, several genes belonging to the TnSMu2 gene cluster (SMU.1334c -- SMU.1359) were downregulated in the *lytS* mutant. These genes are annotated as encoding a series of gene products involved in bacitracin and gramicidin synthesis \[[@B56]\], but more recently have been shown to be responsible for nonribosomal peptide and polyketide (NRP/PK) biosynthesis of a pigment that enhances aerobic growth and tolerance to H~2~O~2~ challenge in *S. mutans* UA159 \[[@B45]\]. The altered expression of one or more of these genes may explain, in part, the increased ROS accumulation that was observed in the *lytS* mutant when challenged with H~2~O~2~ (Figure [5](#F5){ref-type="fig"}). Furthermore, it was previously found that a two-component system responsible for positive regulation of the NRP/PK genes was located on the TnSMu2 genomic island of UA140 but not in UA159 \[[@B45]\]. This observation, combined with the microarray results performed here (Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2) suggest that LytST may have taken over some of the regulatory functions of this non-core-genome two-component system that is missing in UA159. Interestingly, H~2~O~2~ has also been shown to be a potent stimulator of competence and eDNA release in *S. sanguinis*\[[@B57]\], *S. gordonii*\[[@B57],[@B58]\], and *S. pneumoniae*\[[@B59]\]. Although the effects of H~2~O~2~ on *S. mutans* competence, cell lysis, and eDNA release have not been directly measured, it has been shown that growth under aerobic conditions promotes competence in *S. mutans*\[[@B47]\], and that expression of competence-related genes is upregulated during aerobic growth \[[@B11]\]. The results presented here have demonstrated that expression of *comYB*, a gene encoding a component of the DNA-binding uptake system in *S. mutans*\[[@B47]\] was upregulated 2-fold in early exponential phase and 22-fold in late exponential phase in the *lytS* mutant (Additional file [1](#S1){ref-type="supplementary-material"}: Table S1 and Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The significance of high-level *comYB* expression in the *lytS* mutant at late exponential phase is unclear, given that maximal *S. mutans* competence develops in actively-growing populations \[[@B60],[@B61]\]. Accordingly, upregulation of *comYB* expression did not correlate with increased transformability of the *lytS* mutant under the conditions tested in this study (Figure [3](#F3){ref-type="fig"}). However, it was found that the *lrgA* mutant displayed a significant reduction in competence. It has been recently reported that only a subpopulation of *S. mutans* culture lyses in response to CSP, and this lysis event is controlled in part by the CipB bacteriocin and the CipI immunity protein \[[@B62]\]. Subsequent microarray analysis of a *cipI* (immunity protein) mutant showed that both *lytST* and *lrgAB* expression were highly upregulated in the *cipI* mutant \[[@B63]\]. These results, combined with the fact that LrgA/B has been shown to be involved in regulating cell lysis and eDNA release in *S. aureus*\[[@B21],[@B29]\], lends strong support to the idea that LrgA plays an important role during competence, possibly by altering membrane permeability or by modulating murein hydrolase activity. The *S. mutans comY* operon consists of nine co-transcribed genes, of which the first eight genes are either essential to or significantly affect competence \[[@B46]\]. The ninth gene of this operon is predicted to encode acetate kinase (AckA), an enzyme that catalyzes the inter-conversion of acetyl-phosphate and acetate \[[@B46],[@B64]\]. For micro-organisms with an inefficient or incomplete TCA cycle such as *S. mutans*, AckA-mediated conversion of acetyl-phosphate to acetate is thought to be a critical mechanism of generating ATP \[reviewed in \[[@B65]\]\]. Since *ackA* (*comYI*) was previously found to be upregulated in *S. mutans* during aerated growth \[[@B11]\], it is possible that LytST is involved in the regulation of energy generation through the phosphate acetyltransferase (Pta)-AckA pathway during aerobic growth and/or during oxidative stress. In this respect, it has recently been reported that an *S. mutans pta* mutant was more susceptible to both acid and oxidative stresses \[[@B66]\]. The ability of *S. mutans* to combat H~2~O~2~ stress is critical for its survival in the oral cavity, yet H~2~O~2~ detoxifying mechanisms and their regulation have not been extensively-characterized in this organism, limited primarily to the ScnRK and VicRK two-component systems \[[@B67],[@B68]\], *ropA*\[[@B69]\], *brpA*\[[@B70]\], *luxS*\[[@B71]\] and genomic island TnSMu2 \[[@B45]\]. H~2~O~2~ has been shown to have potent antibacterial effects on *S. mutans*\[[@B72]\], and it is thought that H~2~O~2~ produced by other oral streptococcal species serves as an antagonist against *S. mutans*. For example, *S. sanguinis* and S*. gordonii* have been shown to produce H~2~O~2~ via pyruvate oxidase under aerobic growth conditions, and this H~2~O~2~ production allows them to compete effectively against *S. mutans* when co-cultured under aerobic growth conditions \[[@B57]\]. It is therefore possible that the *S. mutans* LytST regulon mediates a pleiotropic protective response against these H~2~O~2~-producing niche competitors. On-going and future studies by our group will focus on experimental testing of this hypothesis. Conclusions =========== In summary, the LytST two-component system has been shown to have a pleiotropic effect on gene expression in *S. mutans*. This is congruent with microarray analyses of *lytS* mutants in *S. aureus*\[[@B38]\] and *S. epidermidis*\[[@B40]\]. However, unlike in other organisms, we have been able to identify a pattern of LytS-mediated gene expression that suggests a role for this regulon in responding to oxidative/H~2~O~2~ stress. Although we have not yet been able to identify the external signal to which LytS responds, it is likely linked to an oxidative stress-sensing mechanism, such as H~2~O~2~-mediated membrane damage (ie. lipid peroxidation) via its large number of transmembrane domains, or oxygen/ROS interactions with its predicted cytoplasmic GAF domain, a ubiquitous signaling domain that has been shown to detect changes in the redox state of bound iron or oxygen in *Mycobacterium tuberculosis*\[[@B73]-[@B75]\]. Establishing mechanistic links between the LytST regulon, H~2~O~2~ resistance, and competence regulation will provide valuable new insights into *S. mutans* survival and virulence in the oral cavity. Methods ======= Bacterial strains, media, and growth conditions ----------------------------------------------- For all experiments, frozen glycerol stocks of *S. mutans* UA159 and its isogenic *lytS* (SAB111; Δ*lytS*::NPKm^r^), *lrgA* (SAB113; Δ*lrgA*::NPSp^r^), *lrgB* (SAB119; Δ*lrgB*::NPEm^r^), and *lrgAB* (SAB115; Δlrg*AB*::ΩKm^r^) mutants \[created previously in \[[@B37]\] were freshly streaked for isolation on either Todd Hewitt Yeast Extract (THYE) or Brain Heart Infusion (BHI), containing selective antibiotic as appropriate: kanamycin (Km) -- 1000 μg/ml, erythromycin (Em) -- 10 μg/ml, spectinomycin (Sp) - 1000 μg/ml). With the exception of SAB115 (*lrgAB* mutant), all mutants were created using non-polar (NP) antibiotic-resistance markers \[[@B37]\]. Unless otherwise indicated, all *S. mutans* cultures were grown as static cultures in BHI or THYE broth at 37°C and 5% CO~2~. Analysis of *lrgAB* expression ------------------------------ To measure the effects of oxygen and glucose on *lrg* expression, overnight THYE cultures of UA159 and the *lytS* mutant (n = 3 biological replicates each, grown at 0 RPM, 37°C and 5% CO~2~) were each inoculated to an OD~600~ = 0.02 into THYE containing either 11 mM or 45 mM glucose. For "low O~2~" cultures, 2 L culture flasks each containing 400 ml media were grown at 0 RPM, 37°C, and 5% CO~2~. For aerobic cultures, 500 ml culture flasks each containing 100 ml media were grown at 37°C and 250 RPM. Total RNA was isolated from all cultures sampled at exponential (EP; OD~600~ = 0.2 -- 0.4) and stationary (SP; OD600 = 1.4 -- 1.7) growth phase, with an RNeasy Mini kit (Qiagen) and FASTPREP (MP Biomedicals) using previously-described methods \[[@B76]\]. Real-time reverse-transcriptase PCR and data analysis using *lrgA* and 16S primers was performed using previously described primers \[[@B37]\] and methods \[[@B77]\]. Fold-change expression of *lrgA* and 16S under each growth condition (11 mM low-O~2~, 11 mM aerobic, 45 mM low-O~2~, 45 mM aerobic) was calculated by dividing the gene copy number of each test sample by the average gene copy number of UA159 EP. Data was then normalized by dividing each *lrgA* fold-change expression value by its corresponding 16S fold-change expression value. RNA microarray analysis of UA159 and *lytS* mutant -------------------------------------------------- To assess the effect of LytS on global gene expression, overnight BHI cultures of UA159 and *lytS* mutant (n = 3 biological replicates per strain) were diluted to an OD~600~ = 0.02 in BHI, and grown as static cultures at 37°C and 5% CO~2~. Total RNA was isolated from each culture at early-exponential (OD~600~ = 0.15) and late exponential phase (OD~600~ = 0.9), using previously-published methods \[[@B77]\]. RNA microarray analysis was performed using *S. mutans* UA159 microarrays provided by The Institute for Genomic Research, and previously-described methods and data analysis \[[@B11],[@B70],[@B78]\]. In brief, 2 μg total bacterial RNA was used in each reverse-transcription and cDNA labeling reaction (performed as described in \[[@B70],[@B78]\]), and a single preparation from each culture was hybridized per microarray slide in a Maui hybridization chamber (BioMicro Systems, Salt Lake City, UT). The resulting microarray slides were scanned, analyzed, and normalized using TIGR Spotfinder software (<http://www.tigr.org/software/>), and in-slide replicate analysis was performed with the TIGR microarray data analysis system (MIDAS; <http://www.tigr.org/software/>). Statistical analysis was carried out with BRB array tools (<http://linus.nci.nih.gov/BRB-ArrayTools.html/>) with a cutoff *P* value \< 0.005 for the early exponential-phase data and *P* \< 0.001 for the late exponential phase data. To validate the microarray results, real-time quantitative RT-PCR was performed on a subset of the differentially-expressed genes, as described previously \[[@B77],[@B79]\]. All real-time PCR primers were designed with Beacon Designer 4.0 software (Premier Biosoft International, Palo Alto, CA), and standard curves for each gene were prepared as published elsewhere \[[@B80]\]. The microarray data obtained from these studies have been deposited to NCBI's gene expression omnibus (GEO) \[[@B81]\] (GEO Accession \#GSE39470) and comply with MIAME guidelines \[[@B82]\]. Quantitative competence assays ------------------------------ To compare the ability of UA159 and its isogenic *lytS*, *lrgA*, *lrgB*, and *lrgAB* mutants to take up exogenously-added plasmid DNA, a quantitative competence assay was performed on n = 4-6 biological replicates of each strain using a previously-published protocol \[[@B83]\] with the following modifications: Overnight cultures of each strain were diluted to an OD~600~ = 0.02 in BHI, and grown in a 96-well plate to an OD~600~ = 0.15 prior to addition of 500 ng plasmid DNA with and without 100 ng CSP. Plasmid pAT28 (encoding spectinomycin resistance; \[[@B84]\]) was used to assess transformation efficiency in UA159, *lytS*, *lrgB*, and *lrgAB* mutants. Because the *lrgA* mutant was generated with a spectinomycin-resistance cassette \[[@B37]\], plasmid pORi23 \[encoding erythromycin resistance; \[[@B85]\]\] was used to assess transformation efficiency in UA159 and *lrgA* mutant. After 2.5 h incubation in the presence of plasmid DNA +/- CSP, cultures were serially diluted and plated on BHI agar with and without selective antibiotic. CFU/ml of each culture were enumerated after 48 h growth at 37°C and 5% CO~2~, and transformation efficiencies were calculated as the percentage of transformants (CFU/ml on BHI + selective antibiotic) among total viable cells (CFU/ml on BHI). H~2~O~2~ assays --------------- To assess of the ability of UA159, *lytS,* and *lrgAB* mutants to grow in the presence of H~2~O~2~, overnight cultures of each strain (n = 6 biological replicates) were each diluted 40-fold into BHI. 1 ml aliquots of each diluted culture were either untreated or challenged with 1 mM H~2~O~2~. Aliquots of each (500 μl per well, 2 wells total) were then immediately transferred to an optically-clear 48-well tissue culture plate (Costar 3548), which was incubated for 20 h at 37°C (aerobic atmosphere) in a Biotek Synergy microplate reader. OD~600~ measurements of each well were recorded at 2 h intervals. Oxidative stress measurements ----------------------------- To assess intracellular oxidative stress in UA159 and *lytS* mutant, single isolated colonies of each strain (n = 3-6 biological replicates per strain) were inoculated into culture tubes containing 4 ml BHI, and grown in "low-O~2~" conditions (37°C, 0 RPM, 5% CO~2~). After 20 h growth, 2 × 1 ml aliquots of each culture were harvested by centrifugation in a microcentrifuge (3 min at 13,000 RPM). The culture supernatants were discarded, and cell pellets were each resuspended in 1 ml Hanks Buffer (HBSS) containing 5 μM chloromethyl 2′,7′-dichlorofluorescein diaceate (CM-H~2~DCFDA; Invitrogen Molecular Probes), a cell-permeable fluorescent compound that is oxidized in the presence of H~2~O~2~ and other reactive oxygen species (ROS) and is considered a general indicator of cellular oxidative stress \[[@B52],[@B53]\]. Cell suspensions were incubated at 37°C for 60 min to "load" the cells with CM-H~2~DCFDA, followed by centrifugation (3 min at 13,000 RPM). Supernatants were discarded, and cell pellets were washed once with HBSS prior to resuspension in 1 ml HBSS or in 1 ml HBSS containing 5 mM H~2~O~2~. Each cell suspension was transferred into triplicate wells (200 μl per well) of an optically-clear 96 well plate (Costar 3614), and the plate was transferred to a Biotek Synergy microplate reader. Fluorescence in relative fluorescence units (RFU; using 492-495 nm excitation and 517-527 nm emission) and OD~600~ readings of each well were recorded after 30 min incubation at 37°C. Statistical analysis -------------------- All statistical analyses, unless otherwise indicated, were performed using Sigmaplot for Windows 11.0 software (Build 11.0.0.75, Systat Software, Inc.). Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= SJA carried out the RNA microarray experiments and associated data analysis, performed all real-time PCR studies, participated in the conception and design of the study, and helped draft the manuscript. MDQ carried out all of the RNA isolations for comparing the effects of glucose and oxygenation on *lrgAB* expression. ER optimized and carried out all of the quantitative competence assays. RAB participated in the design and coordination of the study, and helped draft the manuscript. KCR participated in the conception and design of the study, performed the H~2~O~2~ assays, intracellular ROS measurements, and drafted the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ###### Additional file 1 **Table S1.**Genes differentially expressed by loss of LytS at early-exponential phase (P\< 0.005). ###### Click here for file ###### Additional file 2 **Table S2.**Genes differentially expressed by loss of LytS at late exponential phase (P\< 0.001). ###### Click here for file Acknowledgements ================ This work was supported by a University of Florida HHMI-Science for Life Undergraduate Research Award to M. D. Q., NIH-NIDCR grants R03 DE019179 (KCR) and R01 DE13239 (RAB). We thank Christopher Browngardt for technical assistance in editing microarray data.

{#sp1 .340} {#sp2 .341} {#sp3 .342} {#sp4 .343} {#sp5 .344} {#sp6 .345} {#sp7 .346} {#sp8 .347} {#sp9 .348} {#sp10 .349} {#sp11 .350} {#sp12 .351} {#sp13 .352} {#sp14 .353} {#sp15 .354} {#sp16 .355} {#sp17 .356} [^1]: Locke\'s Essay on the Human Understanding. Abridged by J. Murray, LL.D. Dublin, 1852.

Sequence data are available from GenBank, Fishbol, and BOLD as indicated in the article text. Sequences longer than 200 bp were uploaded in GenBank (accession numbers: Germany KJ531289 to KJ531379; France KJ535741 to KJ535783; Spain KJ623816 to KJ623830; Portugal MF067430 to MF067499) and BOLD database in the case of COI sequences (UK and ROI KJ510424 KJ531384 and KJ563141-KJ645864). Introduction {#sec001} ============ Seafood fraud is more common than most consumers think and many studies have highlighted the fact that species substitution is especially frequent in certain seafood products, such as those labelled as Atlantic Bluefin tuna, European hake or Atlantic cod \[[@pone.0196641.ref001]--[@pone.0196641.ref003]\]. The consequences of this malpractice not only involve the economic deception of consumers \[[@pone.0196641.ref004]\], but may also have a negative impact on the sustainability of marine resources \[[@pone.0196641.ref005]\]. However, the diversity and number of fish commonly traded globally as seafood is so vast, that much remains to be done to understand the true ecological costs of mislabelling \[[@pone.0196641.ref006]\]. One of the essential elements in the fight against seafood fraud is legislation. European Union (EU) labelling regulations are aimed at providing information to consumers such as commercial and scientific names, thus assuring their traceability and identification throughout the value chain (EU 1379/2013), however in this regulation the type of fishery or aquaculture product determine the mandatory information required in the labels and, therefore, may decrease the expected effects \[[@pone.0196641.ref007]\]. Recent studies suggest that seafood mislabelling has generally decreased in European countries due to the existence and enforcement of these labelling regulations and the use of appropriate species identification methodologies \[[@pone.0196641.ref001],[@pone.0196641.ref008]\]. This can also be linked to the EU involvement in funding projects dealing with this problem from the very beginning of the EU framework programme \[[@pone.0196641.ref009]\], putting Europe at the forefront of the authenticity tests development, especially regarding seafood \[[@pone.0196641.ref010]\]. Tunas are among the most desirable marine fish worldwide, with a global tuna and tuna-like species catch that peaked at 7.7 million tonnes in 2014 \[[@pone.0196641.ref011]\]. Skipjack (*Katsuwonus pelamis*) and Yellowfin (*Thunnus albacares*) were the tuna species most captured with about 3 and 1.5 million tonnes, respectively \[[@pone.0196641.ref010]\]. In contrast, the captures of Bluefin tuna (three species: *T*. *thynnus*, *T*. *orientalis* and *T*. *maccoyii*) during the same period did not exceed 40,000 tonnes. The conservation status of the different tuna species and stocks is also variable but worrying: several stocks are overfished (31%) and near to that threshold (17%), whereas 52% remain at a healthy level of abundance \[[@pone.0196641.ref012]\]. However, market demand has not decreased and the tuna fishing fleet maintains their capacity \[[@pone.0196641.ref010]\]. Atlantic Bluefin tuna (*T*. *thynnus*) deserves special attention since the strong market demand on this species during the last decades nearly provoked a collapse in the populations, such as East Atlantic and Mediterranean, which forced the reduction of the total allowable catch (TAC) for the Mediterranean fishery since 2007. This measure allowed the recovery of the stock \[[@pone.0196641.ref013]\]. Since the most desirable species are not always available for the market, strong economic forces may result in some degree of substitution, fraud or mislabelling \[[@pone.0196641.ref014]\]. It is not easy to find a global mislabelling rate for tuna; different studies have shown different levels. In general, factors such as country, type of retailer, sampling target or year may explain these differences. Pardo et al. \[[@pone.0196641.ref015]\] suggest an average 18% misdescription for tuna, Gordoa et al. \[[@pone.0196641.ref016]\] found 37% of fresh and frozen tuna in Spain at points of sale and 48% in restaurants. In some other cases, these values were extremely high such as 95% found by Oceana in Brussels restaurants for Bluefin tuna \[[@pone.0196641.ref017]\]; however, studies have typically varied in their sampling strategies, and therefore remain poorly comparable. Transnational evaluation of seafood fraud could reveal trends among countries or geographic areas, which ultimately could help to design coordinated measures to reduce the global incidence of mislabelling. However, transnational studies are scarce; some examples were reported in North America, with samples taken from USA and Canada \[[@pone.0196641.ref018]\]. As part of the Labelfish project, 1563 seafood samples of different categories and processing degrees were collected across 19 cities and six European countries, revealing an average mislabelling rate of 4.93% for the European retail sector \[[@pone.0196641.ref007]\]. Later, in 2015 the EU Commission organised a coordinated plan to analyse 3906 samples of fish, mostly white fish, in 27 Member States and 2 EFTA (European Free Trade Association) Member States. These samplings and analyses resulted in identifying an overall mislabelling of white fish in Europe of 6% \[[@pone.0196641.ref019]\]. This study benefits from the previous Mariani et al. \[[@pone.0196641.ref007]\] sampling, focusing only in the tuna products. The objective was to gain deeper understanding of the patterns and drivers of tuna mislabelling across Europe by examining the factors affecting mislabelling rates of these products in six European countries. In particular, the analysed factors included the influence of processing and species labelling in mislabelling rates, and the type of substitution which characterizes the fraud in tuna products. Materials and methods {#sec002} ===================== Sampling {#sec003} -------- Commercial samples of tuna products were purchased in markets of 18 different cities in Europe belonging to 6 countries (France, Germany, Ireland, Portugal, Spain, and United Kingdom) between 2013 and 2014. Locations were chosen in order to have a good coverage and geographical replication for each country ([S1 Table](#pone.0196641.s001){ref-type="supplementary-material"}). In each city, the sampling was aimed to cover a wide metropolitan area and a wide range of types of retailers, including supermarkets, traditional markets and specialized fishmongers. When several products were purchased in a single store these were chosen with different brands or types of processing. The most abundant types of tuna products (fresh, frozen and canned tuna) have been sampled in all countries, while in some Southern regions in Europe (in Spain, Portugal and France), other types of convenience food containing tuna were also sampled and analysed, such as salads and precooked products. 545 samples were successfully analysed: 225 were fresh and frozen (unprocessed), 268 canned (processed) and 52 miscellaneous (processed). The number of samples analysed per country were: 87 in Spain, 71 in Portugal, 93 in France, 53 in ROI, 154 in UK, and 87 in Germany ([S1 Table](#pone.0196641.s001){ref-type="supplementary-material"}). Samples were obtained in their original packaging and were transported to the laboratory on the day of purchase, where they were stored at -20°C, or a small piece of tissue was removed and preserved in absolute ethanol. Packaging was retained or photographed and all label information was registered. Assessment of compliance with European seafood labelling legislation {#sec004} -------------------------------------------------------------------- Determination of tuna commercial products mislabelling was carried out taking into consideration the adequate European regulation: - (EC) No 852/2004 where it is established the definition of processed and unprocessed food (i.e. fresh and frozen tuna fall into the category of unprocessed fishery and aquaculture products, while canned tuna into the processed ones). - EEC 1536/92 where it is stated that preserved tuna and bonito must be prepared exclusively from certain fish species (i.e. tuna cans should contain only any *Thunnus* species or *Katsuwonus pelamis*), mixing of species is not allowed in each tin (unless muscular structure has disappeared), only commercial names are required - EU1379/2013 indicating the required information to be presented to consumers, among others commercial and scientific names (the latter except for canned and other prepared products such as salads). - Also, all member states have translated these European regulations into national legislation, the main aspect to be considered is the specific denomination that each country establishes to designate different tuna products, including fresh, frozen and canned tuna. A summary of these denominations is presented in [S2 Table](#pone.0196641.s002){ref-type="supplementary-material"}. DNA extraction, amplification and analysis {#sec005} ------------------------------------------ Sample screening involved five European laboratories with extensive experience in seafood authenticity. Each lab also carried out blind-sample ring trials to ascertain consistency of the methods used to identify fish species (full details of these experiments are presented in [S1 File](#pone.0196641.s008){ref-type="supplementary-material"}). A summary of the methods employed in this study is presented in [S3 Table](#pone.0196641.s003){ref-type="supplementary-material"}, where the specific DNA extraction commercial kits, primers used, molecular regions and size of the amplicons, DNA sequencing procedure and sequence analysis are indicated. Different mitochondrial DNA target regions were used in the laboratories involved in the study: cytochrome b, cytochrome oxidase I and control region. Tuna species in fresh and frozen products were identified using larger fragments, 464 bp for cytochrome b marker, 650 bp for cytochrome oxidase I and 450 bp for the control region marker \[[@pone.0196641.ref020],[@pone.0196641.ref021]\]. In the case of canned tuna most laboratories used cytochrome b markers of shorter size, 187 and 176 bp \[[@pone.0196641.ref009],[@pone.0196641.ref022]\], except for France, which used a short control region fragment of 150 bp \[[@pone.0196641.ref008]\]. Primers and PCR protocols are specified in [S4 Table](#pone.0196641.s004){ref-type="supplementary-material"}. Once checked by agarose electrophoresis in 1--2% agarose gels, PCR products were sequenced. Subsequent DNA sequence analysis was performed as indicated in [S3 Table](#pone.0196641.s003){ref-type="supplementary-material"}. Briefly, sequences were edited using Chromas (Technelysium), Bioedit \[[@pone.0196641.ref023]\] and GeneDoc \[[@pone.0196641.ref024]\] and matched against NCBI database using the nucleotide BLAST (Basic Alignment Search Tool) and the BOLD (Barcode of Life) database for the COI sequences \[[@pone.0196641.ref025]\]. Species were identified using a 99% minimum match criterion, with the exception of *Thunnus albacares* and *T*. *obesus*, where the threshold was 100% \[[@pone.0196641.ref026]\]. For an unequivocal identification, laboratories from Spain, France and Germany also constructed Neighbor-Joining trees using MEGA software \[[@pone.0196641.ref027]\] with their own reference sequences using Tamura-Nei distances. Sequences longer than 200 bp were uploaded in GenBank (accession numbers: Germany KJ531289 to KJ531379; France KJ535741 to KJ535783; Spain KJ623816 to KJ623830; Portugal MF067430 to MF067499) and BOLD database in the case of COI sequences (UK and ROI KJ510424 KJ531384 and KJ563141-KJ645864) Samples containing a different species than the one declared in the label were considered mislabelled. When only the commercial name was present, mislabelling records were obtained by following each country's list of approved commercial designation for tunas ([S2 Table](#pone.0196641.s002){ref-type="supplementary-material"}). When the scientific name was present, it was the one taken as a reference to compare with the analytical result. Statistical analysis {#sec006} -------------------- Software GraphPad Prism was used to perform pairwise comparisons between data sets using Chi-square with Yate's correction and correlation analysis for the dependence between number of samples and mislabelling rate. Results and discussion {#sec007} ---------------------- 545 tuna samples--of which 225 fresh & frozen, 268 canned and 52 miscellaneous (e.g. dried, roes, salads etc.)--were successfully sequenced and identified (for more detailed information about the results of previous tuna species identification ring trials see [S1 File](#pone.0196641.s008){ref-type="supplementary-material"}); 37 of them were mislabelled, making an overall mislabelling rate of 6.79% ([Fig 1](#pone.0196641.g001){ref-type="fig"}). This study is not only the largest sampling effort reported for tuna products but also one that involves six different European countries. Previously reported mislabelling rates for tuna were very variable, on average 18% \[[@pone.0196641.ref015]\], mainly because these rates may change over time and also because the type of retailer or provenance of samples influence dramatically the results obtained. The much lower value found here may be an indication of the impact of labelling legislation and control, the mass media coverage of food fraud with the consequence of consumer being more aware of food labels \[[@pone.0196641.ref008]\]. {#pone.0196641.g001} The mislabelling rate was not significantly different (Chi-square = 0.1045; df = 1; P = 0.3733) between fresh & frozen and canned tuna, 6.70% and 7.84%, respectively, while the miscellaneous products showed 1.92% mislabelling (not significative differences among them). Previous works have reported higher mislabelling rates, recently Gordoa et al. \[[@pone.0196641.ref016]\] have shown that fresh and frozen tuna in Spain was mislabelled up to 37% at points of sale, while in restaurants the mislabelling rate was even higher, 48%. In a previous report from Oceana \[[@pone.0196641.ref028]\], tuna samples obtained at different points of the value chain in different states of the USA showed that 58% of tuna products were mislabelled. However, this overall data may not be comparable directly, because the specific type of tuna products (i.e. species) have a relevant influence on the final mislabelling rate. [Fig 1A](#pone.0196641.g001){ref-type="fig"} shows that small differences between mislabelling rates were observed among Portugal, France, Ireland, UK and Germany (not significant), with France showing the lowest mislabelling rate of the study. On the other hand, Spain showed the highest mislabelling rates, with significant differences with those of France and UK (Chi-square = 3.0760; df = 1; P = 0.0397 and Chi-square = 3.2840; df = 1; P = 0.0350, respectively). However, these overall mislabelling rates did not reflect the mislabelling situation of different tuna products: i.e. while some products exhibited 1.92% mislabelling rate (for example the category "miscellaneous"), fresh and frozen tuna products were mislabelled at 6.70% ([Fig 1B](#pone.0196641.g001){ref-type="fig"}). In fact, we have observed significant differences for fresh and frozen tuna for Spain (25%) compared with Ireland (0%, Chi-square = 3.2250; df = 1; P = 0.0363), UK (3.7%, Chi-square = 7.3940; df = 1; P = 0.0033) and Germany (4.7%, Chi-square = 5.1300; df = 1; P = 0.0118). Canned samples presented an overall mislabelling rate of 7.84%, in this case differences in canned tuna among countries, ranging from 3.45% (France) to 13.04% (Germany) were not significant ([Fig 1C](#pone.0196641.g001){ref-type="fig"}). Additionally, no significant correlation was found between the number of samples taken in each country for each category or the seafood consumption ratio per habitant and the mislabelling rate (data not shown), therefore other factors should be considered when interpreting the results obtained. The analysis of the influence of the type of labelling in the final mislabelling rate can be observed in [Fig 2](#pone.0196641.g002){ref-type="fig"} and [S5 Table](#pone.0196641.s005){ref-type="supplementary-material"}. Although a significant correlation couldn't be established among the number of samples labelled as tuna and the mislabelling rate in a particular country (data not shown), the influence of sampling tuna product with Atlantic Bluefin tuna label can be clearly seen in the changes in mislabelling rates: when these products are excluded in the mislabelling rate calculation, the differences among countries were not significant. In fact, while overall mislabelling rate drops down to 1% in the case of labelling just tuna, it goes up (88%) in the case of Atlantic Bluefin tuna labelled products. These results agree with the results found by Vandamme et al. \[[@pone.0196641.ref029]\] that reported a low mislabelling rate for tuna in sushi of about 10%, but which rises up to 18% when tuna species is considered in the labelling. Likewise, Gordoa et al. \[[@pone.0196641.ref016]\] reported a 73% of mislabelling for Atlantic Bluefin tuna and points towards economic gain as the main reason for mislabelling tuna. Atlantic Bluefin mislabelling has been highlighted as an example of inverse relationship between low volume catches and high proportion of substitution; offer and demand do not match and the result is a very high mislabelling rate \[[@pone.0196641.ref030]\]. {#pone.0196641.g002} In the case of canned tuna, including species in the label provoked an increase in the mislabelling ([Fig 3](#pone.0196641.g003){ref-type="fig"} and [S6 Table](#pone.0196641.s006){ref-type="supplementary-material"}), and significant differences could be observed in overall values, from 1% mislabelling rate for Tuna labelling up to 10% mislabelling rates when species are indicated in the label (Chi-square = 4.381, p = 0.0182). General names such as tuna, which include any species of the genus *Thunnus* and *K*. *pelamis*, were associated with very low mislabelling rates (0%), while indicating the species in the can resulted in a higher mislabelling rate (17--60%) ([S6 Table](#pone.0196641.s006){ref-type="supplementary-material"}). In general, these mislabelling rates were lower than those found for fresh or frozen tuna products. {#pone.0196641.g003} Current labelling legislation in EU establishes the obligation to indicate commercial name and species in the case of fresh, frozen, smoked and dried seafood products (EU1379/2013). However, we have found many fresh and frozen tuna products, from 29% in Portugal up to 100% in Ireland, which were still labelled with the generic name tuna ([S5 Table](#pone.0196641.s005){ref-type="supplementary-material"}). Although we did not consider them mislabelled in this study, these results indicate a poor implementation of labelling rules across Europe. In the case of canned products, legislation allows the use of generic names, such as tuna, and as it can be seen here, samples with the generic name tuna have shown very low mislabelling rates. These differences in labelling requirements between fresh/frozen and canned tuna are difficult to understand from a consumer point of view as the same need for information should be required. In fact, the campaign "one fish, one name" advocates for the establishment of more specific names for every type of seafood product \[[@pone.0196641.ref031]\] and the main objective is to empower consumers to make more accurate purchasing decisions. The implementation of this approach will help to better protect resources and to fight illegal fishing practices. [Fig 4](#pone.0196641.g004){ref-type="fig"} and [S7 Table](#pone.0196641.s007){ref-type="supplementary-material"} show the type of substitution observed in the analysed samples. Frozen and fresh samples exhibited two main types of substitutions, affecting the products labelled as *T*. *albacares* and *T*. *thynnus*. In the case of *T*. *thynnus* (7 samples mislabelled), commercially labelled as Atlantic Bluefin tuna, thon Rouge or atún Rojo, the levels of substitution by *T*. *albacares* or *T*. *obesus* were similar (43%). More mislabelled samples were found for *T*. *albacares* (Yellowfin, thon Albacore, atun de aleta amarilla), in this case only one species, *T*. *obesus*, was found to substitute this species. In this latter case, unintentional substitution may take place due to occurrence of mixed schools of both species, which are very similar as juveniles \[[@pone.0196641.ref032]\], and can be captured at the same time. {#pone.0196641.g004} In terms of species, substitution results in canned tuna were different from those of fresh and frozen tuna. Canned tuna labelled as Yellowfin, Light tuna, thon albacore and atún Claro were replaced by *T*. *alalunga* (50% of samples, all of them from Spain), *T*. *obesus* (20%, none of them were samples from Spain, national legislation allows in this country to label *T*. *obesus* as atún Claro) and *K*. *pelamis* (20% of the samples) and *E*. *alleteratus* (10%). This is an example of lack of coherence in the European legislation: in one country, it is permitted to label *T*. *albacares* or *T*. *obesus* as Light tuna (Spain), while in the other European countries is not allowed. One might expect that this may have an impact on the mislabelling rates found for Light tuna in Spain, however, mislabelling rates for this product were similar to other countries where only *T*. *albacares* can be used in Light tuna can production. The results shown in [S6 Table](#pone.0196641.s006){ref-type="supplementary-material"} indicate that percent of samples labelled generically as tuna has an impact in the mislabelling rate of the country for canned tuna, since this type of products present mislabelling rates close to 0%. Samples of *K*. *pelamis* were only relevant in UK, Germany, France and Ireland, while in countries like Spain and Portugal is very difficult to find canned tuna labelled as *K*. *pelamis*. In the case of *K*. *pelamis* labelled cans bought in the UK, all mislabelled samples were substituted by *T*. *albacares/T*. *obesus*. This type of substitution has been referred by some authors as reverse substitution, a cheaper species is substituted by an expensive one, and that may be an indication of hiding a product from a IUU fishery practice \[[@pone.0196641.ref016]\]. Another substitution was found in Germany where *T*. *alalunga* was substituted by *K*. *pelamis*. These results may indicate that different value chains, fresh/frozen tuna and canned tuna rely in different providers of raw material, besides the cultural aspects of each particular market (i.e. Spanish consumers value light appearance meat in canned tuna while this feature may be not as relevant in other European markets). It has been reported that identification of the *Thunnus* species with mtDNA markers may present some problems due to low interspecific variability and introgression issues. One of the problems is the low genetic distance found between *T*. *alalunga* and *T*. *orientalis*. There is also a low percentage of specimens of Atlantic Bluefin Tuna (ABFT) which exhibit almost the same sequence as Pacific Bluefin Tuna (PBFT) using some mtDNA markers and vice versa \[[@pone.0196641.ref026]\]. However, in our samples there were not any of these cases (i.e. a sample labelled as ABFT but being identified as PBFT) and although the current legislation would consider this example mislabelled (fresh and frozen tuna) this problem does not affect our mislabelling results. There is also introgression between ABFT and *T*. *alalunga*, 2--3% of ABFT specimens showing mitochondrial sequences of *T*. *alalunga*, but again we did not find any samples with this situation. Therefore, we conclude that in the present study these issues are not affecting our mislabelling results. After looking into tuna product labelling across a significant portion of the largest global player in seafood trade (i.e. the European Union), it is apparent that this high-demand and widely marketed food category epitomises all the key challenges of global seafood sustainability and traceability. First, most of the products rely on catches from distant and/or tropical waters and a range of processing avenues, all of which poses the logistic challenges of a long and complex supply chain. Secondly, "tuna" is one of the general "umbrella" terms under which many species with diverse biological traits continue to be traded, especially in geographic areas exporting high volumes of seafood to Europe \[[@pone.0196641.ref033]\]. Collectively, the opportunities for species substitution, be it for deliberately maximising financial gain or through sheer logistic errors/mismanagement, remain high; so does the exposure of consumers to a lack of transparency that prevents environmentally conscious purchasing decision and/or may result in inadvertent consumption of unhealthy products (e.g. high heavy metal content, \[[@pone.0196641.ref034]\]). Progress in this area can be achieved through a robust international policy of accurate species-level labelling and coordinated governance efforts. Supporting information {#sec008} ====================== ###### Tuna samples collected in six European countries between 2012 and 2014. (DOCX) ###### Click here for additional data file. ###### Commercial denominations of tuna in EU and six member states (ES, PT, UK, ROI, FR, GER). (DOCX) ###### Click here for additional data file. ###### DNA analysis methods used in this study by the participant laboratories. (DOCX) ###### Click here for additional data file. ###### Primers sequences and PCR protocols used in the study. (DOCX) ###### Click here for additional data file. ###### Number of samples of fresh and frozen tuna analysed and mislabelling results split by type of label. (DOCX) ###### Click here for additional data file. ###### Number of samples of canned tuna analysed and mislabelling results split by type of label. (DOCX) ###### Click here for additional data file. ###### Species which appear as substitute in the mislabelled samples. (DOCX) ###### Click here for additional data file. ###### Tuna species identification ring trials. (DOCX) ###### Click here for additional data file. We acknowledge Anxela Aldrey, Roswitha Koch and Rainer Kündiger for their technical assistance. The work was funded by LABELFISH project, supported by the EU Atlantic Area Programme Project Number 2011-1/163 and the UK Department for Environment, Food & Rural Affairs grant FA0116. [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

All relevant data are within the manuscript and its Supporting Information files. Introduction {#sec001} ============ The faithful transmission of genetic material during each cell division is vital for the survival of all living organisms. The kinetochore, a multi-subunit protein complex that assembles onto a specialized region of the chromatin called the centromere, is essential for proper chromosome segregation \[[@pgen.1008477.ref001]\]. In *Saccharomyces cerevisiae*, kinetochores are assembled around a central histone H3 variant known as Cse4^CENP-A^, which marks the centromere. Kinetochore assembly is initiated by Mif2^CENP-C^, an evolutionarily conserved protein, which directs a hierarchical assembly of proteins to form the Constitutive Centromere-Associated Network (CCAN), whose structure has recently been determined \[[@pgen.1008477.ref002]--[@pgen.1008477.ref004]\]. Like most other kinetochore subunits, CCAN associates with the centromere throughout the cell cycle, and contains: Mif2, the Ctf19-Okp1-Mcm21-Ame1 (COMA) complex \[[@pgen.1008477.ref005]\], Chl4-Iml3 \[[@pgen.1008477.ref006]\], Ctf3-Mcm16-Mcm22 (CMM) \[[@pgen.1008477.ref007]\] and Cnn1-Wip1-Mhf1/2 \[[@pgen.1008477.ref008]\]; of these, Mif2 and Ame1-Okp1 play an early and essential role \[[@pgen.1008477.ref009], [@pgen.1008477.ref010]\]. Ctf19-Mcm21 directly binds to Ame1-Okp1 to form the COMA complex, followed by the sequential assembly of Chl4-Iml3, Ctf3-Mcm16-Mcm22 and Cnn1-Wip1. Moreover, mutants of the non-essential subunits of CCAN in *Saccharomyces cerevisiae* have been shown to impair faithful chromosome segregation \[[@pgen.1008477.ref011]--[@pgen.1008477.ref014]\]. Once fully assembled, CCAN provides a platform that recruits the outer kinetochore proteins, such as the KMN network (Knl1/Mis12 complex/Ndc80 complex), which connects the microtubules to facilitate chromosome movement \[[@pgen.1008477.ref015], [@pgen.1008477.ref016]\]. While much has been learned about the composition and assembly of the kinetochore, much less is known about how various post-translational modification pathways may regulate its activity \[[@pgen.1008477.ref017]\]. An earlier genetic screen in the yeast *Saccharomyces cerevisiae* identified *SMT3* as a high-copy suppressor of a temperature-sensitive mutant of *mif2-3*, an essential protein required for proper chromosome segregation \[[@pgen.1008477.ref018]\]. *SMT3* encodes the yeast **S**mall **U**biquitin-like **MO**difier (**SUMO**), which similarly suppresses the temperature-sensitivity of the CENP-C^Mif2^ mutant in chicken cells \[[@pgen.1008477.ref019]\]. SUMO is a member of the ubiquitin-like protein family, and is highly conserved throughout all eukaryotes \[[@pgen.1008477.ref020]\]. Sumoylation, like ubiquitination, utilizes a three-step enzymatic cascade to modify target proteins, which begins with an E1 activating enzyme (Aos1-Uba2 in *S*. *cerevisiae*), followed by an E2 conjugating enzyme (Ubc9) and ends with several E3 ligases (Siz1, Siz2 and Mms21 in *S*. *cerevisiae*), which catalyze the covalent attachment of SUMO to its target proteins \[[@pgen.1008477.ref021]\]. Sumoylation, being a highly dynamic and reversible post-translational modification, is removed from its target proteins by a group of enzymes known as the Ulp/SENP (ubiquitin-like protease/sentrin-specific protease) family of proteases, which cleave the isopeptide bond between SUMO and its substrate. The founding members of the Ulp/SENP family of enzymes are *S*. *cerevisiae* Ulp1 and Ulp2 \[[@pgen.1008477.ref022], [@pgen.1008477.ref023]\], whereas six SENPs, SENP1--3 and SENP5--7, have been found in humans \[[@pgen.1008477.ref024]\]. Interestingly, *SMT4*, another suppressor of *mif2-3* \[[@pgen.1008477.ref018]\], was found to encode the Ulp2 protease \[[@pgen.1008477.ref023], [@pgen.1008477.ref025]\]. This suggests that homeostasis of intracellular sumoylation, possibly at the kinetochore, may be needed to suppress the conditional lethality of the *mif2-3* mutant. Moreover, the *ulp2Δ* mutant exhibits defects in mitotic progression and chromosome segregation \[[@pgen.1008477.ref023], [@pgen.1008477.ref025], [@pgen.1008477.ref026]\], and was recently reported to specifically accumulate aneuploidy of chromosome I \[[@pgen.1008477.ref027]\]; this further suggests an important role for Ulp2 in maintaining proper chromosome segregation. The mechanism by which Ulp2 promotes proper chromosome segregation appears to be conserved; mutation of Ulp-4, an ortholog of Ulp2 in *C*. *elegans*, results in a chromosome segregation defect \[[@pgen.1008477.ref028]\], while the knockdown of SENP6, the human ortholog of Ulp2, also affects chromosome segregation and causes mis-localization of the inner kinetochore complex CENP-H/I/K \[[@pgen.1008477.ref029]\]. Altogether, these findings suggest an important role for Ulp2^SENP6^, and ultimately a role for SUMO homeostasis, in maintaining proper chromosome segregation, possibly via Mif2^CENP-C^ mediated kinetochore assembly. We previously identified the substrates of Ulp2 via a proteome-wide approach, showing that Ulp2 specifically targets protein complexes at three distinct chromosomal regions, which include the nucleolar RENT complex, the MCM complex and the inner kinetochore CCAN complex \[[@pgen.1008477.ref030]\]. Remarkably, the loss of Ulp2 was found to increase CCAN sumoylation by nearly 20-fold, including Mcm21, Mcm16, Mcm22, Ame1 and Okp1, indicating that Ulp2 specifically targets CCAN \[[@pgen.1008477.ref030]\]. This was followed by two studies, which together demonstrated that Ulp2 targets its nucleolar substrates via a dual-substrate recognition mechanism \[[@pgen.1008477.ref031], [@pgen.1008477.ref032]\]; Ulp2 localizes to the nucleolus through its binding to the Csm1 nucleolar protein, where it then specifically targets poly-sumoylated substrates through its C-terminal SUMO-interacting motif (SIM). Interestingly, a mutation in Ulp2's SIM resulted in a synergistic growth defect when it was combined with a mutation in a C-terminal Conserved Region (CCR) of Ulp2 (a.a. 931--934), whose function had not been defined \[[@pgen.1008477.ref031]\]. Here we characterize this CCR of Ulp2 as a kinetochore-targeting motif that recruits Ulp2 to the kinetochore by directly binding to the Ctf3^CENP-I^-Mcm16^CENP-H^-Mcm22^CENP-K^ (CMM) complex. Ulp2 then utilizes its SIM to target hyper-sumoylated CCAN subunits, preserving the SUMO homeostasis of the kinetochore machinery. Thus, Ulp2 promotes accurate chromosome segregation through the tight regulation of CCAN sumoylation to maintain SUMO homeostasis of the kinetochore. Results {#sec002} ======= Ulp2's CCR and SIM are both required for maintaining CCAN sumoylation in *HF-SMT3* cells {#sec003} ---------------------------------------------------------------------------------------- In our prior study, we showed that the *ulp2-781Δ* mutant, but not the *csm1Δ* mutant, elevates the sumoylation of both the inner kinetochore and nucleolar proteins \[[@pgen.1008477.ref032]\]. This suggests that in addition to Csm1-binding, Ulp2^781-1034^ contains additional sequence elements that direct Ulp2 to desumoylate CCAN. Sequence alignments of Ulp2's fungal orthologs revealed three conserved hydrophobic residues in the CCR of Ulp2 (a.a. 931--934) ([Fig 1A](#pgen.1008477.g001){ref-type="fig"}, upper panel), which, when mutated to alanine and combined with the *ulp2-SIM*^*3A*^ mutation, resulted in a synergistic growth defect \[[@pgen.1008477.ref031]\]. To determine the effect of these *ulp2* mutations on kinetochore sumoylation, we used the *HF-SMT3* (*6×His-3×Flag-SMT3*) strain and the quantitative SUMO proteomic method developed in our previous studies \[[@pgen.1008477.ref030], [@pgen.1008477.ref033]\] ([Fig 1A](#pgen.1008477.g001){ref-type="fig"}, lower panel). We found that the *ulp2-CCR*^*3A*^ mutation results in an almost five-fold increase in the amount of sumoylated Mcm21 and Okp1 ([Fig 1B](#pgen.1008477.g001){ref-type="fig"}), while the sumoylation of Ulp2's substrates in the nucleolus and at origins of DNA replication are not appreciably affected ([S1 Table](#pgen.1008477.s003){ref-type="supplementary-material"}). Because sumoylated CCAN exists at a low level in wild-type cells that is often below the MS detection limit, we next compared CCAN sumoylation levels in the *ulp2- SIM*^*3A*^*CCR*^*3A*^ double mutant to its levels in the *ulp2-SIM*^*3A*^ mutant, where sumoylated Ulp2 substrates accumulate to higher levels \[[@pgen.1008477.ref031]\]. We found that the *ulp2-CCR*^*3A*^ mutant causes CCAN sumoylation to increase by over four-fold in the *ulp2-SIM*^*3A*^ strain background ([Fig 1B](#pgen.1008477.g001){ref-type="fig"} and [S2 Table](#pgen.1008477.s004){ref-type="supplementary-material"}), similar to its effect in the wild-type background. {ref-type="supplementary-material"}--[S6](#pgen.1008477.s008){ref-type="supplementary-material"} Tables. In each case, the fold-changes in the sumoylated CCAN subunit are shown for each of the indicated strains. CCAN subunits not identified in the MS experiments are indicated by \#. Asterisks (\*) indicate where an insufficient number of peptides are available for statistical analysis.](pgen.1008477.g001){#pgen.1008477.g001} We next examined the effect of the *ulp2-SIM*^*3A*^ mutation and found that it caused a moderate increase in the amount of sumoylated Ame1, Mcm21, Okp1 and Mcm16, indicating a role for Ulp2's SIM in facilitating CCAN desumoylation by Ulp2 ([Fig 1C](#pgen.1008477.g001){ref-type="fig"}, [S3 Table](#pgen.1008477.s005){ref-type="supplementary-material"}). Similarly, this effect is also observed in the *ulp2-CCR*^*3A*^ background, as sumoylated CCAN subunits, including Ame1, Mcm21, Okp1, Mcm16 and Mcm22, accumulated to higher levels in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant compared to the *ulp2-CCR*^*3A*^ single mutant ([Fig 1C](#pgen.1008477.g001){ref-type="fig"}, [S4 Table](#pgen.1008477.s006){ref-type="supplementary-material"}). In fact, the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant drastically increases the sumoylation of multiple CCAN subunits compared to the wild-type strain ([Fig 1D](#pgen.1008477.g001){ref-type="fig"}, [S5 Table](#pgen.1008477.s007){ref-type="supplementary-material"}). Moreover, sumoylation of CCAN reaches a level that phenocopies that of the *ulp2Δ* mutant, indicating that Ulp2's CCR and SIM play a partially redundant role in directing Ulp2 to desumoylate CCAN ([Fig 1D](#pgen.1008477.g001){ref-type="fig"}, [S6 Table](#pgen.1008477.s008){ref-type="supplementary-material"}). CCAN abundance is not appreciably affected in the *ulp2-CCR*^*3A*^ and *ulp2-SIM*^*3A*^ mutants {#sec004} ----------------------------------------------------------------------------------------------- The strains used in the above proteomic approach made use of HF-Smt3, which allows for the enrichment of total sumoylated proteins, but the HF tag on Smt3 compromises poly-sumoylation to an unknown extent \[[@pgen.1008477.ref030], [@pgen.1008477.ref033]\]. A previous study showed that poly-sumoylated protein could be targeted for degradation, which was perturbed by the HF tag on Smt3 \[[@pgen.1008477.ref032]\]. Additionally, our proteomic approach specifically measures the relative change in the amount of proteins that are conjugated to SUMO; thus the poly-sumoylation status or the protein levels of CCAN subunits could not be determined. To address these concerns, we took advantage of Ulp1-C580S, a catalytically inactive form of Ulp1, which exhibits a strong affinity to SUMO itself \[[@pgen.1008477.ref034], [@pgen.1008477.ref035]\]. We first immobilized recombinant Ulp1^403-621^-C580S onto CNBr-activated resin, and then tested its ability to purify endogenous sumoylated proteins from yeast cell extracts, which resulted in the recovery of approximately 50% of SUMO conjugates ([Fig 2A](#pgen.1008477.g002){ref-type="fig"}). To facilitate the detection of CCAN in the *ulp2-CCR*^*3A*^, *ulp2-SIM*^*3A*^ and *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutants, an endogenous Protein A tag was fused to the C-terminus of each CCAN subunit (Ame1, Mcm21, Okp1, Mcm16 and Mcm22). As shown in [Fig 2B](#pgen.1008477.g002){ref-type="fig"}, the expression level of Mcm21 was unaffected by any of the *ulp2* mutations. Furthermore, a higher molecular weight species of Mcm21 is enriched in the elution, and is referred to as sumoylated Mcm21. Both the *ulp2-SIM*^*3A*^ and *ulp2-CCR*^*3A*^ single mutants caused a modest but appreciable increase in the abundance of sumoylated Mcm21, while the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant caused a drastic accumulation in the levels and species of sumoylated Mcm21 ([Fig 2B](#pgen.1008477.g002){ref-type="fig"}), demonstrating partially redundant roles for Ulp2's SIM and CCR in suppressing excessive Mcm21 sumoylation. {#pgen.1008477.g002} Similar to Mcm21, several species of sumoylated Ame1 and Okp1 were detected in wild type cells, which are modestly elevated in the *ulp2-SIM*^*3A*^ and *ulp2-CCR*^*3A*^ mutants, but more drastically accumulate in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant ([Fig 2C and 2D](#pgen.1008477.g002){ref-type="fig"}). A slower migrating species of Ame1 was detected in cell lysate, but was determined to be unrelated to SUMO, as it is insensitive to Ulp1 cleavage ([S1 Fig](#pgen.1008477.s001){ref-type="supplementary-material"}). However, the higher molecular weight species of Ame1 is referred to as sumoylated Ame1, as it is enriched via the Ulp1-C580S resin and can be cleaved by Ulp1 ([S1 Fig](#pgen.1008477.s001){ref-type="supplementary-material"}). Unlike the COMA subunits (Ame1, Okp1 and Mcm21), the levels of sumoylated Mcm16 and Mcm22 increase modestly in the *ulp2-CCR*^*3A*^ mutant, but in the *ulp2-SIM*^*3A*^ mutant they accumulate to a higher level comparable to the level in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant ([Fig 2E and 2F](#pgen.1008477.g002){ref-type="fig"}). This behavior differs from the MS findings above for these proteins, which could be attributed to the fact that the untagged WT Smt3 strain was used here. For example, hyper-sumoylated Mcm16 and Mcm22 could have already reached a maximum level in the *ulp2-SIM*^*3A*^ mutant, which prevents the detection of any additional effect from *ulp2-CCR*^*3A*^. We also did not investigate the other CCAN subunits Ctf3 and Ctf19, whose sumoylation was not detected by MS possibly due to their lower levels. In all cases, the protein levels of unmodified CCAN subunits are not grossly altered in any of the *ulp2* mutants, suggesting that these effects are not due to a change in CCAN expression. Overall, the findings here are in general agreement with the MS results performed using HF-Smt3 cells ([Fig 1](#pgen.1008477.g001){ref-type="fig"}), despite accurate quantification of sumoylated CCAN by MS was limited by their low abundance, especially in wild type cells. The CMM complex mediates the association of Ulp2 to the kinetochore {#sec005} ------------------------------------------------------------------- Prior studies have demonstrated that CCAN assembles at centromeres in a hierarchical order ([Fig 3A](#pgen.1008477.g003){ref-type="fig"}) \[[@pgen.1008477.ref009], [@pgen.1008477.ref010], [@pgen.1008477.ref036]\], and considering that Ulp2's CCR seems to play a specific role in desumoylating CCAN, we tested whether Ulp2 binds to the kinetochore through its CCR. Using an immobilized Ulp2^873-1034^ (Ulp2-CCR) affinity column ([S1 Fig](#pgen.1008477.s001){ref-type="supplementary-material"}), we found that the wild-type Ulp2-CCR resin binds to Mcm21 in yeast cell extracts, and that this binding is abolished by the *ulp2-CCR*^*3A*^ mutation ([Fig 3B](#pgen.1008477.g003){ref-type="fig"}). Interestingly, deletion of Ctf19 or Mcm16 also abolished the binding between Ulp2 and Mcm21 without affecting the abundance of Mcm21 ([Fig 3C](#pgen.1008477.g003){ref-type="fig"}), indicating that Mcm21 does not directly bind to Ulp2. Because Mcm16 is required for the Ulp2-Mcm21 interaction, we next investigated whether deleting Chl4 and Cnn1 had any effect. Deletion of Cnn1, which acts downstream of Mcm16 in CCAN assembly, did not disrupt the binding between Ulp2 and Mcm21, whereas the deletion of Chl4, which helps to recruit Mcm16 to Mcm21, abolished the binding ([Fig 3D](#pgen.1008477.g003){ref-type="fig"}). In contrast, deletion of Chl4 or Cnn1 did not disrupt binding between Ulp2's CCR and Mcm16 ([Fig 3E](#pgen.1008477.g003){ref-type="fig"}), further implying that Mcm16 may directly interact with Ulp2. Moreover, the deletion of any individual subunit of CMM (Ctf3-Mcm16-Mcm21) eliminated Ulp2's interaction with the other two CMM subunits ([Fig 3F--3H](#pgen.1008477.g003){ref-type="fig"}), indicating that an intact CMM complex is needed to interact with Ulp2. In all cases, the *ulp2-CCR*^*3A*^ mutation reduced or abolished its binding to CMM, confirming the specificity of this interaction. Taken together, these findings suggest that Ulp2 interacts with the CMM complex, which in turn mediates Ulp2's interaction with the other CCAN sub-complexes. How Ulp2 may interact with the fully assembled kinetochore remains to be determined. {ref-type="supplementary-material"}).](pgen.1008477.g003){#pgen.1008477.g003} Ulp2's CCR is sufficient for its interaction with the CMM complex {#sec006} ----------------------------------------------------------------- Considering the role of the conserved motif (a.a. 931--934) in Ulp2's CCR in mediating the interaction between Ulp2 and CMM ([Fig 3](#pgen.1008477.g003){ref-type="fig"}), we next sought to determine the minimal region in Ulp2 that is sufficient for binding to CMM. A synthetic peptide containing the conserved motif (Ulp2^896-937^) was found to bind to Ctf3 in yeast cell lysates, while a synthetic peptide (Ulp2^896-937^) containing the *CCR*^*3A*^ mutation does not bind ([Fig 4A](#pgen.1008477.g004){ref-type="fig"}), confirming the specificity of this interaction. To further determine whether the binding between Ulp2 and CMM is direct, we expressed the CMM complex in SF9 insect cells, eliminating any interference from other yeast proteins, and used Tandem Mass Tag (TMT) based quantitative mass spectrometry (MS) to analyze specific proteins that bound to resins immobilized with either Ulp2-CCR or Ulp2-CCR^3A^ peptides ([Fig 4B](#pgen.1008477.g004){ref-type="fig"}). As shown in [Fig 4C](#pgen.1008477.g004){ref-type="fig"} and [S7 Table](#pgen.1008477.s009){ref-type="supplementary-material"}, Ctf3, Mcm16 and Mcm22 of the CMM complex were the only proteins that specifically bind to the Ulp2-CCR peptide resin. In fact, Ctf3, Mcm16 and Mcm22 were found to be more than three-fold enriched by the wild-type Ulp2-CCR peptide resin compared to the Ulp2-CCR^3A^ peptide resin ([Fig 4D](#pgen.1008477.g004){ref-type="fig"}), even though more Ulp2-CCR^3A^ peptide resin was used for the pull-down. Taken together, these results demonstrate that Ulp2's CCR directly binds to the CMM complex. {ref-type="fig"}).](pgen.1008477.g004){#pgen.1008477.g004} To address whether the interaction between Ulp2 and CMM plays a role in recruiting Ulp2 to the kinetochore assembled at the centromere, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) were performed to measure the amount of DNA bound to endogenously tagged Ulp2. This reveals that Ulp2 specifically binds to the centromere-III region but not the *CUP1* region ([Fig 4E](#pgen.1008477.g004){ref-type="fig"}). This centromere-specific binding of Ulp2 is partially reduced by *ulp2-CCR*^*3A*^, similar to that caused by *mcm16Δ* ([Fig 4E](#pgen.1008477.g004){ref-type="fig"}). This partial effect is in agreement with the partial role of Ulp2's CCR in facilitating CCAN desumoylation (Figs [1](#pgen.1008477.g001){ref-type="fig"} and [2](#pgen.1008477.g002){ref-type="fig"}), and it further suggests that Ulp2's SIM and protease domain could be responsible for the remaining centromere binding activity. Consistent with the role of Mcm16 in recruiting Ulp2 to the centromere, deletion of Mcm16 led to an elevated level of sumoylated Mcm21 ([Fig 4F](#pgen.1008477.g004){ref-type="fig"}), while deletion of Ctf19, which disrupts the formation of the Ctf19-Mcm21 sub-complex \[[@pgen.1008477.ref005]\], caused a complete loss of sumoylated Mcm21. Thus, localization of Ulp2 to the centromere via Mcm16 plays a role in keeping the sumoylation of Mcm21 low. The CMM complex plays two roles in maintaining SUMO homeostasis of the inner kinetochore {#sec007} ---------------------------------------------------------------------------------------- Considering the biochemical defects of the *ulp2* mutants in CCAN binding and desumoylation, we next evaluated its potential defect in chromosome segregation by performing a quantitative mating assay to measure the rate of chromosome-III loss in the *ulp2* mutants \[[@pgen.1008477.ref037]\]. As seen in [Fig 5A](#pgen.1008477.g005){ref-type="fig"} and [S8 Table](#pgen.1008477.s010){ref-type="supplementary-material"}, compared to wild-type cells, the *ulp2-SIM*^*3A*^ and *ulp2-CCR*^*3A*^ mutations result in an 8-fold and 4-fold respective increase in the rate of chromosome-III loss, while the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant causes a synergistic 64-fold increase in the rate of chromosome loss. These results indicate that Ulp2's CCR and SIM play a partially redundant role in preventing chromosome loss. {ref-type="supplementary-material"}). B) Growth of various *ulp2* and *mcm16Δ* mutants, following the acute removal of the complementing *ULP2* plasmid by 5-fluoroorotic acid (5-FOA). C) Quantitative MS to measure the effect of *mcm16Δ* on intracellular sumoylation in the *ulp2Δ* mutant (also see [S9 Table](#pgen.1008477.s011){ref-type="supplementary-material"}). D-E) Western blot analysis to observe the effect of *mcm16Δ* on Mcm21 and Ame1 sumoylation in the *ulp2Δ* mutant, following the enrichment of sumoylated proteins via the Ulp1-C580S affinity resin. F) Western blot analysis to observe the effect of *mcm16Δ* on the sumoylation of Mcm21 in various *ulp2* mutants following the enrichment of sumoylated proteins via Ulp1-C580S affinity resin.](pgen.1008477.g005){#pgen.1008477.g005} Given that CMM directly binds to Ulp2 ([Fig 4](#pgen.1008477.g004){ref-type="fig"}), we next asked whether a mutation in this complex would result in a chromosome loss defect similar to that of the *ulp2-CCR*^*3A*^, and further cause a synergistic increase in chromosome loss when it is combined with the *ulp2-SIM*^*3A*^. To address this, a quantitative mating assay was performed, revealing that the *mcm16Δ* mutant caused a 4-fold increase in the rate of chromosome loss ([Fig 5A](#pgen.1008477.g005){ref-type="fig"} and [S8 Table](#pgen.1008477.s010){ref-type="supplementary-material"}). Surprisingly, the *mcm16Δ* mutant did not further increase the rate of chromosome loss when combined with *ulp2-SIM*^*3A*^; instead, loss of Mcm16 in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant drastically reduced the rate of chromosome loss from 64-fold to 6-fold. To explore this further, we examined the effect of *mcm16Δ* on the growth of various *ulp2* mutants. As in our previous work \[[@pgen.1008477.ref031]\], mutations to Ulp2's SIM and CCR result in a growth defect when the complementing wild-type Ulp2 is acutely removed by plasmid shuffling ([Fig 5B](#pgen.1008477.g005){ref-type="fig"}, rows 2--5). Interestingly, *mcm16Δ* strongly suppressed both the growth defect of the *ulp2Δ* mutant ([Fig 5](#pgen.1008477.g005){ref-type="fig"}, rows 1 and 6) and that of the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant ([Fig 5](#pgen.1008477.g005){ref-type="fig"}, rows 5 and 10). These findings raise the possibility that Mcm16 plays another role that is distinct from recruiting Ulp2 (Figs [3](#pgen.1008477.g003){ref-type="fig"} and [4](#pgen.1008477.g004){ref-type="fig"}). To explore other functions of Mcm16, we performed quantitative MS in the *ulp2Δ* background to determine whether the loss of Mcm16 has any effect on sumoylated CCAN. As seen in [Fig 5C](#pgen.1008477.g005){ref-type="fig"} and [S9 Table](#pgen.1008477.s011){ref-type="supplementary-material"}, we observed approximately 2--4 folds more sumoylated Ame1, Mcm21 and Okp1 in the *ulp2Δ* single mutant than the *ulp2Δ mcm16Δ* double mutant, besides the expected absence of sumoylated Mcm16 in the *ulp2Δ mcm16Δ* double mutant. To confirm this, we purified total sumoylated proteins using the Ulp1-C580S pull-down method ([Fig 2A](#pgen.1008477.g002){ref-type="fig"}). As shown in [Fig 5D and 5E](#pgen.1008477.g005){ref-type="fig"}, *mcm16Δ* reduced the hyper-sumoylated species of Mcm21 and Ame1 in the *ulp2Δ* mutant background, whereas the amount of sumoylated Mcm21 and Ame1 was increased in the wild-type *ULP2* background. Moreover, *mcm16Δ* caused a modest reduction of hyper-sumoylated Mcm21 in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant ([Fig 5F](#pgen.1008477.g005){ref-type="fig"}, lanes 5 and 8), although little effect was observed in the *ulp2* single mutants. Overall, these findings indicate that Mcm16 plays two opposing roles in regulating kinetochore sumoylation: 1) recruiting Ulp2 to desumoylate Mcm21 and Ame1, and 2) facilitating the sumoylation of Mcm21 and Ame1, which is more clearly observed in cells lacking Ulp2. It should be noted that these opposing roles of Mcm16 are not contradictory to each other; instead, they suggest that separation-of-function mutations in the CMM complex would be needed to understand these roles further. The homeostasis of CCAN sumoylation is critical for accurate chromosome segregation {#sec008} ----------------------------------------------------------------------------------- Our results suggest that an accumulation of hyper-sumoylated CCAN leads to growth defects and elevated chromosome loss in the *ulp2* mutants ([Fig 5](#pgen.1008477.g005){ref-type="fig"}). If so, the *smt3-allR* mutant, in which formation of poly-SUMO chains is blocked \[[@pgen.1008477.ref038]\], should suppress the chromosome loss defect of the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant. Interestingly, the mutation of *smt3-allR* alone resulted in a drastic 57-fold increase in the rate of chromosome-III loss, which is comparable to that of the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant. This suggests that poly-sumoylation is needed to prevent chromosome loss in wild-type cells. Remarkably, combining the *smt3-allR* and *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutations led to a marked reduction in the rate of chromosome loss by \~10-fold (from \~60-fold to 6-fold) ([Fig 6A](#pgen.1008477.g006){ref-type="fig"} and [S8 Table](#pgen.1008477.s010){ref-type="supplementary-material"}), raising the hypothesis that CCAN sumoylation must be maintained at a precise level to ensure accurate chromosome segregation. To explore this further, we next used the Ulp1-C580S pull-down approach to investigate the effect of *smt3-allR* on the levels of CCAN sumoylation. As shown in [Fig 6B](#pgen.1008477.g006){ref-type="fig"}, *smt3-allR* has little detectable effect on the amount of sumoylated Mcm21 in the wild-type background, indicating that poly-sumoylated Mcm21 in wild-type cells, if present, exists at a level below the detection limit of this assay. However, *smt3-allR* appears to reduce the amount of the slowest migrating species of Mcm21 in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant ([Fig 6B](#pgen.1008477.g006){ref-type="fig"}), suggesting that these are the poly-sumoylated species of Mcm21. However, multiple higher molecular weight species of Mcm21 still persist in the *smt3-allR ulp2-SIM*^*3A*^*CCR*^*3A*^ triple mutant ([Fig 6B](#pgen.1008477.g006){ref-type="fig"}). Because *smt3-allR* is expected to eliminate all branched poly-SUMO chains, these higher molecular weight species of Mcm21 are likely attributed to mono-sumoylation of multiple lysines on Mcm21, although we cannot exclude the possibility that Smt3-allR can still form linear chains whose existence have yet to be confirmed. Similarly, *smt3-allR* does not have an appreciable effect on the abundance of sumoylated Ame1, Okp1, Mcm16 and Mcm22 in the wild-type background ([Fig 6C--6F](#pgen.1008477.g006){ref-type="fig"}), indicating that poly-sumoylated species of these proteins are below the detection limit of this assay. As observed above and here (Figs [2](#pgen.1008477.g002){ref-type="fig"} and [6C--6F](#pgen.1008477.g006){ref-type="fig"}), multiple slower migrating sumoylated species of these CCAN subunits are readily detected in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant, which remain in the *smt3-allR ulp2-SIM*^*3A*^*CCR*^*3A*^ triple mutant, suggesting that each of these CCAN subunits is likely mono-sumoylated on several lysine residues. Interestingly, multiple mono-sumoylated species of Mcm16, Mcm22, Okp1 and Mcm21 appear to accumulate to a higher level in the *smt3-allR ulp2-SIM*^*3A*^*CCR*^*3A*^ triple mutant than in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant. One possible explanation is that *smt3-allR* could channel the unstable poly-sumoylated proteins into the more stable mono-sumoylated proteins, although alternative explanation could also exist; for example, the gel electrophoretic mobility pattern for poly-sumoylated proteins may differ from that of multiply mono-sumoylated species. Regardless of whether poly-SUMO chains could form, a relatively large number of SUMO moieties remain attached to CCAN in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant, which creates a situation where a high local concentration of SUMO at the inner kinetochore could act as a negative feedback signal to recruit Ulp2. Using this feedback mechanism, Ulp2 prevents excessive sumoylation at the inner kinetochore. {ref-type="supplementary-material"}). B-F) The effects of *smt3-allR* on the sumoylation patterns of CCAN subunits in wild-type *ULP2* and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant backgrounds.](pgen.1008477.g006){#pgen.1008477.g006} Regulation of CCAN sumoylation during the cell cycle and in response to DNA replication stress {#sec009} ---------------------------------------------------------------------------------------------- To further explore the functions of Ulp2, we performed tetrad dissections of diploid yeast containing *ULP2/ulp2-707Δ* and *ULP2/ulp2-SIM*^*3A*^*CCR*^*3A*^ heterozygous mutants. As seen in [Fig 7A](#pgen.1008477.g007){ref-type="fig"}, neither of these *ulp2* mutants has an appreciable effect on spore viability or cell growth, unlike the *ulp2Δ* mutant \[[@pgen.1008477.ref023]\]. To determine whether the *ulp2-SIM*^*3A*^*CCR*^*3A*^ double mutant has aneuploidy similar to that of the *ulp2Δ* mutant \[[@pgen.1008477.ref027], [@pgen.1008477.ref039]\], we applied TMT-based quantitative MS to measure the abundance of proteins expressed from each chromosome. As summarized in [S2 Fig](#pgen.1008477.s002){ref-type="supplementary-material"} and [S10](#pgen.1008477.s012){ref-type="supplementary-material"} and [S11](#pgen.1008477.s013){ref-type="supplementary-material"} Tables, chromosomal protein expression levels were not significantly altered between the wild-type and *ulp2-SIM*^*3A*^*CCR*^*3A*^ haploid spores, while elevated levels of protein expression are seen from chromosome I, III and XI in the *ulp2Δ* cells. However, the expected two-fold increase was not observed from any of these chromosomes, likely due to a heterogeneous distribution of aneuploidy in the *ulp2Δ* mutant, which rapidly develops survivors and was found to be genetically unstable, undergoing adaptive evolution and losing its state of aneuploidy \[[@pgen.1008477.ref039]\]. {#pgen.1008477.g007} The observation that the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant has relatively normal growth comparable to the wild-type strain prompted us to investigate its growth under stresses. Interestingly, these *ulp2* mutants are hypersensitive to hydroxyurea (HU) but not to benomyl ([Fig 7B](#pgen.1008477.g007){ref-type="fig"}), suggesting that the accumulation of hyper-sumoylated CCAN in the *ulp2* mutant does not impair kinetochore function during chromosome segregation; instead, it may interfere with the kinetochore in response to DNA replication stress, possibly when the centromeres are replicated. Next, we examined the level of sumoylated Mcm21 during different cell cycle stages. Sumoylated Mcm21 appears to be modestly higher in G1, lower in the S and G2-M phases in both WT and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant ([Fig 7C and 7D](#pgen.1008477.g007){ref-type="fig"}). Interestingly, HU treatment caused a reduction in sumoylated Mcm21 in WT cells, but a marked accumulation of hyper-sumoylated Mcm21 in the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant ([Fig 7E and 7F](#pgen.1008477.g007){ref-type="fig"}). The reason for these distinct responses to HU treatment is presently unknown; although they indicate that sumoylation of Mcm21 is particularly sensitive to DNA replication stress. A remaining question is the role of kinetochore assembly on CCAN sumoylation. To address this, we used the *ndc10-1* mutant, which was shown to disrupt kinetochore assembly at non-permissive temperature \[[@pgen.1008477.ref040]\]. Sumoylated Mcm21 is drastically reduced in the *ndc10-1* mutant following a temperature shift to 37 °C for 6 hours ([Fig 7E](#pgen.1008477.g007){ref-type="fig"}); and FACS analysis shows that the *ndc10-1* mutant accumulates over 2C DNA content, likely as a result of chromosome mis-segregation. This finding, together with the observation that Mcm16 also contributes to sumoylation of Mcm21 ([Fig 5C](#pgen.1008477.g005){ref-type="fig"}), suggests that sumoylation of Mcm21 likely occurs on the fully assembled kinetochore at the centromere where Ulp2 is recruited (see [Fig 4E](#pgen.1008477.g004){ref-type="fig"}). Discussion {#sec010} ========== Prior studies have implicated that protein sumoylation plays an evolutionarily conserved, yet poorly understood role in regulating kinetochore function. Among the enzymes that catalyze reversible sumoylation, the Ulp2^SENP6^ family of enzymes appears to play a specific role in regulating chromosome segregation \[[@pgen.1008477.ref023], [@pgen.1008477.ref025], [@pgen.1008477.ref028], [@pgen.1008477.ref029], [@pgen.1008477.ref041], [@pgen.1008477.ref042]\]; however, the mechanism has been elusive. Our results here show that Ulp2 targets the inner kinetochore CCAN complex through two distinct and partially redundant mechanisms ([Fig 8](#pgen.1008477.g008){ref-type="fig"}): one that utilizes a newly identified kinetochore-targeting motif (previously referred to as CCR) of Ulp2, which recruits it to the kinetochore via the CMM complex, and the second that applies a negative feedback mechanism to selectively target hyper-sumoylated CCAN subunits via the SIM motif of Ulp2. Importantly, a failure in either mechanism results in an elevated chromosome loss rate, while a loss of both leads to a synergistic increase in chromosome loss. Interestingly, the *smt3-allR* mutant, which reduces the level of poly-sumoylation, results in an elevated rate of chromosome loss in cells containing wild-type Ulp2 ([Fig 6](#pgen.1008477.g006){ref-type="fig"}); however, the same mutation suppresses the chromosome loss defect of the *ulp2* mutant where CCAN becomes aberrantly poly-sumoylated. Moreover, hyper-sumoylated Mcm21 accumulates in this *ulp2* mutant in response to DNA replication stress, resulting in impaired growth ([Fig 7](#pgen.1008477.g007){ref-type="fig"}). Thus SUMO homeostasis at the kinetochore is critical to ensure accurate chromosome segregation likely via centromere replication. {#pgen.1008477.g008} Our previous study showed that Ulp1 is responsible for the bulk of intracellular desumoylation, while in contrast Ulp2 is highly specific \[[@pgen.1008477.ref030]\]. This finding was followed by two recent studies, which showed that Ulp2 targets its substrates using a dual substrate recognition mechanism \[[@pgen.1008477.ref031], [@pgen.1008477.ref032]\]. The findings here further extend this, revealing a new negative feedback mechanism by which Ulp2 targets the kinetochore, where it selectively targets hyper-sumoylated CCAN subunits to maintain SUMO homeostasis. Notably, the *smt3-allR* mutation, which eliminates the formation of branched poly-SUMO chains \[[@pgen.1008477.ref043]\], does not appreciably alter the level of sumoylated CCAN subunits in cells containing wild-type Ulp2, unlike the effect of the *ulp2-SIM*^*3A*^ mutation, which eliminates this negative feedback mechanism altogether. Thus, the binding between the SIM and branched poly-SUMO chains alone is insufficient to account for the negative feedback mechanism of Ulp2. Instead, the fact that several CCAN subunits are modified by multiple SUMO moieties in the *ulp2 smt3-allR* mutant ([Fig 7](#pgen.1008477.g007){ref-type="fig"}) suggests that mono-sumoylation of multiple lysines in the CCAN complex could create a high local concentration of SUMO at the kinetochore, collectively triggering Ulp2's negative feedback mechanism. Consistent with this idea, sumoylation of CCAN requires a functional Ndc10, suggesting that the assembled kinetochore is being targeted. Considering that SUMO has been found to modify multiple subunits of many protein complexes \[[@pgen.1008477.ref033], [@pgen.1008477.ref044]--[@pgen.1008477.ref046]\], it is conceivable that Ulp2 might employ a similar negative feedback mechanism to target other substrates, particularly when their sumoylation is allowed to accumulate. The findings here suggest that homeostasis of kinetochore sumoylation, specifically by Ulp2, is critical for accurate chromosome segregation. However, the specific function of sumoylation at the kinetochore or the specific role that sumoylated CCAN plays remain key questions for future investigation. The observation that several CCAN subunits can be sumoylated at multiple lysine residues or via poly-SUMO chains raises the possibility that these sumoylation events may act collectively to regulate the kinetochore, aside from acting as Ulp2's negative feedback signal. Concerning the nature of this function, a previous study showed that the knockdown of SENP6 in human cells led to the mis-localization of the CENP-H/I/K complex \[[@pgen.1008477.ref029]\], the human ortholog of *S*. *cerevisiae* CMM. However, deletion of Mcm16, a core subunit of the CMM complex, does not fully recapitulate the chromosome segregation defect of the *ulp2* mutant ([Fig 5A](#pgen.1008477.g005){ref-type="fig"}), suggesting that the role that Ulp2 plays in regulating the kinetochore is not limited to maintaining the function of CMM in yeast. Instead, the fact that *mcm16Δ* strongly suppresses the chromosome loss defect of the *ulp2* mutant appears to be consistent with the requirement for Mcm16 in generating an aberrantly hyper-sumoylated kinetochore in the *ulp2* mutant. On the other hand, the reduction of poly-sumoylation in the *smt3-allR* mutant results in a substantial increase in chromosome loss rate ([Fig 6](#pgen.1008477.g006){ref-type="fig"}), indicating that poly-sumoylation plays an important role in maintaining accurate chromosome segregation in wild-type cells. Although the *smt3-allR* mutation does not appreciably affect the amount of sumoylated CCAN subunits in unperturbed wild-type cells ([Fig 6](#pgen.1008477.g006){ref-type="fig"}), we cannot exclude the possibility that poly-sumoylated CCAN may accumulate transiently and is thus too low to be detected in unperturbed cells. Strikingly, the combination of the *smt3-allR* and *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutations led to a mutual suppression of the chromosome loss defect seen in the individual mutants ([Fig 6A](#pgen.1008477.g006){ref-type="fig"}), further supporting the idea that the level of sumoylation at the CCAN complex must be tightly regulated to ensure accurate chromosome segregation. What might the function of sumoylated CCAN be? Given the relatively low stoichiometry of sumoylated CCAN subunits, it seems appropriate to consider them an unstable intermediate, which trigger a biological response only when needed, with Ulp2 preventing their accumulation under normal circumstances. Interestingly, the *ulp2* mutants are hypersensitive to DNA replication stress, but not to benomyl that specifically perturbs chromosome segregation ([Fig 7B](#pgen.1008477.g007){ref-type="fig"}). Moreover, DNA replication stress strongly perturbs the amount of sumoylated CCAN ([Fig 7E and 7F](#pgen.1008477.g007){ref-type="fig"}). Although still speculative, these findings suggest that sumoylated CCAN may act during centromere replication when the kinetochore is expected to undergo a dynamic remodeling. If so, how the kinetochore-centromere interface is perturbed in the *ulp2* mutant remains an important subject for future investigations. Materials and methods {#sec011} ===================== *S*. *cerevisiae* strain and plasmid construction {#sec012} ------------------------------------------------- Standard *S*. *cerevisiae* genetic methods were used to generate strains and plasmids in this study ([S12 Table](#pgen.1008477.s014){ref-type="supplementary-material"}). All integrated mutations in yeast strains and plasmids were confirmed by PCR and DNA sequencing. Yeast growth assay {#sec013} ------------------ Yeast growth was measured using a 5-Fluoroorotic acid (5-FOA) sensitivity assay as previously described \[[@pgen.1008477.ref031]\]: Briefly, cells were grown in 4 mL of synthetic complete medium lacking leucine (SC-Leu, US Biological) until OD~600~ \~1, followed by normalization to an OD~600~ of 0.5. Cells were then diluted five-fold serial dilutions in a sterile 96-well plate with sterile dH~2~O. 5 μL of each dilution was then spotted on either SC-Leu plates or 5-FOA plates (SC supplemented with 0.1% 5-FOA). Both plates were incubated at 30°C for two days, and images were acquired using a Bio-Rad ChemiDoc MP imaging system. Similarly, wild-type and various mutant cells were grown in YPD, 10-fold serial diluted and then spotted on plates containing either hydroxyurea or benomyl (see [Fig 7](#pgen.1008477.g007){ref-type="fig"}). Chromosome loss assay {#sec014} --------------------- Chromosome loss rates were measured using a quantitative mating assay as previously described \[[@pgen.1008477.ref037]\]. Briefly, *ulp2* mutants, expressed in haploid *MATα ARG2 LEU2 ura3* strains, were mated to a tester strain (HZY601: *MATα arg2 URA3*) by mixing \~1×10^7^ log-phase cells of each strain on a filter membrane (0.8 μm MCE Membrane Filter, MF-Millipore), followed by incubation at 30°C for 5 h on a YPD plate (1% Yeast extract, 2% Peptone and 2% Dextrose). Cells were washed and resuspended in 1 mL of sterile dH~2~O. The total number of viable cells was determined by taking 1% of the cell population and plating them onto SC-Arg plates. Diploid cells were selected by plating 10--100% of the remaining cells onto SC-Arg-Ura plates, such that the final numbers of cells growing on SC-Arg-Ura plates were between 100 and 200. Chromosome loss rates were determined by taking the number of Arg^+^ Ura^+^ Leu^−^colonies divided by the total number of viable colonies. For each experimental strain, 95% confidence intervals, of the median chromosome loss rates, were calculated using 16 isolates from each strain. Protein purification and preparation of CNBr-activated resin {#sec015} ------------------------------------------------------------ Ulp1^403-621^-C580S affinity resin was generated by cloning Ulp1^403-621^-C580S into a LIC 2C-T plasmid containing an N-terminal, TEV-cleavable, 6×His-MBP fusion tag. This construct was transformed into *Escherichia coli* Rosetta-2(DE3) pLysS (Novagen) cells, and grown in 2 liters of LB (Luria Broth) media containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol. Proteins were expressed using IPTG induction (0.2 mM IPTG) for 16h at 18°C, with cells being induced at a starting OD~600~ of \~0.6. Cells were lysed in PBSN buffer (1.06 mM KH~2~PO~4~, 5.6 mM K~2~HPO~4~, 154 mM NaCl, 10% Glycerol, 0.2% NP-40, pH 7.4) with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 200 μM benzamidine, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin A). Ulp1^403-621^-C580S was purified via Ni-NTA resin (Qiagen), followed by dialysis and anion exchange chromatography (monoQ 5/50 GL) on an ÄKTA pure FPLC system. Elution fractions were pooled and concentrated to a final protein concentration of 15 mg/mL; proteins were then conjugated to CNBr-activated resin (GE Healthcare) according to manufacturer protocols. Ulp2-CCR resin was prepared in the same manner as Ulp1^403-621^-C580S resin with the following modification: Ulp2^873-1034^ WT or mutant variants were cloned into a LIC 2G-T plasmid containing an N-terminal, TEV-cleavable, 6×His -GST fusion tag. Analysis of CCAN sumoylation and protein-protein interactions {#sec016} ------------------------------------------------------------- To analyze sumoylated CCAN proteins, whole cell extracts were generated by glass bead beating, followed by the enrichment of sumoylated proteins via Ulp1-C580S affinity resin. Specifically, yeast cells were grown in 100 mL of YPD or SC-Leu medium to an OD~600~ of 1.0. Cells were harvested and washed with 10 mL of PBS buffer (PBS, 10% Glycerol, pH 7.4), supplemented with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 200 μM benzamidine, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin A), 20 mM N-Ethylmaleimide and 20 mM Iodoacetamide. Cell pellets were resuspended in 1 mL of PBSN buffer, containing 2 mM MgCl~2~, protease inhibitors and 100 μg DNaseI (Grade II, Roche). Whole cell extracts were generated at 4°C via glass bead-beating (500 μL of glass beads, 10 cycles of 30s break with 2 min rest period) and followed by centrifugation at 15,000 ×*g*. Sumoylated proteins were purified by incubating soluble whole cell extracts with 20 μL Ulp1^403-621^-C580S resin for 2h at 4°C. Resin was washed six times with PBSN buffer, and bound proteins were eluted by boiling in 25 μL of 2×LDS sample buffer (NuPAGE LDS Sample Buffer, Invitrogen). Samples were then reduced using 50 mM DTT and run on SDS-PAGE and analyzed by western blotting using an α-Protein A 1° antibody (Sigma P3775), α-Smt3 1° antibody (rabbit polyclonal antibody made via Covance, Inc.), and an α-rabbit HRP 2° antibody (Sigma). To test if the slower migrating species of Ame1 are sumoylated ([S1 Fig](#pgen.1008477.s001){ref-type="supplementary-material"}), cells expressing Protein A tagged Ame1 were harvested and lysed as above with the following modifications: Omission of N-ethylmaleimide and Iodoacetamide from the cell washing buffer, and the addition of 2 mM DTT and 2.5 μg of recombinant Ulp1 to ensure that SUMO conjugates have been cleaved off of target proteins \[[@pgen.1008477.ref035]\]. To analyze the interactions between CCAN subunits and Ulp2-CCR, pull-down assays were performed using either wild type or mutant variants of Ulp2-CCR resin. Pulldown assays were performed in the same manner as described above for the enrichment of sumoylated proteins using Ulp1^403-621^-C580S. To detect binding between Ctf3 and CCR peptides, biotinylated peptides of UIp2^896-937^-CCR and UIp2^896-937^-CCR^3A^ (0.2 mM from EZbio) in 1 mL PBS were preincubated with 100 μL of NeutrAvidin-agarose resin and washed with 1 mL of PBSN. Whole cell extracts containing Ctf3-Protein A were incubated with 10 μL of peptide resin and washed with 5×1 mL of PBSN buffer. Bound proteins were eluted using 25 μL of 2×LDS sample buffer and then run on SDS-PAGE followed by Protein a western blot as described above. ChIP analysis of Ulp2 {#sec017} --------------------- To analyze the localization of Ulp2 to centromere, ChIP was carried out as described previously \[[@pgen.1008477.ref047]\]. Briefly, yeast cultures (50 mL for each immunoprecipitation) were grown to an OD~600~ of 0.8 and cross-linked for 15 min with 1% formaldehyde at room temperature followed by the addition of 125 mM glycine to quench the reaction. Whole cell lysates were prepared via glass bead-beating as described above and sonicated to shear the genomic DNA to an average size of 500 bp. Immunoprecipitation was performed using Dynabeads Protein G and anti-Flag antibody M2 (5 μL for each IP, Sigma) and then the Input and IP material were purified using QIAquick PCR Purification kit (QIAGEN). The Input was diluted 1:100 and IP samples were diluted 1:10 in water followed by qPCR using SYBR Green 2x master mix (KAPA Biosystems) on a Roche LightCycler 480. Genomic DNA prepared from wild-type cells was serially diluted to make a standard curve of each primer pair for calculation. Fold enrichment values were calculated as percentage of total Input DNA. Primer pairs used here: CEN3-Forward ATCAGCGCCAAACAATATGGAAAA, CEN3-Reverse GAGCAAAACTTCCACCAGTAAACG, CUP1-Forward AACTTCCAAAATGAAGGTCA and CUP1-Reverse GCATGACTTCTTGGTTTCTT. Cell cycle arrest and FACS analysis {#sec018} ----------------------------------- To analyze sumoylation of Mcm21 during cell cycle ([Fig 7](#pgen.1008477.g007){ref-type="fig"}), wild-type and *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant (containing *bar1Δ*) were arrested in the G1 phase by 15 nM alpha-factor for 3.5 hours. Cells were then washed twice with pre-warmed fresh YPD media and then suspended in two volumes of pre-warmed fresh YPD media to allow re-entry into the S phase. At each time point, aliquots of cells were harvested for Ulp1^403-621^-C580S pull-down analysis as described above. In parallel, 300 μL cell culture of each time point was fixed by 700 μL ethanol, treated by Protease K and RNAse A, and then stained by Sytox Green dye for FACS analysis using a BD LSRFortessa cell analyzer. 200 mM HU or 7.5 μg/mL nocodazole was added to various yeast cultures for 3 hours to arrest cells in the early S or G2-M phase, respectively. These arrested cells were then similarly processed for WB and FACS analyses. Preparation of sumoylated proteins from yeast whole cell extracts and TMT labeling of peptides for LC-MS/MS analysis {#sec019} -------------------------------------------------------------------------------------------------------------------- Total sumoylated proteins from yeast strains containing HF-Smt3 were purified as previously described \[[@pgen.1008477.ref031]\]. To generate yeast whole cell extracts for Tandem Mass Tag (TMT) labeling, yeast cells were grown up in YPD medium at 30°C to an OD~600~ of 1.0. Cells were pelleted by centrifugation at 4000 ×*g* at 4°C, followed by glass bead beating for 10 min in lysis buffer (200 mM NaOH, 100 mM Phosphate Buffer pH 8.0, 2% SDS). Samples were neutralized using 200 mM HCl and boiled at 65°C for 10 min. Protein concentrations were then measured by Bradford reagent and equal amounts of protein (\~200 μg) from each extract were digested using 2 μg of trypsin protease at 37°C. Protein digests were acidified using Trifluoroacetic acid (TFA) to a final concentration of 0.5%. Digested peptides were desalted using C18 Sep-Pak cartridges (Waters) and dried down by speed-vac at 30°C. Thermo Scientific TMT10plex labels were used to label tryptic peptides generated from yeast whole cell extracts. Specifically, TMT10-126, TMT10-127N, TMT10-128C, TMT10-129N, TMT10-130C were used to differentially label samples. Briefly, 20 μg of peptides from yeast whole cell extract were labeled with an individual TMT10 tag for 2h at room temperature. Samples were then combined and dried down by speed-vac at 30°C. Peptides were then re-suspended in 80% acetonitrile 20% dH2O and subjected to offline fractionation as previously described \[[@pgen.1008477.ref033]\]. [Fig 5C](#pgen.1008477.g005){ref-type="fig"} used TMT10-127N, TMT10-128C, TMT10-129N and TMT10-130C. [Fig 5D](#pgen.1008477.g005){ref-type="fig"} used TMT10-126, TMT10-127N, TMT10-128C, TMT10-129N and TMT10-130C. Sf9 insect cell expression of Ctf3-Mcm16-Mcm22 and pulldown assay with Ulp2-CCR resin {#sec020} ------------------------------------------------------------------------------------- SF9 insect cell co-expression of Ctf3, Mcm16 and Mcm22 was performed as described in the Baculovirus Expression System (Thermo Fisher Scientific). The co-expression construct was derived from yeast genomic DNA and sequentially cloned and inserted into the pFastbac plasmid using standard restriction enzyme and DNA ligation. Whole cell extracts were generated from 200 mL of SF9 insect cells at \~ 2.0 x 10^6^ cells/mL. Cells were harvested by centrifugation at 400 *×g* at 4°C, followed by washing with 10 mL of wash buffer (PBS, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 200 μM benzamidine, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin A and 100 μg/mL of DNaseI). Cell pelleted were dounced 20 times with a 15 mL dounce homogenizer and spun down at 30,000 *×g* at 4°C. 1 mL of clarified cell extracts were then bound to 25 μL Ulp2-CCR or Ulp2-CCR^3A^ resin for 2 hours at 4°C. Resin was washed with 8 volumes of PBS + 0.2% NP-40 and eluted using 150uL of elution buffer (6M Urea + 50mM phosphate for 1 hour at 4°C. Elutions were reduced, alkylated and digested with Trypsin (1 μg per elution) as described above. Samples were then acidified and labeled using TMT labels as described above. Data analysis for LC-MS/MS data {#sec021} ------------------------------- Data analysis for SILAC labeled samples were performed as previously described \[[@pgen.1008477.ref031]\] with the following exception: A minimum cut-off for 3 unique peptides was not applied. Instead proteins with less than 3 unique peptides were validated by manual inspection. TMT labeled samples were searched via the COMET peptide search engine as part of the Trans Proteomic Pipeline (TPP). The search results were then processed using TPP, where quantification of the TMT reporter ion was analyzed by the Libra software tool. An FDR of less than 1% was applied for peptide identification, and a minimum intensity of the TMT reporter ion was set at 1000. Expression of chromosome specific proteins were quantified by averaging the contributing signal of each TMT reporter ion that originates from proteins belonging to each of the 16 yeast chromosomes. The MS data analysis of SF9 expressed CMM extracts was performed in the same manner as above, with the following modifications: a composite protein database, containing the SF9 insect cell proteome along with the yeast protein sequence of Ulp2, Ctf3, Mcm16 and Mcm22 was used to search the MS data; additionally, a 3 peptide minimum cut-off was applied. Supporting information {#sec022} ====================== ###### A\) Western blot of Protein A tagged Ame1 from whole cell extracts and enriched sumoylated proteins. The upper band of Ame1 in whole cell extract (WCE), as indicated by an asterisk, was insensitive to Ulp1 treatment. In contrast, the eluted sample contains an upper band, which is sensitive to Ulp1 treatment and is thus the sumoylated species of Ame1. B) Conjugation of purified Ulp2-CCR (WT and 3A mutant) proteins to CNBr resin. These resins were then used to examine the binding between the Ulp2-CCR and CCAN subunits (see [Fig 3](#pgen.1008477.g003){ref-type="fig"}). (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare chromosome-specific protein expression between wild type and various *ulp2* mutants. Expression of chromosome specific proteins were quantified by averaging the contributing signal of each TMT reporter ion that originates from proteins encoded by genes on each of the 16 yeast chromosomes. A) Comparison of chromosome-specific expression of proteins between WT and *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant. B) Comparison of chromosome-specific expression of proteins between WT and four independent *ulp2Δ* strains. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in WT and the *ulp2-CCR*^*3A*^ mutant. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in the *ulp2-SIM*^*3A*^ and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutants. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in WT and the *ulp2-SIM*^*3A*^ mutant. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in the *ulp2-CCR*^*3A*^ and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutants. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in WT and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutant. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in the *ulp2Δ* and the *ulp2-CCR*^*3A*^*SIM*^*3A*^ mutants. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare the binding proteins of the wild-type Ulp2-CCR and the Ulp2-CCR^3A^ resins, using SF9-insect cell extracts expressing the yeast CMM complex. \% abundance, standard-error of the mean (SEM), average abundance ratios and the number of positive spectral matches (PSMs) for proteins associating with CCR and CCR^3A^ resin are listed. (DOCX) ###### Click here for additional data file. ###### Quantitative mating results for assaying the rate of chromosome III loss, which are used to generate Figs [5B](#pgen.1008477.g005){ref-type="fig"}, [6A](#pgen.1008477.g006){ref-type="fig"} and [7A](#pgen.1008477.g007){ref-type="fig"}. Median chromosome III loss rates, the 95% confidence interval (CI) and the Fold-change relative to WT are shown. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare sumoylated proteins in the *ulp2Δ* and *ulp2Δmcm16Δ mutants*. Median ratios and the number of positive spectral matches (PSMs) are listed for each protein. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare the % abundance of proteins expressed on each chromosome in wild-type and the *ulp2-SIM*^*3A*^*CCR*^*3A*^ mutants. (DOCX) ###### Click here for additional data file. ###### Quantitative MS to compare the % abundance of proteins expressed on each chromosome in wild-type and several independently prepared *ulp2Δ* mutants. (DOCX) ###### Click here for additional data file. ###### Yeast strains and plasmids used in this study. (DOCX) ###### Click here for additional data file. We would like to thank Yong-qi Gao and other members of the Zhou lab for technical assistance, Dr. Kevin Corbett's lab in advising the expression of the yeast Ctf3-Mcm16-Mcm22 complex in insect cells, and Dr. Sue Biggins for the *ndc10-1* strain. We would also like to thank Drs. Kevin Corbett and Arshad Desai for their critical readings of this work and helpful suggestions. Finally, we thank the anonymous reviewers for their constructive criticisms and helpful suggestions. [^1]: The authors have declared that no competing interests exist.

1. Introduction {#sec1-ijms-19-01027} =============== The mammalian oral mucosal tissue is characterized by permanent morphological and biochemical modification during its lifespan. These changes are substantially regulated by the stage of the keratinization process that involves keratinoblasts. Moreover, the maintenance of the "balance" between keratinoblasts, keratinocytes, and fibroblasts is crucial for the morphological modification of oral mucosa \[[@B1-ijms-19-01027]\]. The basic structure can be modified as the response to changes in environment, saliva contents, as well as drug delivery and administration. The proper morphology of oral mucosa influences the biochemical/metabolic status of the tissue. Therefore, the tissue architecture of oral mucosa and its cellular metabolic status are often recognized as the main factors determining the physiological and/or pathological condition of the oral mucosa \[[@B2-ijms-19-01027]\]. We have recently intensively investigated the structure of oral tissue in pig using fluorescence observation and confocal microscopy. Using the primary cultivation systems we established the co-culture of mucosal keratinocytes and fibroblasts isolated from porcine buccal pouch mucosa. It is also well demonstrated that culture of separated keratinocytes may be achieved only using selective medium with enzymatic separation of tissue \[[@B3-ijms-19-01027]\]. However, the culture system is often composed of both of these cells' populations and therefore only co-culture system of oral mucosa may be successfully implemented in experiments. Our recent studies, using microarray analysis, indicated substantial changes in gene expression during porcine buccal pouch mucosal cells culture \[[@B4-ijms-19-01027]\]. We found that the transcriptomic profile was significantly related to the time period of in vitro culture (IVC). Moreover, using real-time cells proliferation system (RTCA) we found an increased proliferation index of mucosal cells cultured for long-term in vitro. These observation suggested that during long-term buccal pouch mucosal cells cultivation, the cells undergo substantial proliferation and differentiation \[[@B3-ijms-19-01027]\]. The identification of new ontological groups that represent genes significantly up-, and/or down-regulated during cells proliferation in vitro suggested that oral mucosal cells represent tissue form recognized as "metabolic bioreactor". Using microarray technique we analyzed both, known genes that are involved in new metabolic/homeostasis pathways, and new genes that may be markers of well recognized processes. As we know, the cells' morphological composition and metabolic/homeostasis status belong to the main features that describe tissue biology \[[@B5-ijms-19-01027],[@B6-ijms-19-01027],[@B7-ijms-19-01027]\]. Although the morphological architecture of oral mucosa is well recognized using several histological methods, the metabolic versus biochemical status of these cells cultured primary in vitro, is not entirely known. Therefore this study was aimed to investigate the transcriptomic profile of genes involved in the metabolism and homeostasis in porcine buccal pouch mucosal cells during long-term primary in vitro culture. 2. Results {#sec2-ijms-19-01027} ========== Using Affymetrix^®^ Porcine Gene 1.1 ST Array we examined expression of 12,258 porcine transcripts. Genes with fold change higher then abs (2) and wit corrected *p* value lower than 0.05 were considered as differentially expressed. This set of genes consists of 131 different transcripts. The amounts of up and downregulated genes were presented as volcano plots ([Figure 1](#ijms-19-01027-f001){ref-type="fig"}). DAVID (Database for Annotation, Visualization and Integrated Discovery) software was used for extraction of the genes belong to "positive regulation of metabolic process" and "regulation of homeostatic process" gene ontology biological process terms (GO BP). Up and down regulated gene sets were subjected to DAVID searching separately and only gene sets where adjusted *p*-value were lower than 0.05 were selected. Selected sets of genes were subjected to a hierarchical clusterization procedure and presented as heatmaps ([Figure 2](#ijms-19-01027-f002){ref-type="fig"}). Set of the differentially expressed genes belonging to "positive regulation of metabolic process" and "regulation of homeostatic process" GO BP terms category were also presented with symbols, fold changes in expression, Entrez gene IDs and corrected *p* values were presented in [Table 1](#ijms-19-01027-t001){ref-type="table"}. STRING-generated interaction network was created with differentially expressed genes belonging to the "positive regulation of metabolic process" and "regulation of homeostatic process" ontology terms. The results show that there are evidences for *TGFB1* interaction with *REL, CCL2*, and *SPP1* genes. Moreover, evidence show that *REL* can interact with *LYN* and *FCER1G* genes and *SPP1* can interact with *ITGB3*. The results were shown in [Figure 3](#ijms-19-01027-f003){ref-type="fig"}. Furthermore the *CPDB* analysis showed that two pairs of genes: *LYN* and *FCER1G* as well as *ITGB3* and *SPP1* can be found together in complexes categorized in Reactome, PID (Pathway Interaction Database) and BioCarta databases. The results were shown in [Table 2](#ijms-19-01027-t002){ref-type="table"}. In Gene Ontology database genes that formed one particular GO group can also belong to other different GO term categories. By this reason we investigated genes shared between "positive regulation of metabolic process" and "regulation of homeostatic process" GO BP terms. The relations between these genes were showed in [Figure 4](#ijms-19-01027-f004){ref-type="fig"}. RT-qPCR (Real Time- quantitative Polymerase Chain Reaction) analysis was performed, in order to quantitatively validate the microarray analysis. The results were shown as a bar chart ([Figure 5](#ijms-19-01027-f005){ref-type="fig"}). As can be seen in the figure above, while most of the directions of changes in gene expression have been validated, the scale of changes often varies between the methods. This is explainable as the RT-qPCR is a far more quantitative method than Microarrays. These variations are sometimes small (e.g., *SPP1*, *LIF*, *LYN*), while sometimes showing major discrepancies between the two analyses (e.g., *FCER1G*, *ITGB3*, *ETS1*). Nevertheless, in some examples the direction of changes also wasn't validated by RT-qPCR. There were situations, in which only singular samples exhibited this kind of variations (e.g., *REL*, *ETS1*, *CCL2*), while in one example, *TGFB1*, all of the changes have shown different directions. This fact could be explained by difference in precision of those two methods, however it brings major limitation of whole transcriptome screening to light, as it is mainly a qualitative, rather than quantitative study. 3. Discussion {#sec3-ijms-19-01027} ============= The intensity of morphological and biochemical changes of oral mucosa throughout its life is a characteristic feature of the analyzed tissue in mammals. The basic structure, with mutual proportions and interactions between keratinoblasts, keratinocytes, and fibroblasts, may be modified by many external factors, like environment changes, stress, mechanical strain, drug delivery, and administration. Along with the anatomical features, the oral mucosa plays a critical physiological role in distributing masticatory forces, as well as protecting the underlying residual ridge from excessive loading \[[@B8-ijms-19-01027]\]. We can use mucosal biomechanical parameters, as fundamentals to define oral soft tissues behavior, which are closely relevant to clinical applications, including stimuli for tissue remodeling, pressure--pain thresholds, tissue displaceability and residual bone resorption. For example, the oral mucosa was found to be highly deformable and elastic under compression. Additionally, apart from the elastic response, there is a viscous component in this fluid-rich material that increases material viscoelasticity \[[@B9-ijms-19-01027]\]. The processes associated with epithelium regeneration is the base for stem cell research and/or oral cancer investigation. These artificially cultured tissue equivalents are used in transplant surgery for the treatment of a variety of tissue dysfunctions in regions such as the eye, esophagus, or urethra \[[@B3-ijms-19-01027],[@B10-ijms-19-01027]\]. Oral mucosa has been highlighted as a viable alternative source of epidermal cells, due to its easy preparation and suitable features, such as higher cell proliferation rates, lower terminal cell differentiation degrees and an increased biological activity as compared to epidermal keratinocytes. This tissue has another advantage. During harvesting it has been proven to produce less disability, and provides better aesthetic outcome \[[@B11-ijms-19-01027],[@B12-ijms-19-01027]\]. As shown by Guzman-Uribe et al., it is possible to develop dermal-epidermal substitutes from the isolation of cells from oral mucosa for diabetic and healthy subjects using the air-liquid technique \[[@B13-ijms-19-01027]\]. Nevertheless, the dynamic behavior of oral mucosa tissues remain not well understood. Therefore, employing primary cell culture and a microarray approach, we aimed to investigate the transcriptomic profile of buccal pouch mucosal cells during long-term, in vitro culture. Our recent studies are an introduction to issues related to characterization of morphological and biochemical mechanisms of the oral mucosa \[[@B4-ijms-19-01027],[@B14-ijms-19-01027]\]. In the present study, we examined the mRNA expression level changes during IVC of two ontology groups: (1) "Positive regulation of metabolic process" and (2) "Regulation of homeostatic process". This approach will allow us to better understand dynamic behavior of oral mucosa tissues. From all analyzed genes, which showed different expression patterns, a total of 17 genes that belong to both gene ontology biological process terms were studied. In the group of described transcript profiles after microarray assay, we found five genes (*ITGB3*, *TGFB1*, *LYN*, *ETS1,* and *PTGS2*) with similar changes in transcripts expression profile during culture in both GO BP terms. Integrin subunit beta 3 (*ITGB3*) and transforming growth factor beta 1 (*TGFB1*) show increased transcript levels after 7 days of culture. During longer maintenance of the cell culture, after 15 days we observed significant decrease in *ITGB3* and *TGFB1* mRNA levels in both GO BP terms. Interestingly, in the final phase of our culture (after 30 days), heat map clearly shows slightly increased transcript expression. Integrins are heterodimeric cell surface glycoproteins consisting of α and β subunits, that connect the extracellular matrix (ECM) to the cytoskeleton \[[@B15-ijms-19-01027]\]. Mechanical forces on matrix-integrin-cytoskeleton linkages are crucial for cell viability, morphology, and organ function. The production of force depends on the molecular connections from extracellular-matrix-integrin complexes to the cytoskeleton. Therefore, we can conclude that integrins are necessary to perform one of the key functions of the oral mucosa-distributing masticatory forces. Moreover, it is well recognized that integrins play an essential role in creating epithelial cell polarity \[[@B16-ijms-19-01027],[@B17-ijms-19-01027],[@B18-ijms-19-01027]\]. Cell polarity is a fundamental organizing principle in metazoan that is necessary for cell division, differentiation, and morphogenesis. Polarization of epithelia is implicit in the development of lumens, which are essential for glandular tissues to carry out their normal functions, nevertheless loss of cell polarity and subsequent tissue disorganization is a hallmark of diseases such as cancer \[[@B19-ijms-19-01027],[@B20-ijms-19-01027]\]. For example, β1 integrin ablation results in a loss of polarity leading to defective arterial lumen formation and asymmetric cell division in skin epithelia \[[@B21-ijms-19-01027]\]. In other studies, authors using three-dimensional (3D) culture models demonstrated a direct role of β1-integrin in the regulation of epithelial cell polarity. Blocking β1-integrin function in this system caused inversion of apical polarity \[[@B22-ijms-19-01027]\]. Other studies have shown integrins' complex role in epithelial cell differentiation \[[@B23-ijms-19-01027]\]. Some studies indicate pivotal role of integrins in the action of *TGFB1* signaling pathway in epithelial cells \[[@B24-ijms-19-01027],[@B25-ijms-19-01027]\]. The cytokine transforming growth factor beta 1 (*TGFB1*) is a known mediator of fibroblast---myofibroblast differentiation, and it mainly elicits its effects through the SMAD (Mothers against decapentaplegic homolog) signal transduction pathway. It also influences a range of other cellular processes, including migration and proliferation, and its release initiates a sequence of events that are crucial in tissue repair, including chemoattraction of inflammatory cells, induction of angiogenesis, and regulation of inflammatory mediators \[[@B26-ijms-19-01027]\]. The effects of *TGFβ* on transcription can be positive or negative depending on the targeted gene and the cellular context \[[@B27-ijms-19-01027]\]. Cirillo et al. \[[@B28-ijms-19-01027]\] suggested that the TGF-β family of cytokines secreted by cancer-associated fibroblast (CAFs) derived from genetically unstable oral squamous cell carcinomas promote the malignant phenotype by weakening intercellular epithelial adhesion. As shown by authors, members of the TGF-β family of cytokines distinguish CAFs from oral cancer and from normal oral fibroblasts \[[@B28-ijms-19-01027]\]. In other studies, the researchers investigated the role of *TGF-β* in the induction of fibrosis in another oral disorder, namely oral submucous fibrosis (OSF) \[[@B29-ijms-19-01027],[@B30-ijms-19-01027]\]. OSF is a potentially malignant condition of the oral cavity, characterized by a generalized submucosal fibrosis \[[@B31-ijms-19-01027]\]. Fibrosis is caused by abnormal increase in the collagen production, nevertheless the exact mechanism is not known. Isoforms of *TGF-β*, *β1* and *β2* have been defined as a pro-fibrotic growth factors which cause deposition of extracellular matrix (ECM) by increasing the synthesis of matrix protein like collagen and decreasing the degradation by stimulating various inhibitor mechanisms. As detailed by Kamath et al., expression of *TGF-β1* and *TGF-β2* was increased in OSF as compared to normal oral mucosa \[[@B29-ijms-19-01027]\]. Additionally, authors have shown the most prominent role of isoform β1 in the fibrotic pathway. Similar patterns of mRNA expression exhibit also type of cytokine genes: C-C motif chemokine ligand 2 (*CCL2*) and polo like kinase 2 (*PLK2*)---protein kinases that have a role in normal cell division \[[@B32-ijms-19-01027]\]. It should be noted that variable expression of *CCL2* and *PLK2* only concerns "positive regulation of metabolic process" GO term. Proto-oncogenes *LYN* and *ETS1* have also shown similar changes in transcript expression profiles in both GO terms described. Heat maps indicates significant reduction of mRNA expression in day 15 compared with day 7. Subsequently, we demonstrated slightly increased transcript levels in 30 compare with D15, however still significantly lower than in D7 (see [Table 1](#ijms-19-01027-t001){ref-type="table"}). Src-family protein-tyrosine kinases (STKs) belong to a family of nonreceptor-type tyrosine kinases and include at least eight members (including *LYN*) with significant amino acid sequence homology. STKs are known to play crucial roles in the regulation of leukocyte functions, including cell migration, adhesion, phagocytosis, and proliferation \[[@B33-ijms-19-01027]\]. *LYN* is mainly expressed in hematopoietic cells with dual roles both as a positive and a negative signaling molecule in B lymphocytes and myeloid cells \[[@B34-ijms-19-01027]\]. Lim et al. \[[@B35-ijms-19-01027]\] also suggested that this proto-oncogene plays a positive regulatory role in retinoic acid-inducible gene I (*RIG-I*)---mediated interferon expression as a downstream component of IFN-β promoter stimulator-1 (*IPS-1*). As shown by Chen laboratory, *LYN* may be a key candidate gene for the formation of precancerous lesions in oral buccal mucosa \[[@B36-ijms-19-01027]\]. The proto-oncogene *ETS1* is a transcription factor known to regulate the expression of a number of genes involved in extracellular matrix remodeling \[[@B37-ijms-19-01027]\]. Similar to the proto-oncogene *LYN* described above, *ETS1* is a critical B cell transcription factor that prevents plasma cells (PCs) differentiation. Furthermore, as reviewed by Garrett-Sinha, *ETS1* is expressed in T cells, NK cells, and NK T cells and is inducible in other, non-lymphoid cell types in response to certain stimuli \[[@B37-ijms-19-01027]\]. Interactions between *ETS1* and its important regulators *LYN*, which maintains *ETS1* expression to limit the differentiation of autoreactive PCs, were described by a Mayeux et al. \[[@B38-ijms-19-01027]\] study. The researchers observed a significant genetic interaction between *Lyn* and *Ets1* in B cells, resulting in a greater and more rapid production of IgM autoAbs in *Lyn*+/−*Ets1*+/− mice than in *Lyn*+/− or *Ets1*+/− mice. Oikawa and Yamada indicate that generally, expression levels of ETS1 correlate well with the grade of invasiveness and metastasis. Upregulation of this ETS family member expression has been documented in many types of human tumors, including oral cancer \[[@B39-ijms-19-01027]\]. Vairaktaris and coworkers compared the levels of *ETS1* expression in diabetic and normal oral mucosa rat models and subsequently they analyzed expression changes in cancerous stages. The authors have shown elevated expression of this proto-oncogene both in diabetic and normal rats, but in cancerous stages (oral squamous cell carcinoma (OSCC)) expression levels was higher in diabetic than in normal rats indicating that diabetes may contribute to enhanced invasion and metastatic potential by increasing *ETS1* levels \[[@B40-ijms-19-01027]\]. In the hamster model with OSCC, the researchers also observed elevated expression levels of *ETS1* compare with controls \[[@B41-ijms-19-01027]\]. Prostaglandin-endoperoxide synthase 2 (*PTGS2*), also known as cyclooxygenase 2 (*COX2*), is the last example of analyzed genes described in both GO BP terms. PTGS2 is the key, rate-limiting enzyme in prostaglandin biosynthesis from arachidonic acid, and acts both as a dioxygenase and as a peroxidase. Cyclooxygenases exist in at least two isoforms, PTGS1 (*COX1*) and PTGS2 (*COX2*). Unlike *PTGS1*, which is expressed constitutively, *PTGS2* expression is induced by cytokines and growth factors and is upregulated during inflammation \[[@B42-ijms-19-01027]\]. *PTGS2* has been shown to be expressed in most solid tumor types \[[@B43-ijms-19-01027],[@B44-ijms-19-01027]\]. Similar to the previously described genes, few studies analyzed *PTGS2* expression in OSCC. For example, Byatnal and coworkers evaluated *COX2* expression using indirect streptavidin biotin method. The researchers did not describe enzyme in normal oral mucosa. Elevated *COX2* expression was observed in 58 out of 75 specimens of OSCC \[[@B45-ijms-19-01027]\]. Other studies have also shown, employing immune histochemical staining, that upregulated *COX2* expression was found in OSCC and dysplasia compared to normal mucosa subjects \[[@B46-ijms-19-01027]\]. Additionally, Mauro et al. have compared both isoforms, *PTGS1* and *PTGS2*, expression levels by immunohistochemistry and RT-PCR in normal human oral mucosa and three different pathologies (hyperplasia, dysplasia, and carcinoma). As in the previously cited studies, *PTGS2* is not expressed in the normal tissue. Authors demonstrated enzyme expression in hyperplasia, reaches the maximum activation in dysplasia and then starts to be downregulated in carcinoma \[[@B47-ijms-19-01027]\]. *COX1* mRNA and protein have been already detected in normal oral mucosa. In conclusion, our data showed how morphological and biochemical changes of oral mucosal tissue throughout long-term cell culture in vitro are manifested in variable gene expression levels. However, it must be considered that this study is an entry level, in vitro analysis of the tissue of interest. Given the fact, that the cell culture is primarily obtained from the tissue sample, the results need to be accounted for all of the types of cells that are present in the "mix", additionally the microarray approach, used to analyze the full transcriptome of the cells is largely qualitative, which can be seen, as validation of the results with quantitative RT-qPCR, often gives variable results. This might be due to the fact that the microarrays account for multiple available exons forming many variants of the expressed gene, which is not usually the case with RT-qPCR, as it probes for a specific gene sequence. It can also be explained with the mutual interaction between different cDNA species, present in the sample used for microarray analysis, which may lead to highly reproducible, false negative/positive results that impact the probe averages used for calculation of the total gene results. Finally, the analysis is focused only on transcriptome, which does not always reveal the full picture, as processes such as: alternative splicing, translation regulation, and post-translational modification can often change the amount in which the protein product is present, as well as the way in which it acts. Despite that and the fact that the study is in vitro based, which does not always translate into in vivo situation, it gives insight on the basic molecular mechanisms driving oral mucosal cells in vitro behavior, and may serve as reference for the future in vivo and clinical research. We observed differential expression profile of genes involved in two gene ontology groups, namely "positive regulation of metabolic process" (GO: 0009893) and "regulation of homeostatic process" (GO: 0032844). Firstly, we identified new genes, which may be the markers of these processes in oral mucosal cells in pigs. Additionally, differential expression profile of these cells during long-term in vitro culture suggests that the intensity of cellular metabolism and homeostasis is regulated by genes involved in "lifespan regulatory mechanisms". Our recent experiments indicate that formation of proper morphological architecture of oral mucosa belongs to genetically inherited processes. Therefore, we suggested that, at least in vitro, both tissue cellular morphology and metabolic/physiological properties may be dependent on cellular lifespan. However, these results need to be confirmed by further analysis on protein level, possibly focused on particular, isolated cell populations found in the oral mucosal tissue. 4. Materials and Methods {#sec4-ijms-19-01027} ======================== 4.1. Animals {#sec4dot1-ijms-19-01027} ------------ For this study, a total of 35 pubertal crossbred Polish Landrace gilts (young female pigs), bred on commercial local farm were used. They had a mean age of 155 days (range 140--170 days) and the mean weight was 100 kg (95--120 kg). All of the animals were housed under identical conditions and fed the same forage (depending on age and reproductive status). All experiments were approved by Local Ethics Committee of the Poznan University of Life Sciences, Poland (permission no. 32/2012, 30.06.2012). 4.2. Cell Isolation and Culture {#sec4dot2-ijms-19-01027} ------------------------------- After slaughter, samples of buccal pouch mucosa were obtained within 40 min and transported to the laboratory. The excised tissue was washed twice in Dulbecco's phosphate buffered saline (D-PBS) (137 mM NaCl, 27 mM KCl, 10 mM Na~2~HPO~4~, 2 mM KH~2~PO~4~, pH 7.4). The surface of the buccal pouch was surgically removed using sterile surgical blades. The tissue fragments were incubated with 0.05% collagenase I (Sigma Aldrich, Madison, WI, USA) for 40 min at 38 °C in a shaking water bath and then were treated witch 0.5% Trypsin/EDTA (Cascade Biologics, Portland, OR, USA) for 10 min. The cell suspension obtained from this digestion was filtered through mesh to remove non-dissociated tissue fragments. Isolated cells were washed three times by centrifugation (10 min at 200× *g*) with Dulbecco's modified Eagle's medium (DMEM; Sigma Aldrich, Madison, WI, USA) supplemented with gentamicin (20 μg/mL) and 0.1% BSA. The final cell pellet was resuspended in DMEM supplemented with 10% fetal calf serum (FCS; Sigma Aldrich, Madison, WI, USA) and 10 U/mL penicillin G, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B. Cell viability was 90 to 95% as determined by trypan blue staining (Sigma Aldrich, Madison, WI, USA). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO~2~. Once the oral mucosal cell cultures attained 70--80% confluency, they were passaged by washing with PBS, digested with 0.025% Trypsin/EDTA (Cascade Biologics, Portland, OR, USA), neutralized by a 0.0125% trypsin inhibitor (Cascade Biologics, Portland, OR, USA), centrifuged, and resuspended at a seeding density of 2 × 10^4^ cells/cm^2^. The culture medium was changed every three days. Before the collection of cells for the analyzed samples, photos of the culture were taken to monitor the possible changes of morphology ([Figure 6](#ijms-19-01027-f006){ref-type="fig"}). The reference sample photos (24 h) are not shown in the figure, due to the difficulties in obtaining clear photograph of a primary culture in such early stages, due to visual contaminants proprietary to such culture. 4.3. Microarray Expression Analysis and Statistics {#sec4dot3-ijms-19-01027} -------------------------------------------------- Total RNA (100 ng) from each sample was subjected to two rounds of sense cDNA amplification (Ambion^®^ WT Expression Kit). The obtained cDNA was biotin labelled and fragmentated by Affymetrix GeneChip^®^ WT Terminal Labelling and Hybridization (Affymetrix, Santa Clara, CA, USA). Biotin-labelled fragments of cDNA (5.5 μg) were hybridized to the Affymetrix^®^ Porcine Gene 1.1 ST Array Strip (48 °C/20 h). Microarrays were then washed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station. The array strips were scanned employing Imaging Station of the GeneAtlas System. Preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Operating Software (Affymetrix, Santa Clara, CA, USA). The quality of gene expression data was confirmed according to the quality control criteria provided by the software. The obtained CEL files were imported into downstream data analysis software. The primary microarray analysis was performed by the means of Bioconductor and R programming languages. The background was normalized by the Robust Multiarray Averaging (RMA) algorithm. Subsequently the microarray data was merged with a description file. To determine the statistical significance of the analyzed genes, moderated *t*-statistics from the empirical Bayes method were performed. The obtained p-value was corrected for multiple comparisons using Benjamini and Hochberg's false discovery rate. The selection of significantly altered genes was based on a *p*-value beneath 0.05 and expression higher than two fold. Differentially expressed genes were subjected to selection by examination of genes involved in cell migration regulation. The differentially expressed gene list (separated for up- and down-regulated genes) was uploaded to DAVID software (Database for Annotation, Visualization and Integrated Discovery) \[[@B48-ijms-19-01027]\]. Among extracted enriched Gene Ontology Biological Process (GO BP) terms, we focused on "positive regulation of metabolic process" (GO: 0009893) and "regulation of homeostatic process" (GO: 0032844). Interactions between differentially expressed genes/proteins belonging to the "positive regulation of metabolic process" and "regulation of homeostatic process" GO terms were investigated by STRING10 software (Search Tool for the Retrieval of Interacting Genes) \[[@B49-ijms-19-01027]\]. The list of gene names was used as a query for an interaction prediction. The search criteria were based on co-occurrences of genes/proteins in scientific texts (text mining), co-expression, and experimentally observed interactions. The results of such analysis generated a gene/protein interaction network where the intensity of the edges reflected the strength of the interaction score. Additionally we have investigated if product (proteins) of selected differentially expressed genes belongs to a known protein complexes. Gene names were subjected to Max Plank Institute for Molecular Genetics Consensus Path Data Base (GCDB). This database integrates interaction networks in Homo sapiens including binary and complex protein-protein, genetic, metabolic, signaling, gene regulatory and drug-target interactions, as well as biochemical pathways from 32 public resources \[[@B50-ijms-19-01027]\]. In order to further investigate the changes in studied GO terms, we have calculated the z-score (the number of up- regulated genes minus the number of down- regulated genes divided by the square root of the count) analysis with GOplot package \[[@B51-ijms-19-01027]\]. The results shows allowed us to investigate the enrichment of those two GO BP terms. 4.4. Real Time q-PCR Analysis {#sec4dot4-ijms-19-01027} ----------------------------- The RT-qPCR method was performed to confirm the results obtained in the analysis of expression microarrays. Based on the results obtained during the analysis of expression microarrays, three genes were selected from each heatmap: the ones showing highest, lowest, and intermediate-level of expression. Changes in the level of expression of those genes were then examined ([Table 1](#ijms-19-01027-t001){ref-type="table"}). Four biological samples of each gene were used for the analysis. Each biological test was performed in 3 replicates. Reverse transcription was based on the protocols and reagents of SABiosciences (RT^2^ First Stand Kit---330401), using a Veritimer 96 well Thermal Cycler. 1 microgram of each gene's RNA transcript was used for reverse transcription. Real-time PCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), RT^2^ SYBR^®^ Green ROX^TM^ qPCR Master Mix (Qiagen Sciences, Hilden, Germany) and sequence-specific primers ([Table 3](#ijms-19-01027-t003){ref-type="table"}). Glyceraldehyde-3-phosphate dehydrogenase (*GADPH*), β-actin (*ACTB*), and hypoxanthine-guanine phosphoribosyltransferase *1* (*HRPT1*) were used as reference genes. Gene expressions were analyzed using the relative quantification (RQ) method. The q-PCR starters were designed using Primer3Plus software (<http://primer3plus.com/cgi-bin/dev/primer3plus.cgi>). The sequence of the respective genes was taken from the Ensemble database (<http://www.ensembl.org/index.html>), from which only the sequence of exons was exported, as the target sequence of the designed starter was spread across the border of two adjacent exons. This approach was used as a precaution against the possibility of a non-specific DNA template-based product (DNAse contained in the reverse transcription kit that was used as the other precaution). Agarose gel electrophoresis was applied to confirm the specificity of the amplified products. Marta Dyszkiewicz-Konwińska---experimental design, conducting of the experiments, choice of models, and writing of manuscript parts; Mariusz J. Nawrocki---writing of manuscript parts; Yan Huang-experimental design and editorial assistance; Artur Bryja---conducting of the experiments, data analysis, and figure preparation; Piotr Celichowski---data analysis, figure preparation, and writing of manuscript parts; Maurycy Jankowski---conducting of the experiments, data analysis, and language corrections; Katarzyna Błochowiak---revision of medical methodology; Katarzyna Mehr---revision of medical methodology; Małgorzata Bruska---project supervision; Michał Nowicki---project supervision; Maciej Zabel---revision of methodology and writing assistance; Bartosz Kempisty---project supervision, project design, revision of methodology, editorial supervision, and writing of manuscript parts All authors declare they have no conflicts of interest. {#ijms-19-01027-f001} {#ijms-19-01027-f002} {#ijms-19-01027-f003} {#ijms-19-01027-f004} {#ijms-19-01027-f005} {#ijms-19-01027-f006} ijms-19-01027-t001_Table 1 ###### The official gene symbols, fold changes, and adjusted *p*. values of the differently expressed genes that belong to the "positive regulation of metabolic process" and "regulation of homeostatic process". Adjusted *p*-values are presented in brackets next to their respective fold changes. Official Gene Symbol Fold Change D7/D1 Fold Change D15/D1 Fold Change D30/D1 Fold Change D7/D15 Fold Change D7/D30 Fold Change D15/D30 ENTREZ GENE ID ---------------------- ------------------- ---------------------- ---------------------- -------------------- -------------------- --------------------- ---------------- *LMO2* 0.62 (0.004) 0.30 (0.0001) 0.33 (0.0002) 0.48 (0.03) 0.54 (0.03) 1.11 (0.74) 4005 *LIF* 0.37 (0.005) 0.09 (4.45 × 10^−5^) 0.13 (5.79 × 10^−5^) 0.25 (0.01) 0.35 (0.02) 1.40 (0.33) 3976 *REL* 0.91 (0.49) 0.30 (0.0004) 0.46 (0.002) 0.33 (0.02) 0.51 (0.06) 1.53 (0.38) 5966 *ETS1* 0.41 (0.002) 0.16 (0.0001) 0.19 (0.0002) 0.40 (0.03) 0.46 (0.05) 1.17 (0.75) 2113 *LYN* 2.04 (0.005) 0.81 (0.20) 0.83 (0.25) 0.40 (0.03) 0.41 (0.04) 1.03 (0.98) 4067 *RFC4* 1.27 (0.11) 0.57 (0.006) 0.54 (0.004) 0.45 (0.03) 0.43 (0.04) 0.95 (0.94) 5984 *CCL8* 3.04 (0.0005) 0.33 (0.0004) 1.30 (0.0003) 0.11 (0.01) 0.10 (0.01) 0.92 (0.86) 6355 *TGFB1* 0.61 (0.002) 0.27 (8.78 × 10^−5^) 0.50 (0.0005) 0.44 (0.01) 0.81 (0.22) 1.84 (0.07) 7040 *PLK2* 4.25 (0.002) 0.71 (0.20) 2.59 (0.01) 0.17 (0.03) 0.61 (0.31) 3.67 (0.19) 10,769 *ITGB3* 0.82 (0.32) 0.28 (0.001) 0.63 (0.04) 0.34 (0.04) 0.77 (0.42) 2.22 (0.23) 3690 *DUSP5* 0.34 (0.01) 0.06 (0.0004) 0.11 (0.0009) 0.17 (0.04) 0.34 (0.12) 1.97 (0.46) 1847 *PTGS2* 0.66 (0.16) 0.12 (0.0007) 0.22 (0.002) 0.18 (0.03) 0.33 (0.09) 1.86 (0.45) 5743 *SCARB1* 3.01 (0.001) 1.14 (0.40) 1.79 (0.01) 0.38 (0.03) 0.59 (0.13) 1.56 (0.36) 949 *FCER1G* 2.09 (0.001) 0.89 (0.32) 0.87 (0.24) 0.42 (0.03) 0.41 (0.02) 0.98 (0.98) 2207 *SPP1* 4.35 (0.002) 0.40 (0.01) 0.30 (0.005) 0.09 (0.02) 0.07 (0.02) 0.76 (0.77) 6696 *CCL2* 0.92 (0.75) 0.23 (0.002) 0.63 (0.09) 0.25 (0.04) 0.69 (0.40) 2.71 (0.23) 6347 ijms-19-01027-t002_Table 2 ###### The list of proteins complexes from Reactome, PID (Pathway Interaction Database) and BioCarta databases, that included protein products of differently expressed genes that belongs to the "positive regulation of metabolic process" and "regulation of homeostatic process". *p*-Value *q*-Value Complex_Name Source Members_Input_Overlap Members_Input_Overlap_Geneids Size Effective_Size --------------- --------------- ----------------------------------------------------------------- ---------- ----------------------- ------------------------------- ------ ---------------- 5.51 × 10^−6^ 2.94 × 10^−5^ αv/β3 Integrin/Osteopontin PID ITGB3; SPP1 3690; 6696 3 3 1.10 × 10^−5^ 2.94 × 10^−5^ GPVI:FceRI γ:FYN:LYN Reactome FCER1G; LYN 2207; 4067 4 4 1.10 × 10^−5^ 2.94 × 10^−5^ αv/β3 Integrin/Osteopontin/Src PID ITGB3; SPP1 3690; 6696 4 4 1.83 × 10^−5^ 2.94 × 10^−5^ Fc epsilon receptor I/LYN/SYK BioCarta FCER1G; LYN 2207; 4067 5 5 1.83 × 10^−5^ 2.94 × 10^−5^ GPVI:phosphorylated Fc Epsilon R1 γ:FYN:LYN:Collagen type I:SYK Reactome FCER1G; LYN 2207; 4067 5 5 2.75 × 10^−5^ 3.67 × 10^−5^ Antigen/IgE/Fc epsilon R1/LYN/SYK PID FCER1G; LYN 2207; 4067 6 6 3.85 × 10^−5^ 4.40 × 10^−5^ Antigen/IgE/Fc epsilon R1/LYN/SYK/WIP PID FCER1G; LYN 2207; 4067 7 7 ijms-19-01027-t003_Table 3 ###### Primer information and primer sequences used for the RT-qPCR analysis. Gene Number Product Length (bp) 3′-5′ 5′-3′ ---------- ---------------- --------------------- ---------------------- ---------------------- *SCARB1* NM_213967.1 242 ccccatcgtctaccagatcc agtcctgaagaagtggggtg *PTGS2* NM_214321 202 aaaggcctcaatcgaccaga atctgggcgaggcttttcta *DUSP5* XM_003359366 250 tgcacgacccacctacacta gcgagatcacactcctcctc *ITGB3* NM_214002.1 231 ctcatcggccttgctactct agagacacccacaatcctgg *PLK2* XM_003133981 205 agcctgcttccagacaaaaa gaaggaggtagagccgaggt *CCL2* NM_214214.1 112 gaagagtcaccagcagcaag tggcttatggagtcctggac *TGFB1* NM_214015.2 208 accatgccaatttctgcctg gaacgcacgatcatgttgga *CCL8* NM_001164515.1 206 caatggaaagatccccttca ctcgcagtccaggtaggaag *RFC4* XM_013988325 169 atgcatctgatgaacgtgga cgtcttaaagctgcctgagc *LYN* XM_021089363.1 248 agaggccatcaacttcggat tctgcaggtagtcgaaggtg *ETS1* NM_001162886.1 166 atcagctggacaggagatgg gtttacccgccgtcttgtg *REL* XM_005662527.3 238 ccagaaactgtggcaggatt aggctgaggtaccattgtgg *LIF* NM_214402.2 209 gtgccaacgccctctttatt attgaggctcctttggtccc *SPP1* NM_214023 82 agaagttccgcagatccgaa tccgtctcctcactttccac FCR1G NM_001001265.1 111 accctcctctactgtcgact ataagtctcctggttccggg

Introduction {#sec1-1} ============ *Alpinia calcarata* Roscoe (Heenaratta) and *A. galanga* (Linn.) Willd (Aratta) are two important medicinal plants belonging to family Zingiberaceae and are used in Ayurveda and Sri Lankan Traditional Medicine since ages. Matured, dried rhizomes are used in medicine and in many herbal drug stores it is available in powder form too. Among the two plants, *A. calcarata* is more valued since it is one of the ingredients of "*Buddarajakalka*" which is used in the treatment of chronic respiratory tract infections even among children. Therefore, proper identification is necessary before using in medicinal preparations to obtain correct therapeutic effect as per the classical texts. Morphological features do not give clear idea about the identity when they are in powder form. In such conditions, microscopical examinations would be the only choice. Therefore, present work was undertaken to highlight various microscopical characters by which the two plant materials can be identified microscopically when the materials are in powder form.\[[@ref1][@ref2]\] Materials and Methods {#sec1-2} ===================== Plant material {#sec2-1} -------------- *A. calcarata* Roscoe and *A. galanga* (Linn.) Willd mature rhizome samples were collected in the month of May 2011 from local gardens at Panadura, Pannipitiya and Industrial Technology Institute (ITI) premises when they were in flowering condition. Confirmation was further done with the help of floras and other reputed literature.\[[@ref1][@ref3]\] Voucher specimens (HTS/Phcog/197695 and 197696) were deposited at Pharmacognosy laboratory of Herbal Technology Section at ITI. Collected two rhizome samples were cleaned well and cut into one inch pieces. One part stored separately in Formalin-Aceto-Alcohol solution for microscopical detection. Other part is dried under shade for few days and then powdered separately and passed through 40 mesh size and stored in two airtight containers separately for powder microscopical detection.\[[@ref4]\] Microscopic study of powdered plant material {#sec2-2} -------------------------------------------- Pinch of *A. calcarata* Roscoe rhizome powder was placed on the grease-free microscopic slide along with the drop of glycerin and water (1:1) and then it was covered with a clean cover slip, observed under the compound microscope at 10X followed by 40X magnification. Same observation was done for all the samples collected from three different places separately and important identifying characters were drawn with the help of camera lucida.\[[@ref5]\] Same procedure was done for *A. galanga* (Linn.) Willd rhizome powder as well.\[[@ref4]\] Results {#sec1-3} ======= The results shows *A. calcarata* Roscoe powder consist of plenty of simple and compound starch grains of 30 to 50 μ. Some of the starch grains were muller shaped, some were triangular, pear shaped, and most of them were round and oval shaped. In case of *A. galanga* (Linn.) Willd, less number of simple starch grains of 50 to 70 μ size and mostly round and oval shaped and, very few pear shaped, were also found. Parenchyma cells at places studded with aggregation of small silica crystalline matter were found only on *A. calcarata* Roscoe. Few silica crystals were found in the parenchyma cells of *A. galanga* (Linn.) Willd that are diamond shaped 80 to 100 μ in size. Besides, other identification characters are vessels, fibers, and trichomes that are bigger in *A. galanga* (Linn.) Willd with the diameter between 200 to 300 μ. Pitted fibers are more prominent in *A. galanga* (Linn.) Willd and parenchyma cells near vessels are at places studded with small prismatic crystals of Calcium Oxalate and less frequently found cluster crystals of Calcium Oxalate as well \[Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}\]. {#F1} {#F2} Discussion {#sec1-4} ========== Rhizome powder of both *A. calcarata* Roscoe and *A. galanga* (Linn.) Willd were studied in detail to highlight important anatomical characters. Matured *A. calcarata* Roscoe rhizome is smaller in size compared to that of *A. galanga* (Linn.) Willd and due to that most of the anatomical characters show smaller length and breadth. Diagnostic identification characters of *A. calcarata* Roscoe were compound starch grins, triangular-shaped starch grains, and plenty of simple starch grains in one parenchyma cell compared to that of *A. galanga* (Linn.) Willd. Diamond-shaped silica crystals were found only on *A. galanga* (Linn.) Willd rhizome powder. Present study has revealed an easy technique to identify two medicinal plant materials microscopically and the method can also be employed to detect the degree of adulteration in powdered raw medicinal plant materials as well. In addition to the presently used two botanical sources, there are many more similar rhizomes being used as "*Arattaala*" in Sri Lanka that has to be identified in order to standardize the official source. Conclusion {#sec1-5} ========== Microscopical detection is easy, reliable and cost effective tool for detection of adulteration in medicinal plant materials.

1. Introduction {#sec1-nutrients-11-02764} =============== Alcohol intake is one of the seven main risk factors in the world for both deaths and disability adjusted life years (DALYs) \[[@B1-nutrients-11-02764]\]. Its consumption is associated with more than 60 acute and chronic diseases. Some studies, however, point out the protective effects of alcohol in cardiovascular diseases \[[@B2-nutrients-11-02764],[@B3-nutrients-11-02764],[@B4-nutrients-11-02764]\], diabetes \[[@B5-nutrients-11-02764],[@B6-nutrients-11-02764]\], and kidney cancer \[[@B7-nutrients-11-02764]\]. The association between drinking alcohol and the prevalence of metabolic syndrome (MetS) and its components is not consistent. One prospective study found a linear increase in metabolic syndrome risk with an increase in alcohol consumption \[[@B8-nutrients-11-02764]\]. Park showed that alcohol consumption is associated with a higher incidence of MetS in men. However, no such association was found in women \[[@B9-nutrients-11-02764]\]. In a longitudinal study of older people, alcohol consumption was not associated with the prevalence and incidence of MetS compared with abstainers in both sexes. In men, however, there was an adverse effect of alcohol on glycemia, waist circumference, and systolic blood pressure \[[@B10-nutrients-11-02764]\]. Alkerwi et al. found, however, that moderate alcohol intake reduced the prevalence of MetS, while in women the beneficial effect of alcohol was noted at a dose twice lower than that in men \[[@B11-nutrients-11-02764]\]. In a study conducted by Stoutenberg et al., alcohol reduced the risk of MetS in men at every level of intake \[[@B12-nutrients-11-02764]\]. There were also links between moderate and heavy alcohol consumption and a high concentration of fasting glucose and a reverse dose-response association between alcohol and a low concentration of high-density lipoproteins (HDL)-cholesterol. Alcohol intake was not significantly associated with abdominal obesity, hypertension, and elevated triglyceride concentration. Freiberg et al. showed that moderate alcohol consumption was associated with a lower incidence of MetS and a favorable effect on serum lipid concentration and waist circumference \[[@B13-nutrients-11-02764]\]. Patterns of alcohol consumption and their relationships with health may vary in different populations, depending on age, sex, ethnicity, cultural traditions, socioeconomic status and lifestyle \[[@B14-nutrients-11-02764],[@B15-nutrients-11-02764],[@B16-nutrients-11-02764]\]. The results of previous studies have shown that moderate alcohol consumption was in many populations associated with a lower prevalence of cardiovascular disease (CVD), and a lower mortality due to them \[[@B2-nutrients-11-02764],[@B4-nutrients-11-02764]\]. Because the presence of MetS leads to an increased risk of CVD, we hypothesized that the association between alcohol consumption and the prevalence of MetS will be similar to that for CVD, i.e., moderate consumption will be associated with a lower odds ratio of MetS. Here, our aim was to examine the relationship between the current consumption of alcoholic beverages and the prevalence of MetS and its components in men and women from the Świętokrzyskie region in Poland. 2. Subjects and Methods {#sec2-nutrients-11-02764} ======================= 2.1. Study Design and Sample Collection {#sec2dot1-nutrients-11-02764} --------------------------------------- The research material that was analyzed was the data of 13,172 participants of the PONS project (Polish-Norwegian Study), which was carried out in the years 2010 to 2012 in the Świętokrzyskie region in Poland. Men and women aged 37--66 participated in this cross-sectional study regarding health. We have described in detail the recruitment process as well as the research method and techniques used in previous papers \[[@B17-nutrients-11-02764],[@B18-nutrients-11-02764]\]. The project involved biochemical and anthropometric measurements. The sociodemographic data of the participants and information on health and lifestyle were collected in face-to-face interviews using structured questionnaires. Out of the 13,172 people who volunteered for the study, the data of 12,285 participants (66.7% women) were used for further analysis. The remaining 887 subjects were rejected due to a lack of complete data. 2.2. Ethical Approval {#sec2dot2-nutrients-11-02764} --------------------- The Ethics Committee from the Cancer Centre and Institute of Oncology in Warsaw approved the study (No. 69/2009/1/2011). Permission to analyze the above data was issued in 2016 by the Committee on Bioethics at the Faculty of Health Sciences, Jan Kochanowski University, Kielce, Poland, (No. 45/2016). 2.3. Biochemical Profiling and Anthropometric Measurements {#sec2dot3-nutrients-11-02764} ---------------------------------------------------------- All laboratory analyses of fasting plasma glucose, triglycerides (TGs) and high-density lipoprotein cholesterol (HDL-cholesterol) concentrations were performed in one hospital laboratory that meets the standards of the National Reference Laboratory. The glucose concentration in the blood serum was measured using the enzyme method with hexokinase. The concentration of TGs was assessed using the enzymatic method, with phosphoglycerol oxidase and determination of H~2~O~2~ (with peroxidase). The concentration of HDL-cholesterol was obtained using the colorimetric non-precipitation method with polyethylene glycol modified enzymes (PEG method, Kyowa, Medex). The laboratory tests were performed with the Integra 800 instrument (La Roche Diagnostics, Switzerland). The coefficient of variation (CV) for the parameters determined in the internal quality control were 1.7% for glucose, 2.7% for HDL-cholesterol, and 1.5% for TG. The variation coefficient of the CV (V2) corrected set in the external quality control for the methods used in the study was as follows: For glucose, 2.5%, for HDL-cholesterol, 4.6% and for TG, 2.5%. The internal quality control of the tested parameters was based on the following control materials: PreciControl ClinChem Multi 1, reference number: 05947626190; PreciControl ClinChem Multi 2, reference number: 05947774190. A blood pressure (BP) measurement on the right upper limb artery in a sitting position was performed twice. Here, anthropometric measurements included body height measurements, using a stadiometer (SECA GmbH & Co., Hamburg, Germany). To measure body weight, a body composition analyzer (Tanita S.C. 240MA, Tokyo, Japan) was used, and waist circumference was understood as half the distance between the lower edge of the rib and the upper iliac crest, which was measured with a non-elastic metric tape measure. The assumed measuring accuracy was 0.1 cm and 0.1 kg. Body height and weight measurements were used to calculate the body mass index (BMI, kg/m^2^). 2.4. MetS Definition {#sec2dot4-nutrients-11-02764} -------------------- MetS was defined according to the International Diabetes Federation (IDF) Task Force for Epidemiology and Prevention guidelines. MetS classification, according to IDF, unlike other classifications, takes into account ethnic differences of waist circumferences. Due to the lower cut-off points defining abdominal obesity, this classification allows earlier diagnosis of MetS, i.e., with less excess weight. It also identifies individuals with MetS who have the correct BMI more accurately. MetS was diagnosed for subjects with at least three of five of the following components: Abdominal obesity (waist circumference ≥94 cm in men and ≥80 cm in women), elevated BP (systolic BP ≥130 and/or diastolic BP ≥85 mm, Hg or antihypertensive drug treatment, or previously diagnosed hypertension), elevated fasting glucose (≥100 mg/dL or drug treatment of elevated glucose or previously diagnosed type 2 diabetes), hypertriglyceridemia (≥150 mg/dL or drug treatment for elevated TG or previously diagnosed dyslipidemia), and reduced HDL-cholesterol (\<40 mg/dL in men, \<50 mg/dL in women, or drug treatment for reduced HDL-cholesterol or previously diagnosed dyslipidemia) \[[@B19-nutrients-11-02764]\]. The following drugs were included in the treatment of MetS components in the case of hypertension: Beta-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and calcium channel blockers. In the case of type 2 diabetes, oral hypoglycemic agents, insulin, and incretin drugs were administered subcutaneously. In the case of dyslipidemia, statins and fibrates were used. 2.5. Alcohol Consumption {#sec2dot5-nutrients-11-02764} ------------------------ Alcohol intake was obtained using a standardized questionnaire that included questions on the participants usual consumption of vodka, beer, and wine during the previous year. For every participant, a daily intake of pure ethanol was calculated. The calculation took into account the average content of ethanol in each type of drink consumed and the frequency of its consumption. Since there are significant differences in the amount of alcohol consumed by men and women, different categories of consumption in both sexes were used in the analysis. All individuals were divided into five groups: Nondrinkers (participants who did not drink any alcohol during the previous 12 months) and drinkers, in bands of 0.1--10.0, 10.1--20.0, 20.1--30.0, and \>30.0 g of ethanol/day in men, and 0.1--5.0, 5.1--10.0, 10.1--15.0, and \>15 g of ethanol/day in women. 2.6. Sociodemographic and Lifestyle Data {#sec2dot6-nutrients-11-02764} ---------------------------------------- The sociodemographic variables included sex, age (years), education (total number of education years), and marital status (married or in a stable relationship, single, or a widow/widower). Subjects were classified as current, former, and non-smokers. Current smokers were respondents who smoked cigarettes every day during the study, former smokers were those who had not smoked for longer than six months. The rest of the participants comprised the group of non-smokers. As one serving of coffee, one standard 250 mL mug was adopted. Physical activity (PA) was evaluated with the use of the International Physical Activity Questionnaire (IPAQ). Here, the IPAQ was used to collect information on the frequency and duration of all physical activities (intensive, moderate, and walking) done by respondents during the last week, connected with their professional work, housework, active movement, recreation, and sports. Total PA was presented in metabolic equivalents (MET/min/week^−1^) \[[@B20-nutrients-11-02764]\]. 2.7. Statistical Analyses {#sec2dot7-nutrients-11-02764} ------------------------- All continuous variables were expressed as means and standard deviations (X ± SD). The categorical variables were reported as frequencies and percentages (*n*, %). Differences in baseline characteristics between individuals with diagnosed MetS and the control group were evaluated through the Mann--Whitney U test or the chi-square test, depending on the distribution of each feature. The analyses were performed separately for both sexes. The multivariate logistic regression analyses were used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for the prevalence of MetS. Non-drinkers were adopted as the reference group. Analyses were repeated with each MetS component in the binary form, based on standardized cutoff values (0 = normal, 1 = abnormal). Three models were adopted in the logistic regression analysis: Model I---unadjusted; model II---adjusted for age, education, physical activity, coffee consumption (continuous variables) and marital status (married or in a relationship; single or a widow/widower), smoking (current, past or never); and Model III---additionally adjusted for BMI. A *p* value \<0.05 was assumed statistically significant for all calculations. All data were analyzed using the Statistical Package Statistica software (version 13.1, StatSoft, Warsaw, Poland). 3. Results {#sec3-nutrients-11-02764} ========== Men who were diagnosed with MetS were slightly older, more often married or living in stable relationships, had a heavier weight and higher BMI, more often had abdominal obesity, high BP, increased concentration of glucose and TG and a reduced HDL-cholesterol concentration, compared to men from the control group ([Table 1](#nutrients-11-02764-t001){ref-type="table"}). However, they did not differ significantly in their level of education. Men with MetS more often consumed larger amounts of alcohol (\>30.0 g/day), more often were former smokers, but also more non-smokers, compared with men from the control group. Women who were diagnosed with MetS were older, characterized by lower education and were less likely to be married or living in a stable relationship, compared to those in the control group. Women with MetS had significantly greater mass and BMI, more often had abdominal obesity, high BP, increased concentration of glucose and TG, and decreased HDL-cholesterol concentration compared to women in the control group. In the group with MetS there were more non-drinkers, but no significant differences were noted in the percentage of current and former smokers. Both men and women with MetS drank less coffee and were less physically active compared to participants in the control group. In the group of men currently consuming the largest amounts of alcohol, (\>30 g/day) the risk of MetS was significantly higher compared to non-drinkers in all three models ([Table 2](#nutrients-11-02764-t002){ref-type="table"}). In women, with the increase of alcohol consumption MetS risk decreased, but only in the unadjusted model. The inclusion of confounding variables caused that the association was no longer statistically significant. The highest alcohol consumption in men (\>30 g/day) was associated with a greater risk of abdominal obesity, but only in model II, which adjusted for age, marital status, education, smoking, drinking coffee and physical activity ([Table 3](#nutrients-11-02764-t003){ref-type="table"}). The addition of BMI to the model meant that this relationship ceased to be statistically significant. In women, the risk of abdominal obesity was lower at every level of alcohol consumption, but only in the unadjusted model. Consumption of more than 30 g of alcohol per day increased the risk of high blood pressure in men in all three models ([Table 4](#nutrients-11-02764-t004){ref-type="table"}). In women, drinking 0.1 to 10 g of alcohol per day was associated with a reduced risk of high blood pressure compared to non-drinkers in the unadjusted model. In both adjusted models there was no significant relationship between alcohol consumption and BP. The highest alcohol consumption significantly increased the risk of elevated glucose concentration in all three models in men ([Table 5](#nutrients-11-02764-t005){ref-type="table"}). In women in the unadjusted model, consumption of more than 15 g of alcohol/day significantly reduced the risk of elevated glucose concentration, while consumption of 10.1--15.0 g increased the risk in both adjusted models. In men who drink \>10 g alcohol/day, the risk of reduced HDL-cholesterol concentration decreased significantly with the increase in intake in both the unadjusted model and the two adjusted models ([Table 6](#nutrients-11-02764-t006){ref-type="table"}). In women, in the unadjusted model, at every level of alcohol consumption, the risk of decreased HDL-cholesterol concentration was significantly lower than in non-drinkers. In model II, a similar relationship was found in the group of drinkers \>5 g, and in the group of drinkers \>15 g alcohol/day in model III. There were no significant relationships between alcohol consumption and triglyceride concentration in men ([Table 7](#nutrients-11-02764-t007){ref-type="table"}). In women, the concentration of triglycerides was lower at every level of alcohol consumption in comparison to non-drinking only in the unadjusted model. 4. Discussion {#sec4-nutrients-11-02764} ============= The study shows the existence of links between the current alcohol consumption and MetS risk, but the strength and direction of these relationships may differ in both sexes and may be different for individual MetS components. In the analysis, we adopted a half-dose of alcohol for women compared to men due to differences in body composition and alcohol metabolism between men and women. Nevertheless, we did not find in women, similar to men, an increased risk of MetS and its components (except glucose) in the highest categories of alcohol consumption. This may be due to the fact that women drinking more than 10 g of ethanol/day were more than three times less prevalent than men drinking more than 20 g of ethanol/day respectively. The increased risk of MetS in the highest category of alcohol consumption in men, and the lack of such association in women, may also be the result of other behaviors in the women surveyed which are more favorable to health, e.g., a lower percentage of current and former smokers, as well as a healthier diet. The results of other authors are also quite diverse. In the Korean population, consumption of 0.1 to 5.0 g of alcohol/day was associated with a significantly lower prevalence of MetS \[[@B21-nutrients-11-02764]\]. However, Alkerwi et al., in the meta-analysis of observational studies, noted that an intake of \<40 g alcohol/day in men and \<20 g in women significantly reduces the risk of MetS \[[@B11-nutrients-11-02764]\]. Wakabayashi et al. found that the risk of MetS was the lowest in subjects drinking \<22 g of alcohol/day in both sexes \[[@B22-nutrients-11-02764]\]. The results of our analyses indicate that similar associations were found only in women in the unadjusted model. In an earlier study conducted in Poland, it was also not noted that drinking alcohol was associated with a lower risk of MetS in men \[[@B23-nutrients-11-02764]\]. It was only observed that a lower probability of MetS appeared in women in the highest quartile of alcohol consumption. The authors mentioned, however, that the overall amount of alcohol consumed by women in this study was small, as with 90% of them, the consumption did not exceed 3 g of ethanol per day. The results of our research are, at least partially, in line with the results of a meta-analysis of prospective studies, which proved that heavy consumption of alcohol, i.e., \>35 g/day, is associated with higher MetS risk \[[@B24-nutrients-11-02764]\]. Also, Bermudez et al. confirmed in a multivariate analysis that men consuming 28.41--47.33 g alcohol/day had an increased risk of MetS \[[@B25-nutrients-11-02764]\]. Barrio-Lopez et al. found an increased risk of MetS in those who drink ≥7 drinks/week, which was mainly due to beer consumption \[[@B26-nutrients-11-02764]\]. There was no significant link between drinking wine or liquor and MetS. Lee et al. have indicated, however, that the average frequency of drinking is not associated with the prevalence of MetS in any of the sexes, while a factor which positively correlated with some of the MetS components was the frequency of binge drinking \[[@B27-nutrients-11-02764]\]. The results of the research on the relationship between alcohol consumption and individual MetS components are also ambiguous. Freiberg et al. showed a lower risk of abdominal obesity in both drinkers of ≥1 and ≥20 alcoholic drinks per month, compared to drinkers of \<1 alcoholic drink/month \[[@B13-nutrients-11-02764]\]. In turn, Lourenço et al. found an increased risk of abdominal obesity in women who drink 15.1--30 g/day and \>30 g/day and in men who drink \>60g/day, compared to non-drinkers \[[@B28-nutrients-11-02764]\]. This relationship appeared with a much higher alcohol consumption than in the population we studied. Similarly, the adverse effect of alcohol on the waist circumference was observed in older men (i.e., those aged 65--84 years), but in women no similar relationship was observed \[[@B10-nutrients-11-02764]\]. Alcohol consumption may probably contribute to excessive weight gains in some people as a result of increased energy supply from alcoholic beverages \[[@B29-nutrients-11-02764]\]. Schröder et al. have stated that after controlling for energy under-reporting, drinking more than 30 g of ethanol per day significantly increases the risk of exceeding the recommended energy intake for men \[[@B30-nutrients-11-02764]\]. Alcohol can also contribute to abdominal obesity through non-calorie-related mechanisms, such as changes in the concentration of steroid hormones that may cause central fat storage \[[@B31-nutrients-11-02764]\]. Ambiguous associations between alcohol consumption and abdominal obesity may also result from various effects caused by different alcoholic beverages. Vadstrup et al. found that moderate or high consumption of beer and spirits were associated with an increased waist circumference, while consumption of moderate-to-high amounts of wine had the opposite effect \[[@B32-nutrients-11-02764]\]. Yoon et al. observed that the daily intake of ≥30 g of alcohol increased blood pressure in men \[[@B33-nutrients-11-02764]\], which is consistent with the results of the studies in our population. Meta-analysis of trials conducted by Roerecke et al. confirmed that a reduction in alcohol consumption reduces BP in a dose-dependent manner \[[@B34-nutrients-11-02764]\]. For people who drank more than 2 alcoholic drinks a day, a decrease in alcohol consumption was associated with a significant reduction in BP. The authors emphasize that the above associations are less documented in women \[[@B34-nutrients-11-02764]\]. Stoutenberg et al. observed links between moderate and heavy alcohol consumption and a high concentration of fasting glucose in men \[[@B12-nutrients-11-02764]\], while in the Korean population it was found that heavy alcohol consumption was associated with a high glucose concentration in women \[[@B21-nutrients-11-02764]\]. Nygren et al. found that binge drinking and total alcohol consumption were associated with increased glucose concentration in women in Sweden \[[@B35-nutrients-11-02764]\]. On the other hand, in some studies, a beneficial effect of the intake of moderate amounts of alcohol on the risk of diabetes has been observed \[[@B5-nutrients-11-02764],[@B6-nutrients-11-02764]\]. Huang et al. have associated the varied risk of diabetes in the studies of various authors with the type of alcoholic beverages they drink \[[@B36-nutrients-11-02764]\]. These authors have shown that drinking wine can have a much stronger protective effect on the risk of type 2 diabetes than beer or spirits. The increased risk of abnormal fasting glucose concentration in our studies may partly result from the fact that the pattern of consumption of alcoholic beverages in Poland is dominated by beer, which provided more than 50% of alcohol consumed in total \[[@B37-nutrients-11-02764]\]. In the second place there are spirits, providing over 30% of consumed alcohol. The lowest is consumption of wine, which is attributed the greatest health benefits among alcoholic beverages \[[@B38-nutrients-11-02764],[@B39-nutrients-11-02764]\]. Current research suggests that the relationship between alcohol consumption and triglyceridemia is J-shaped, and triglyceridemia is lowest in people who drink 10--20 g/alcohol per day \[[@B40-nutrients-11-02764]\]. In the men we examined, there was a similar (but not significant) tendency in the highest analyzed category of alcohol consumption. In a study previously carried out in Poland, with the increase in alcohol consumption, a reduced likelihood of elevated triglycerides among women was observed \[[@B23-nutrients-11-02764]\]. However, the results of our research are consistent with the results of the meta-analysis carried out in 2011 \[[@B41-nutrients-11-02764]\]. Its authors also did not confirm the significant effect of alcohol on TG concentration. This may be due to the fact that the consumption of moderate amounts of alcohol may increase lipoprotein lipase activity, and thus influence triglyceridemia \[[@B40-nutrients-11-02764]\]. In a long-term 6-year study, a non-linear, umbrella-shaped relationship between alcohol consumption and HDL-cholesterol concentration was reported \[[@B42-nutrients-11-02764]\]. HDL-cholesterol concentration was the lowest in people who consumed moderate amounts of alcohol (1--2 servings/day in men and 0.5--1.0 servings/day in women). The umbrella-shaped relationship was the result of consumption of mainly hard liquor, and not of beer, where the relationship changes in HDL-cholesterol concentration were linear. In cross-sectional analysis, the authors observed a dose-dependent relationship response between higher alcohol consumption and higher baseline HDL-cholesterol levels, which is consistent with the results of our research. Similar results were obtained by most other authors \[[@B21-nutrients-11-02764],[@B23-nutrients-11-02764],[@B24-nutrients-11-02764],[@B33-nutrients-11-02764]\]. Alcohol intake may increase the concentration of HDL-cholesterol, probably by increasing liver production and/or the transport rate of HDL apolipoproteins apoA-I and apoA-II, as well as by increasing cellular cholesterol outflow and cholesterol esterification in plasma \[[@B43-nutrients-11-02764],[@B44-nutrients-11-02764],[@B45-nutrients-11-02764]\]. The limitation of this study is, above all, the cross-sectional design of the study. There is also the risk of imprecisely determining the amount and frequency of alcoholic beverages drunk by the study participants. In addition, among non-drinkers, we were unable to separate recent abstainers from lifetime abstainers. Interpreting the results regarding the relationship between alcohol consumption and MetS risk may also be hindered by the multidirectional effects of alcohol on individual components whose MetS is a compilation. Sun et al. even suggest that it is more appropriate to estimate the impact of alcohol consumption on individual components than on MetS as a whole \[[@B24-nutrients-11-02764]\]. In addition, the obtained results may be influenced by the differences in the utilization of alcohol in the body related to sex \[[@B46-nutrients-11-02764],[@B47-nutrients-11-02764]\], ethnicity \[[@B48-nutrients-11-02764],[@B49-nutrients-11-02764],[@B50-nutrients-11-02764],[@B51-nutrients-11-02764]\], and different effects caused by particular types of alcoholic beverages dominating in the consumption structure of a given population \[[@B32-nutrients-11-02764],[@B36-nutrients-11-02764],[@B38-nutrients-11-02764],[@B39-nutrients-11-02764]\]. The limitation of our work is also the lack of data on the energy value of diets and nutrient intake, which may affect the spread of MetS. The strength of the study is taking into account a large number of confounders associated with MetS and its components, and a large number of participants (over 12,000), who are homogeneous in terms of their age and ethnicity. 5. Conclusions {#sec5-nutrients-11-02764} ============== In men, a current consumption \>30 g of alcohol per day was significantly associated with a higher risk of MetS, high blood pressure, increased glucose concentration, and abdominal obesity. In women, alcohol consumption from 10.1 to 15.0 g was only associated with a greater risk of abnormal glucose concentration. In both sexes, current alcohol consumption was associated with a higher concentration of HDL-cholesterol, while no significant associations between alcohol intake and triglyceride concentration were found. It is difficult to formulate unequivocal recommendations regarding alcohol consumption in MetS prophylaxis due to its different impact on particular MetS components. In order to explain the causal relationship between alcohol consumption and the metabolic syndrome and its components, prospective studies are necessary. The data collection was supported by the Maria Sklodowska-Curie Institute of Oncology in Warsaw (Poland) and the Polish-Norwegian Foundation Research Fund. Conceptualization, E.S., D.K., E.C., D.R., M.G.-O. and S.G. Formal analysis, E.S., E.C., M.G.-O. Funding acquisition, E.S., D.K. and S.G. Methodology, E.S., E.C. and S.G. Project administration, D.R. Software, E.C. Supervision, E.S. and S.G. Writing -- original draft, D.K., D.R. and M.G.-O. The project is supported under the program of the Minister of Science and Higher Education under the name "Regional Initiative of Excellence" in 2019--2022, project number: 024/RID/2018/19, financing amount: 11.999.000,00PLN. The authors declare no conflict of interest. nutrients-11-02764-t001_Table 1 ###### Sociodemographic characteristics and lifestyle habits of men and women. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Parameters Men Women ------------------------------------------------------------------------------------------------ --------------------- ----------------- ----------------- ----------------- ----------------- ----------------- ----------------- **Age (years) (X ± SD)** 55.14 ± 5.50 56.68 (5.24) **\<0.001** ^a^ 54.33 ± 5.33 57.18 ± 4.92 **\<0.001** ^a^ Education (years) (X ± SD) 13.30 ± 3.22 13.13 ± 3.17 **0.084** ^a^ 13.67 ± 3.14 12.66 ± 3.12 **\<0.001** ^a^ Body mass (kg) (X ± SD) 80.06 ± 11.56 90.35 ± 12.31 **\<0.001** ^a^ 67.55 ± 11.23 76.81 ± 12.95 **\<0.001** ^a^ Body mass index (BMI) (kg/m^2^) (X ± SD) 26.71 ± 3.45 30.04 ± 3.68 **\<0.001** ^a^ 26.28 ± 4.56 30.27 ± 4.92 **\<0.001** ^a^ Married or in a relationship: *n* (%)\ 1688 (87.33)\ 1952 (90.66)\ **0.001** ^b^ 3538 (75.28)\ 2564 (73.28)\ **0.040** ^b^ Single or widow/widower: *n* (%) 245 (12.67) 201 (9.34) 1162 (24.72) 935 (26.72) Abdominal obesity: *n* (%) 922 (47.70) 1983 (92.10) **\<0.001** ^b^ 2828 (60.17) 3344 (95.57) **\<0.001** ^b^ Elevated blood pressure or antihypertensive drug treatment: *n* (%) 1318 (68.18) 2049 (95.17) **\<0.001** ^b^ 2558 (54.43) 3181 (90.91) **\<0.001** ^b^ Increased glucose concentration or drug treatment of elevated glucose: *n* (%) 347 (17.95) 1509 (70.09) **\<0.001** ^b^ 345 (7.34) 1898 (54.24) **\<0.001** ^b^ Decreased HDL-cholesterol concentration or drug treatment for reduced HDL-cholesterol: *n* (%) 63 (3.26) 989 (45.94) **\<0.001** ^b^ 296 (6.30) 2254 (64.42) **\<0.001** ^b^ Increased triglycerides concentration or drug treatment for elevated triglycerides: *n* (%) 195 (10.09) 1445 (67.12) **\<0.001** ^b^ 243 (5.17) 2331 (66.62) **\<0.001** ^b^ Alcohol consumption (g/day): *n* (%) Nondrinkers (Men) Nondrinkers (Women) 265 (13.71) 289 (13.42) **0.015** ^b^ 746 (15.87) 693 (19.81) **\<0.001** ^b^ 0.1--10.0 0.1--5.0 1070 (55.35) 1165 (54.11) 3328 (70.81) 2432 (69.51) 10.1--20.0 5.1--10.0 358 (18.52) 399 (18.53) 413 (8.85) 267 (7.63) 20.1--30.0 10.1--15.0 154 (7.97) 151 (7.01) 98 (2.09) 55 (1.57) \>30.0 \>15.0 86 (4.45) 149 (6.92) 112 (2.38) 52 (1.49) Current smokers: *n* (%) 444 (22.97) 436 (20.25) **\<0.001** ^b^ 858 (18.26) 646 (18.46) **0.734** ^b^ No smokers: *n* (%) 789 (22.97) 662 (30.75) 2489 (52.96) 1823 (52.10) Former smokers: *n* (%) 700 (36.21) 1055 (49.00) 1353 (28.78) 1030 (29.44) Coffee consumption (portion/day) (X ± SD) 1.88 ± 1.94 1.62 ± 1.85 **\<0.001** ^a^ 2.05 ± 1.86 1.68 ± 1.72 **\<0.001** ^a^ Physical activity (METs/min/week^−1^) (X ± SD) 4984.0 ± 4154.9 4319.6 ± 3742.7 **\<0.001** ^a^ 4600.6 ± 3532.2 4204.0 ± 3381.3 **\<0.001** ^a^ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- *n*---number of participants; X ± SD---arithmetic mean ± standard deviation; the numbers in bold indicate statistically significant results; ^a^ Mann--Whitney U test; ^b^ chi-square test. HDL: High-density lipoproteins. MetS: Metabolic syndrome. MET: Metabolic equivalent. nutrients-11-02764-t002_Table 2 ###### Odds ratios (OR) and 95% confidence intervals (CI) for MetS. MetS Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* -------------------------- -------------------------------- ------------- ------------------------------- ----------- -------------------------------------- ----------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 0.99 (0.83--1.20) 0.986 0.99 (0.82--120) 0.920 0.99 (0.81--1.23) 0.973 10.1--20.0 g 1.02 (0.82--1.27) 0.846 1.05 (0.84--1.32) 0.646 1.01 (0.78--1.29) 0.963 20.1--30.0 g 0.90 (0.68--1.19) 0.456 0.98 (0.74--1.31) 0.896 0.94 (0.68--1.29) 0.697 \>30.0 g of alcohol/day **1.59 (1.16--2.17)** **0.004** **1.73 (1.25--2.39)** **0.001** **1.54 (1.08--2.19)** **0.016** Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g **0.79 (0.70--0.88)** **\<0.001** 0.98 (0.87--1.11) 0.796 0.98 (0.86--1.12) 0.772 5.1--10.0 g **0.69 (0.57--0.83)** **\<0.001** 1.02 (0.84--1.25) 0.788 1.11 (0.90--1.37) 0.345 10.1--15.0 g **0.60 (0.43--0.85)** **0.004** 0.92 (0.68--1.41) 0.920 1.12 (0.76--1.65) 0.579 \>15.0 g of alcohol/day **0.50 (0.35--0.71)** **\<0.001** 0.74 (0.52--1.06) 0.100 0.72 (0.49--1.06) 0.100 Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model III---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results. Ref.: Reference level. nutrients-11-02764-t003_Table 3 ###### Odds ratios and 95% confidence intervals for abdominal obesity. Abdominal Obesity Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* --------------------------------------- -------------------------------- ----------- ------------------------------- ----------- -------------------------------------- ------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 0.98 (0.80--1.20) 0.829 0.98 (0.79--1.21) 0.841 1.06 (0.79--1.43) 0.698 10.1--20.0 g 1.04 (0.81--1.32) 0.780 1.07 (0.84--1.37) 0.590 0.95 (0.67--1.35) 0.790 20.1--30.0 g 1.05 (0.77--1.43) 0.746 1.17 (0.85--1.60) 0.344 1.24 (0.79--1.93) 0.352 \>30.0 g of alcohol/day 1.42 (0.99--2.03) 0.054 **1.77 (1.07--2.92)** **0.025** 1.35 (0.68--2.66) 0.389 Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g **0.83 (0.72--0.95)** **0.008** 1.01 (0.87--1.16) 0.938 0.91 (0.74--1.11) 0.350 5.1--10.0 g **0.71 (0.58--0.87)** **0.001** 1.00 (0.80--1.24) 0.969 0.99 (0.73--1.34) 0.959 10.1--15.0 g **0.58 (0.40--0.83)** **0.003** 0.91 (0.62--1.32) 0.614 1.07 (0.64--1.80) 0.786 \>15.0 g of alcohol/day **0.62 (0.44--0.89)** **0.009** 0.97 (0.67--1.40) 0.857 0.87 (0.52--1.45) 0.595 Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model III---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results. Ref.: Reference level. nutrients-11-02764-t004_Table 4 ###### Odds ratios and 95% confidence intervals for elevated blood pressure. Elevated Blood Pressure or Antihypertensive Drug Treatment/Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* -------------------------------------------------------------------------------- -------------------------------- ------------- ------------------------------- ------------- -------------------------------------- ----------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 0.95 (0.75--1.21) 0.674 0.90 (0.71--1.15) 0.409 0.90 (0.70--1.15) 0.396 10.1--20.0 g 1.25 (0.93--1.67) 0.138 1.28 (0.95--1.73) 0.104 1.25 (0.92--1.69) 0.152 20.1--30.0 g 1.07 (0.74--1.55) 0.706 1.15 (0.79--1.67) 0.464 1.11 (0.76--1.63) 0.577 \>30.0 g of alcohol/day **2.57 (1.53--4.30)** **\<0.001** **2.76 (1.64--4.65)** **\<0.001** **2.53 (1.49--4.30)** **0.001** Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g **0.76 (0.67--0.87)** **\<0.001** 0.90 (0.78--1.03) 0.117 0.88 (0.77--1.02) 0.084 5.1--10.0 g **0.68 (0.55--0.82)** **\<0.001** 0.92 (0.75--1.14) 0.450 0.95 (0.76--1.17) 0.614 10.1--15.0 g 0.72 (0.50--1.03) 0.072 1.09 (0.75--1.58) 0.665 1.17 (0.80--1.71) 0.424 \>15.0 g of alcohol/day 0.79 (0.56--1.13) 0.204 1.11 (0.77--1.61) 0.569 1.09 (0.75--1.59) 0.650 Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model II---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results. Ref.: Reference level. nutrients-11-02764-t005_Table 5 ###### Odds ratios and 95% confidence intervals for increased glucose concentration. Increased Glucose Concentration or Drug Treatment of Elevated Glucose/Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* ------------------------------------------------------------------------------------------- -------------------------------- ----------- ------------------------------- ----------- -------------------------------------- ----------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 0.99 (0.82--1.19) 0.919 1.00 (0.82--1.20) 0.964 1.00 (0.82--1.21) 0.971 10.1--20.0 g 1.15 (0.92--1.43) 0.225 1.22 (0.97--1.52) 0.087 1.19 (0.95--1.50) 0.133 20.1--30.0 g 1.14 (0.86--1.50) 0.372 1.26 (0.95--1.68) 0.113 1.25 (0.93--1.67) 0.135 \>30.0 g of alcohol/day **1.57 (1.16--2.14)** **0.004** **1.70 (1.24--2.32)** **0.001** **1.58 (1.15--2.18)** **0.005** Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g 0.88 (0.78--1.01) 0.061 1.04 (0.91--1.18) 0.597 1.04 (0.91--1.19) 0.569 5.1--10.0 g 1.17 (0.72--1.09) 0.242 1.16 (0.94--1.44) 0.159 1.24 (0.99--1.54) 0.056 10.1--15.0 g 1.17 (0.82--1.66) 0.398 **1.65 (1.14--2.38)** **0.008** **1.84 (1.26--2.70)** **0.002** \>15.0 g of alcohol/day **0.65 (0.44--0.96)** **0.032** 0.85 (0.57--1.27) 0.436 0.86 (0.60--1.29) 0.466 Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model III---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results. Ref.: Reference level. nutrients-11-02764-t006_Table 6 ###### Odds ratios and 95% confidence intervals for decreased HDL-cholesterol concentration. Decreased HDL Concentration or Drug Treatment for Reduced HDL-Cholesterol/Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* ----------------------------------------------------------------------------------------------- -------------------------------- ------------- ------------------------------- ----------- -------------------------------------- ------------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 0.97 (0.79--1.19) 0.778 0.97 (0.79--1.20) 0.777 0.97 (0.78--1.20) 0.779 10.1--20.0 g **0.71 (0.55--0.91)** **0.007** **0.73 (0.56--0.94)** **0.016** **0.70 (0.54--0.91)** **0.008** 20.1--30.0 g **0.57 (0.41--0.80)** **0.001** **0.61 (0.43--0.86)** **0.005** **0.60 (0.42--0.85)** **0.004** \>30.0 g of alcohol/day **0.51 (0.35--0.75)** **0.001** **0.51 (0.35--0.76)** **0.001** **0.47 (0.32--0.70)** **\<0.001** Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g **0.79 (0.70--0.89)** **\<0.001** 0.93 (0.82--1.06) 0.261 0.93 (0.82--1.05) 0.249 5.1--10.0 g **0.60 (0.49--0.74)** **\<0.001** **0.81 (0.65--0.99)** **0.045** 0.83 (0.67--1.02) 0.083 10.1--15.0 g **0.46 (0.31--0.69)** **\<0.001** **0.66 (0.44--0.99)** **0.048** 0.68 (0.44--1.03) 0.070 \>15.0 g of alcohol/day **0.41 (0.27--0.61)** **\<0.001** **0.55 (0.36--0.82)** **0.004** **0.54 (0.35--0.82)** **0.004** Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model III---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results; Ref.: Reference level. nutrients-11-02764-t007_Table 7 ###### Odds ratios and 95% confidence intervals for increased triglycerides concentration. Increased Triglycerides Concentration or Drug Treatment for Elevated Triglycerides/Alcohol Consumption Model I Unadjusted OR (95% CI) *p* Model II Adjusted OR (95% CI) *p* Model III Adjusted + BMI OR (95% CI) *p* -------------------------------------------------------------------------------------------------------- -------------------------------- ----------- ------------------------------- ------- -------------------------------------- ------- Men Nondrinkers (ref.) 1.00 1.00 1.00 0.1--10.0 g 1.03 (0.85--1.24) 0.794 1.04 (0.86--1.26) 0.703 1.04 (0.85--1.27) 0.687 10.1--20.0 g 1.07 (0.85--1.33) 0.580 1.06 (0.85--1.34) 0.594 1.03 (0.82--1.31) 0.775 20.1--30.0 g 0.99 (0.75--1.32) 0.965 1.02 (0.76--1.37) 0.880 1.01 (0.75--1.36) 0.959 \>30.0g of alcohol/day 1.30 (0.95--1.77) 0.097 1.29 (0.95--1.77) 0.107 1.19 (0.87--1.64) 0.277 Women Nondrinkers (ref.) 1.00 1.00 1.00 0.1--5.0 g **0.85 (0.76--0.96)** **0.011** 1.02 (0.90--1.16) 0.794 1.02 (0.89--1.16) 0.795 5.1--10.0 g **0.74 (0.61--0.91)** **0.003** 1.01 (0.82--1.24) 0.934 1.04 (0.84--1.28) 0.713 10.1--15.0 g **0.62 (0.42--0.91)** **0.014** 0.90 (0.61--1.33) 0.592 0.93 (0.62--1.39) 0.734 \>15.0 g of alcohol/day **0.55 (0.37--0.80)** **0.002** 0.74 (0.50--1.09) 0.125 0.74 (0.50--1.10) 0.133 Model I---unadjusted; model II---adjusted for age, marital status, education, smoking, coffee consumption and physical activity; model III---adjusted for age, marital status, education, smoking, coffee consumption, physical activity and BMI. The numbers in bold indicate statistically significant results; Ref.: Reference level.

This is the second in a **Series** of four articles about health statistics Health statistics, often viewed as a dry, dull necessity, are at the centre of several worldwide health controversies. Five factors are fuelling the tension between the supply and demand for high quality health information. First, the need for greater accountability and transparency from governments and international agencies is increasing the demand. Civil society groups, the donor community, scientists, and the public want to benchmark progress and performance of public health and medicine. An important example of this tendency is the emphasis on monitoring, including the health-related Millennium Development Goals (MDGs)[@bib1], [@bib2] and the creation of the Healthcare Commission in the UK to independently monitor the National Health Service (NHS).[@bib3] Second, the media, civil society, and the general public are more sceptical about both statistical and scientific claims.[@bib4] An example is the public dismay about the confusing messages on fat in the diet over the past two decades.[@bib5] Evidence of government manipulation of data during the severe acute respiratory syndrome (SARS) epidemic added to the decline in trust.[@bib6] Third, many representatives of the technical and scientific community and the general public are becoming increasingly sophisticated consumers of information. The scope of relevant information is expanding from simple descriptive epidemiology about health to dimensions of public health and medicine such as quality, efficiency, and equity. Consumers often need more detail including quantification of uncertainty. Increased communication and access to different views through the internet are driving scepticism and sophistication, which in turn adds to the broad demand for transparency and accountability. Fourth, as outlined in the first paper in this series,[@bib7] leaders of various global-health programmes including WHO, many public-private global health initiatives such as the Global Fund to fight AIDS, Tuberculosis, and Malaria, and other development agencies, feel the imperative to produce more information on their programmes and the outcomes of their investments for public consumption. Yearly reports, websites, and other publications are regarded as necessary for sustaining political and financial support for their programmes, and for maintaining political priority with governments in developing countries. One result of this response has been an explosion of proposed indicators that should be measured. For example, the Drug Action Programme at WHO has 98 indicators for monitoring structure, process, and outcome[@bib8] and the HIV department has 35 indicators.[@bib9], [@bib10] An internal review presented to the WHO Director General in 2002 found that WHO recommended 3500 indicators covering all programme areas of the organisation. For most of these indicators, no measurement strategy has been proposed and no measurements have been produced. Fifth, as demand for health information grows, primary data collection platforms in most developing countries are not rapidly improving.[@bib7] However, the information technology revolution has not yet had a major effect on platforms for primary data collection in health systems in most developing countries.[@bib11] In this paper, I explore good practice for health statistics from a worldwide and national perspective. Although there are many dimensions to improvement of health statistics, I concentrate on three issues that are central to fostering good practice in the use of health statistics: focusing on priority indicators; correct use of crude, corrected, or predicted statistics; and the need for explicit data audit trails. Taken together, addressing these issues would catalyse better worldwide health statistics practice and stimulate increased national interest in strengthening fundamental data platforms. I believe that increased focus on the production, analysis, and dissemination of priority health indicators will create demand for valid, reliable, and comparable health information, which will stimulate national efforts to strengthen platforms for primary data collection. I do not discuss in detail the interventions that might be necessary to strengthen such platforms. The MDG health-related indicators are used throughout the paper to draw attention to the difficulties with present indicators in terms of conceptualisation, implementation, and measurement. Although the MDG health-related indicators have been developed for high-level policy use, they represent the issues that generally apply to priority health indicators. Technical terms used in this paper are explained in the [panel](#box1){ref-type="boxed-text"} .PanelDefinitions of technical terms**Indicator**A variable measured to monitor progress or assess what works and what does not.**Validity**Validity refers to the extent to which a measurement is capturing what it is intended to measure. There are different types of validity such as face validity, content validity, criterion validity (denoting predictive validity and concurrent validity), and construct validity (denoting convergent and discriminant validity).**Reliability**Reliability refers to the repeatability or consistency of a set of measurements or measuring instrument; for example, test-retest reliability where a test and a retest are compared.**Comparability**Measurements are comparable if the same value means the same thing in the settings being compared. Two thermometers, one in Farenheit and one in Celsius, can both be valid and reliable but they do not give comparable results.**Out-of-sample**Prediction about ranges of values that are not in the investigator\'s sample (ie, that the investigator\'s data set does not cover).**Out-of-time**Prediction about individuals, populations, etc, in time outside the time range of the investigator\'s sample.**Forecasting**Forecasting is the process of estimation in unknown situations. Predicting is a more general term and connotes estimating for any time series, cross-sectional, or longitudinal data. Forecasting is commonly used when discussing time series data.**Farcasting**Farcasting is trying to predict the value of a variable in a place that may be far away but is not a future value.**Prior**The prior is a reflection of some information the investigator has before the observations in the data set (the investigator should state explicitly how the information on the prior was obtained). The prior is the sum of what is known about the relationship under study. Focusing on priority indicators {#cesec10} =============================== With thousands of indicators recommended but few measured well, the worldwide health community needs to focus its efforts on improving measurement of a small set of priority areas. Prioritisation of indicators is important for two reasons. The first is cost. Human resources in the measurement field are extremely scarce, both nationally and internationally. The second is visibility. Indicators drive policy attention and resources nationally and locally. This inevitable dynamic means that health problems with priority indicators will receive more attention than those that are not measured or not measured as well. Prioritisation requires several questions to be answered. What is the proposed indicator intended to measure? {#cesec20} --------------------------------------------------- Indicators can be classified into six categories on the basis of what it is they measure: health outcomes, risk factors, intervention coverage, structure, process, and non-health-related results. All these types of indicators have important uses in different contexts. Part of the challenge in assessment of indicators is to understand at what level they will be used. At the highest level, such as the MDG indicators, the audience is extremely broad including the technical community, governments, and the general public. What is the public-health significance of the indicator? {#cesec30} -------------------------------------------------------- Public-health importance is probably greatest for indicators of health outcomes, intervention coverage, and perhaps risk factors. Many health outcomes are of public-health importance, but because health problems are changing with new challenges, such as the epidemic of non-communicable diseases in developing countries and emergence of pandemic influenza, assessment of public-health significance must be regularly revisited. Coverage of interventions is also important because only through the delivery of effective interventions to those in need can health outcomes be improved. Finally, risk factors, such as tobacco consumption, can be so strongly linked to health outcomes that they are effectively measures of future health outcomes in themselves. Not all outcomes or all intervention-coverage indicators will meet a public-health importance criterion. [Table 1](#tbl1){ref-type="table"} provides my assessment of the public-health importance of every health-related MDG indicator including official supplemental indicators. The analysis in [table 1](#tbl1){ref-type="table"}, including data availability for each indicator, is strictly based on the official UN MDG website;[@bib12] there could well be other surveys and analyses that are not used for MDG monitoring. The 45 indicators in the table could probably be decreased to 16 that have high public-health importance and contribute unique information. For example, for a set of priority indicators, overall coverage of clean water is probably enough instead of urban and rural breakdowns. Arguably, urban and rural breakdowns do not provide a deep insight into differences in clean water inequalities across countries. A more useful indicator for this comparison might be the amount of clean water coverage in the bottom wealth quintile.Table 1MDG health-related indicators (number and name) reported to the General Assembly and officially reported supplemental series**Type of indicatorPublic-health importance of the indicatorMeasurement strategyData availabilityPredominant type of statistic**1990200020031990--20054. Prevalence of underweight children under 5 years of age4a. Children under 5 moderately or severely underweight, percentage[\*](#tbl1fn1){ref-type="table-fn"}Risk factorHighHousehold surveys with anthropometric measurements with some inconsistency of age groups measured8%31%5%8%Corrected4b. Children under 5 severely underweight, percentageRisk factorAdds little value to 4aHousehold surveys with anthropometric measurements, some inconsistency of age groups measured0%29%3%5%Corrected5. Proportion of population below minimum level of dietary energy consumption5a. Undernourished as percentage of total population[\*](#tbl1fn1){ref-type="table-fn"}Risk factorLow; adds little to prevalence of underweightDetails are not available0%0%0%13%Predicted5b. Undernourished, number of peopleRisk factorLow; same information content as 5aDetails are not available0%0%0%13%Predicted13. Under-5 mortality rate[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeHighVital registration in countries with complete systems, complete birth histories, or children ever born and children surviving questions on household surveys98%100%98%25%Corrected and predicted14. Infant mortality rate[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeLow; redundant, correlation coefficient with under-5 mortality rate in 2000 is 0·99Vital registration in countries with complete systems, complete birth histories, or children ever born and children surviving questions on household surveys99%100%100%26%Corrected and predicted15. Proportion of 1 year-old children immunised against measles[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageMedium/low; represents less than 10% of the intervention package for child survivalHealth service provider registries in the public sector, household surveys84%98%98%83%Crude and corrected16. Maternal mortality ratio[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeHighVital registration in countries with complete systems, sibling histories collected in household surveys82%100%0%17%Predicted17. Proportion of births attended by skilled health personnel[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageHigh; but not the only intervention needed to reduce maternal mortalityHousehold surveys, definition of skilled varies across countries0%34%7%6%Corrected18. HIV prevalence among pregnant women aged 15--24 years18a. AIDS estimated deathsHealth outcomeHighVital registration in countries with complete systems, modeling of mortality based on estimated seroprevalence in other countries0%0%67%8%Predicted18b. HIV prevalence rate, aged 15--49, percentageHealth outcomeHighAntenatal clinic (ANC) serosurveillance in sentinel sites, household serosurveys. ANC sero-surveillance appears to overestimate population prevalence0%0%78%9%Corrected and predicted18c. HIV/AIDS prevalence rate for pregnant women 15--24 attending antenatal care in clinics in capital city[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeLow; represents only a partial fraction of national prevalence, 18b is the true quantity of interestCapital city ANC serosurveillance, because of variability in representativeness of sentinel clinics and demographic significance of capital city, comparability limited0%5%5%29%Crude18d. HIV/AIDS prevalence rates, men, estimated from national population surveysHealth outcomeInput to accurate measurement of 18bHousehold serosurveys0%0%3%0%Corrected18e. HIV/AIDS prevalence rates, women, estimated from national population surveysHealth outcomeInput to accurate measurement of 18bHousehold serosurveys0%0%3%0%Corrected19. Condom use to overall contraceptive use among currently married women aged 15--49 years[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageLow; not a good measure of condom use in high-risk sexual intercourseHousehold surveys6%29%4%8%Corrected19a. Condom use, men, aged 15--24 years at last high-risk sex[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageMedium; condom use for any age-group for high-risk sex would be the quantity of interestHousehold surveys but validity of reported rates of high-risk sex not established0%6%3%1%Corrected19b. Condom use, women, aged 15--24 years at last high-risk sex[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageCorrelation coefficient with 19a in 2000 is 0·85Household surveys but validity of reported rates of high-risk sex not established0%7%3%1%Corrected19c. HIV knowledge, men aged 15--24 years who know that a healthy-looking person can transmit HIVIntervention coverageLow; small component of 19eHousehold surveys0%7%0%27%Corrected19d. HIV knowledge, men aged 15--24 years who know that a person can protect himself from HIV infection by consistent condom useIntervention coverageLow; small component of 19eHousehold surveys0%4%0%1%Corrected19e. HIV knowledge, men aged 15--24 years with comprehensive correct knowledge of HIV/AIDS, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageLow; poorly established link and partial relationship to unsafe sexual practicesHousehold surveys0%5%3%1%Corrected19f. HIV knowledge, women aged 15--24 years who know that a healthy-looking person can transmit HIVIntervention coverageLow; small component of 19eHousehold surveys0%32%0%3%Corrected19g. HIV knowledge, women aged 15--24 years who know that a person can protect himself from HIV infection by consistent condom useIntervention coverageLow; small component of 19eHousehold surveys0%37%0%3%Corrected19h. HIV knowledge, women aged 15--24 years with comprehensive correct knowledge of HIV/AIDS, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageLow; poorly established link and partial relationship to unsafe sexual practicesHousehold surveys0%27%3%2%Corrected19i. Contraceptive use among currently married women aged 15--49 years, any method, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageLow; unclear relationship to preventing HIV transmissionHousehold surveys6%29%5%8%Corrected19j. Contraceptive use among currently married women aged 15--49 years, condom, percentageIntervention coverageLow; less relationship to transmission potential than 19a or 19bHousehold surveys7%29%4%8%Corrected19k. Contraceptive use among currently married women aged 15--49 years, modern methods, percentageIntervention coverageLow; weak relationship to decreasing HIV transmissionHousehold surveys5%29%4%8%Corrected20. Ratio of school attendance of orphans to school attendance of non-orphans aged 10--14 years20a. AIDS orphans (one or both parents), currently livingNon-health outcomeLow for public health but could be important for HIV related social policyModelled relationships based on estimated HIV seroprevalence and mortality0%0%25%3%Predicted20b. Orphans (both parents) aged 10--14 school attendance rate as % of non-orphans attendance rate, where HIV is \>1%[\*](#tbl1fn1){ref-type="table-fn"}Non-health outcomeLow; not HIV specific, in nearly all countries result is 100%Household surveys0%27%3%3%Corrected21. Prevalence and death rates associated with malaria21a. Malaria death rate per 100 000, ages 0--4 years[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeHighVital registration in countries with complete systems; in nearly all endemic countries, based on verbal autopsy data for demographic surveillance sites, or epidemiological models0%100%0%6%Predicted21b. Malaria death rate per 100 000, all ages[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeLow; death rates over the age 0--4 are very lowVital registration in countries with complete systems; in nearly all endemic countries, based on verbal autopsy data for demographic surveillance sites, or epidemiological models0%100%0%6%Predicted21c. Malaria prevalence, notified cases per 100 000 population[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeLow; notified cases are not a measure of prevalenceAdministrative data collected at public facilities0%55%0%4%Crude22. Proportion of population in malaria-risk areas using effective malaria prevention and treatment measures22a. Malaria prevention, use of insecticide-treated bed nets in population \<5, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageHighHousehold surveys0%18%3%2%Corrected22b. Malaria treatment, percentage of population \<5 with fever being treated with antimalarial drugs[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageModerate; resistance makes 'effective antimalarial drugs' better indicatorHousehold surveys, validity not established.0%18%4%2%Corrected23. Prevalence and death rates associated with tuberculosis23a. Tuberculosis death rate per 100 000[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeHighVital registration in countries with complete vital registration, models for all other countries98%98%98%31%Predicted23b. Tuberculosis prevalence rate per 100 000 population[\*](#tbl1fn1){ref-type="table-fn"}Health outcomeHighNo measurement strategy; modelled estimates based on case-notifications98%98%98%31%Predicted24. Proportion of tuberculosis cases detected and cured under directly observed treatment success (DOTS)24a. Tuberculosis, DOTS detection rate, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageHighHealth service provider registries for detected cases, no measurement strategy for denominator0%63%95%40%Predicted24b. Tuberculosis, DOTS treatment success, percentage[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageHighHealth service registries0%76%92%42%Corrected25. Proportion of population using solid fuels[\*](#tbl1fn1){ref-type="table-fn"}Risk factorModerate; real quantity of interest is indoor air pollutionHousehold surveys1%6%61%6%Predicted30. Proportion of population with sustainable access to an improved water source, urban and rural30a. Water, percentage of population with access to improved drinking water sources, rural[\*](#tbl1fn1){ref-type="table-fn"}Risk factorModerate; not clear that separate urban and rural indicators necessaryHousehold surveys: some issues in the consistent definition of "improved"7%0%0%11%Predicted30b. Water, percentage of population with access to improved drinking water sources, totalRisk factorHighHousehold surveys: some issues in the consistent definition of "improved"71%0%0%11%Predicted30c. Water, percentage of population with access to improved drinking water sources, urban[\*](#tbl1fn1){ref-type="table-fn"}Risk factorModerate; correlation with rural in 2002 is 0·69Household surveys: some issues in the consistent definition of "improved"84%0%0%12%Predicted31. Proportion of population with access to improved sanitation, urban and rural31a. Sanitation, percentage of population with access to improved sanitation, rural[\*](#tbl1fn1){ref-type="table-fn"}Risk factorModerate; not clear that separate urban and rural indicators necessaryHousehold surveys72%0%0%11%Predicted31b. Sanitation, percentage of population with access to improved sanitation, totalRisk factorHighHousehold surveys67%0%0%10%Predicted31c. Sanitation, percentage of population with access to improved sanitation, urban[\*](#tbl1fn1){ref-type="table-fn"}Risk factorModerate; not clear that separate urban and rural indicators necessaryHousehold surveys76%0%0%11%Predicted46. Proportion of population with access to affordable essential drugs on a sustainable basis[\*](#tbl1fn1){ref-type="table-fn"}Intervention coverageHighNo measurement strategy0%0%0%0%No dataAll official MDG indicators combined30%44%29%15%[^1] How well does the indicator measure the quantity of interest? {#cesec40} ------------------------------------------------------------- Since indicators can drive policy attention, resources, and implementation, every effort should be made to measure or at least estimate the real quantity of interest. For health-outcome indicators, does the proposed indicator measure the actual health outcome, a component of the outcome, or a proxy that is believed to be correlated to health outcome? In [table 1](#tbl1){ref-type="table"}, indicator 18c "HIV/AIDS prevalence rate for pregnant women 15--24 attending antenatal care in clinics in capital city", could be an important measure for the local HIV programme in the capital city, but this indicator is a poor measure of the real quantity of interest in the MDG framework---ie, national prevalence. Use of partial or proxy measures runs the danger that they slowly become the de facto quantity of interest in policy debates, distracting attention from the original objective of a policy or programme. Even when an indirect or proxy measure is the only option, mapping from the units of that measure into units of the quantity of interest is preferable---eg, ratios of children surviving to children ever born mapped into child mortality. For intervention coverage indicators, the ideal is to measure the proportion of potential health gain that is delivered for a specific health problem.[@bib13] The target for MDG4 is to reduce child mortality; the ideal intervention coverage would measure the proportion of child mortality reduction that could be achieved through the whole collection of affordable interventions that are delivered. Compared with this standard, measurement of only one intervention, measles immunisation in the MDGs, is capturing less than 5% of what can be delivered. If only one intervention is to be measured, preference should be given to the intervention that would have the biggest effect on the outcome if delivered. Because intervention coverage indicators have the potential to drive managerial attention and resource allocation, claims that part indicators such as measles immunisation are good proxies for the rest of child survival interventions are unproven. The potential for indicator-driven policy also argues for the use of composites capturing the coverage of a set of interventions targeting a health problem. Is the indicator value readily interpretable? {#cesec50} --------------------------------------------- There should be a monotonic relation between the value of the indicator and what is desired (all other things being equal). The child death rate is a monotonic indicator; lower rates are always better than are higher ones. However, the caesarean section rate is not a monotonic indicator. Too low and too high rates are equally bad, and there is no consensus on the optimum rate. Is there a practical measurement strategy? {#cesec60} ------------------------------------------ An indicator should not be a priority unless a measurement strategy that will produce valid, reliable, timely, and comparable measurements has been developed. This tenet does not mean that a measurement strategy has been implemented, otherwise, we would be trapped measuring only what has already been measured. Rather, it means that a plan has been developed. Although validity and reliability are familiar notions, comparability and timeliness have often not received adequate attention in the development of measurement strategies. Comparability is crucial---why measure an indicator over time and across countries if the measurements cannot be compared? Murray and colleagues[@bib14] emphasise the difference between validity and comparability by noting that two thermometers, one in Celsius and one in Fahrenheit, can both be valid and reliable but 26 degrees on each is not comparable. Unfortunately only nine of the 16 high-importance MDG indicators have a reasonably well developed measurement strategy that will yield valid, reliable, and comparable measurements. [Table 1](#tbl1){ref-type="table"} also emphasises that data availability overall for these health-related indicators is extremely poor, ranging from 0% for access to essential drugs to 100% for tuberculosis rate, although tuberculosis rate is almost entirely predicted from a model. For the health-related MDG indicators, overall availability of any type of statistics is 15% for 1990--2005. The 15% figure refers only to developing countries for all of the health-related MDG indicators. Full analysis of existing data sources in countries would probably expand this availability, but existing data are often not fully used within countries or for worldwide comparative studies. How should equity dimensions of an indicator be captured? {#cesec70} --------------------------------------------------------- Gwatkin[@bib15] has argued that average levels for an indicator can mask widening inequalities. To measure inequalities over time, disaggregation is necessary. Ideally, the total inequality of a health indicator across the population could be measured.[@bib16] Total inequality can be thought of as between-group inequality plus within-group inequality.[@bib17] For many indicators, however, only between-group inequality can be readily measured. The choice of groups for indicator disaggregation, such as rich and poor, urban and rural, occupation, race, or ethnic origin could profoundly change the comparisons of inequality across populations or over time. The disaggregation of an indicator that is proposed for monitoring inequalities should accord with evidence that this disaggregation captures the largest fraction of variance in the indicator across the populations being analysed. Correct use of crude, corrected, or predicted statistics {#cesec80} ======================================================== Boerma and colleagues[@bib7] have discussed the platform for primary data collection that provides the basis for all health statistics: essential registration systems, sample or sentinel registration systems, household surveys, censuses, budgets, and data collected by health-service providers. Primary data collection is only the first step in the generation of valid, reliable, and comparable statistics. The subsequent steps such as correcting for known biases or predicting out of sample or out of time can generate many different measurements. Health statistics can be divided into three types: crude, corrected, and predicted. Crude health statistics {#cesec90} ----------------------- Crude health statistics are the measurements of indicators that come directly from primary data collection with no adjustments or corrections. These figures are subject to many drawbacks, including incomplete ascertainment, non-representativeness, instrument bias, misclassification, and distortion. Incomplete ascertainment or incomplete coverage is a crucial difficulty for vital registration systems and data from health-service providers.[@bib18] Poor people and other disadvantaged groups often have the greatest health problems and do not get captured in these systems. For household surveys or other sampled data, non-representativeness can be a profound difficulty. For example, monitoring of HIV seroprevalence in antenatal clinics is often undertaken in clinics with known high prevalence.[@bib19] For many measurements and self-reported items on surveys, the instrument itself can be biased---eg, self-reported weights are systematically under-reported by women.[@bib20] Where events need to be classified into categories such as deaths according to the international classification of diseases and injuries, misclassification or inconsistent classification is a common drawback.[@bib21], [@bib22], [@bib23] Finally, where the stakes are high, producers of data might intentionally, or be encouraged to, distort data. Despite their restrictions, crude health statistics are proposed and used for monitoring. [Figure 1](#fig1){ref-type="fig"} shows the MDG indicator malaria prevalence (notified cases per 100 000) plotted against the prevalence of malaria parasites in blood as systematically reviewed by the MARA (Mapping Malaria Risk in Africa) project for sub-Saharan Africa.[@bib24] Although the prevalence of clinical cases of malaria would not necessarily be perfectly correlated with parasitaemia, the absence of any relation even on log scale suggests that because of variations in ascertainment this MDG indicator cannot be used for any interpretation. In some cases, such as complete vital registration systems, crude statistics can be unbiased and appropriate for analytical use. Since bias in crude statistics is expected, the burden of proof should be to show that crude data are in fact unbiased before they are used for comparative purposes.Figure 1MDG indicator malaria prevalence versus average parasite seroprevalence from MARA systematic review for selected sub-Saharan African countries Corrected health statistics {#cesec100} --------------------------- Corrected health statistics are measurements of indicators for which two types of analytical effort might have been undertaken: mapping to the quantity of interest and correction for a range of known biases. Mapping into the quantity of interest includes all the measurements where the primary data collected are on an indirect result of the event under study. For example, census or survey data on the responses of mothers regarding the number of children they have ever borne and the number that are alive are used in many countries to calculate mortality in children younger than 5 years. This mapping is based on certain assumptions and models;[@bib25], [@bib26] mapping into the quantity of interest introduces uncertainty because of parameter uncertainty, residual unexplained variance, and model choice. Correction for known bias ranges from routine procedures such as use of sample weights in household survey analysis to more complex procedures that could include analytical models. Demographers use methods[@bib27] to estimate the incompleteness of vital registration data and then apply these to obtain corrected mortality rates. Less formal or structured methods are routinely used to correct HIV seroprevalence data in antenatal clinics to generate national estimates to address non-representativeness.[@bib28] Correction for known bias is very important if valid, reliable, and comparable health statistics are to be generated; however, substantial scope for legitimate disagreement between analysts can be introduced. Often the details of efforts to correct for known bias are not in the public domain, which has stifled open debate on the best approaches to do such corrections. Corrections for known bias could in fact introduce more error into health statistics; therefore, open debate should be encouraged. The most technical approach to correcting for known bias is to systematically review and use all available primary data and attempt to reconcile differences between data sources.[@bib29], [@bib30], [@bib31] Systematic review and data reconciliation should not be confused with more qualitative triangulation.[@bib32] [Figure 2](#fig2){ref-type="fig"} provides another example of this approach by summarising all the available data on child mortality in Ghana and the corrected figures based on these data.[@bib27], [@bib33] An additional example of this approach is the work to develop internally consistent figures for incidence, prevalence, and death in the global burden of disease project.[@bib34], [@bib35] Software such as DISMOD II is used to identify inconsistencies between different data sources, which the analyst must then reconcile. The advantage of systematic reviews and data reconciliation is that all relevant information is used to correct for known bias. The disadvantage is that they can require substantially more analytical work.Figure 2All available empirical estimates of mortality rates for children younger than 5 years for Ghana and best estimatesCENi=1971 Census, indirect. DHSd93=1993 Demographic and Health Survey, direct. DHSi93=1993 Demographic and Health Survey, indirect. DHSd98=1998 Demographic and Health Survey, direct. DHSi98=1998 Demographic and Health Survey, indirect. DHSd03=2003 Demographic and Health Survey, direct. DHSi03=2003 Demographic and Health Survey, indirect. GDHSd88=1988 Ghana Demographic and Health Survey, direct. GDHSi88=1988 Ghana Demographic and Health Survey, indirect. GFSd80=1980 Ghana Fertility Survey, direct. GFSi80=1980 Ghana Fertility Survey, indirect. Predicted health statistics {#cesec110} --------------------------- Predicted statistics are based on a model relating the quantity of interest to covariates. Two types of predicted statistics are widely used. The first is forecasting, whereby a relation is established during a period of observation and then used to predict out of time into the future. A common use of forecasting is to update corrected statistics to a more recent period to produce series of comparable statistics for a base year, such as the MDG indicator data on maternal mortality ratio in 2000, or many statistics in the MDG database for indoor air pollution, water supply, or tuberculosis prevalence. Predictions are also frequently used to generate figures in settings where no primary data and thus no corrected statistics are available. Since the methods are identical to those for forecasting, prediction out of sample but in the same time period has been termed farcasting. An example of farcasting is MDG estimates of solid fuel use that have been based for many countries on models relating solid fuel use to GDP per capita in those countries with data. Predicted statistics have uncertainty resulting from model choice and parameter and unexplained variance in the model. [Figure 3](#fig3){ref-type="fig"} provides trends for maternal mortality from 1990 to 2000 for Afghanistan, Angola, Pakistan, Nigeria, South Africa, Burundi, and Algeria. These trends are not based on any empirical measurements and are strictly from predictive models. Nevertheless, the trends are different and show striking fluctuations. In Pakistan, the maternal mortality ratio is predicted to be increasing, although in Nigeria the ratio is predicted to be declining. Since these trends are not based on any real findings, to infer different amounts of progress on this MDG target would be completely without basis. The MDG database does not provide uncertainty for the trends in this indicator or other predicted indicators.Figure 3Trends in maternal mortality based strictly on predicted statistics for seven countries There is a grey zone where the corrections to primary data are substantial enough that they can become difficult to distinguish from predicted statistics. In a Bayesian context, predictions provide priors and the primary data with correction allow these priors to be updated. There are, however, cases where the recorded data give little information beyond the prior. When to use what {#cesec120} ---------------- Health statistics are necessary inputs to planning and strategic decision making, programme implementation, monitoring progress towards targets, and assessment of what works and what does not. Crude statistics that are biased have no role in any of these steps. They are, however, an important input to research and are the basis for corrected statistics with greater validity, reliability, and comparability. There is no cogent reason why crude statistics with known biases should be used when corrected statistics are available or can be developed. Nevertheless, several agencies report crude statistics. For example, the US Centers for Disease Control and Prevention report on levels of obesity by state with self-reported weights and heights in the behavioural risk factor surveillance system survey, which are known to underestimate obesity by roughly 50%.[@bib20] By reporting crude statistics, the onus for correction of known bias to allow for interpretation is shifted to the user. Although this approach makes the task easier for the data generator, it is predicated on a strange premise: the users, including policymakers and the concerned public, are in a better position to adjust for known bias than is the health statistician. In fact, the failure to correct for known bias underlies some of the most egregious examples of health statistics. Publication of government data in WHO\'s communicable disease global atlas[@bib36] on notified cases and deaths for rabies, cholera, sexually transmitted infections, and malaria are notable examples of the promotion of crude statistics. WHO claims that the atlas "is bringing together for analysis and comparison standardized data and statistics for infectious diseases at country, regional, and global levels".[@bib36] The style of presentation with maps encourages cross-country comparisons, but simple inspection shows that because of ascertainment bias, misclassification, and non-representativeness, the numbers are not valid, reliable, or comparable---eg, those for rabies for Iran and Pakistan differ by two orders of magnitude. Political arguments, such as the importance of respecting national sovereignty, are often given to explain the use of crude figures. But political pedigree is not a legitimate argument to favour one statistic over another. Frequently governments do have the best primary data and corrected statistics, but the preference for data from the government must be based on the merits of the data, not the politics. The importance of distinguishing political pedigree from substantive arguments about the reliability, validity, and comparability of health statistics is just as true nationally, when geographic subunits are being compared, as it is worldwide. For forward-looking decision making, advocacy for funds, planning, strategic decisions, research, and development investments, people who make decisions need the best available information. We cannot argue that we should allocate no resources to an area of need because good primary data and corrected statistics are not available. For these decisions, when corrected statistics are not available, predicted statistics can play an important part. The publication of the world population prospects every 2 years by the UN Population Division[@bib37] is a good example of the use of predicted statistics. This publication provides population, mortality, and fertility statistics for all countries from 1950 to 2050. For some countries, much of the trend from 1950 up to now is based on corrected statistics, but for many countries much or all of the sequence is based on predictions. Because the world population prospects is revised every 2 years, the entire set of figures from 1950 to 2050 is revised when new data become available. Even though much of the sequence is based on predictions, these basic demographic figures are crucial for various planning and strategic decisions. For monitoring progress towards agreed targets and assessment of what is effective and what is not, the case for using predicted statistics is unconvincing. Nationally or locally, the reason for monitoring is to find out whether present policies and programmes are leading us in the right direction or to identify when unexpected factors are changing trends. If data for monitoring are predicted and the prediction model explains much of the observed variance, the predicted statistics can correctly identify the true trend in many cases. However, the prediction is not sensitive to the actual policies and programmes that have been pursued. That statistics used for monitoring have zero probability of detecting an unexpected trend does not seem right. Imagine enrolling your child in a school, which then hands you the results of your child\'s examination scores for the end of the year on the first day of attendance. These results have been forecast by a very good model and might be right most of the time. However, the grades or assessment are not affected in any way by what your child does during the year. A test for the use of statistics for monitoring and assessment should be that the statistics in question have a reasonable probability of detecting real changes in the quantity of interest. This concern is great when we intend to assess what works and what does not; here predicted statistics should have no role. Because predicted statistics have a zero probability of detecting changes from an abrupt adjustment in policy, they should not be used for this type of monitoring and assessment. Predicted statistics, however, have been substantially used in monitoring. For example, 20 of the health-related MDG indicators in [table 1](#tbl1){ref-type="table"} use some predicted statistics. The World Health Report in 2000 for health system performance used predicted statistics extensively.[@bib38] Two types of argument have been made to support the use of predicted statistics for monitoring. First, several countries in greatest need will have little or no data to report, and if no statistics are reported the topic will lose policy attention. Second, worldwide advocacy needs information on global monitoring. Although the use of predicted statistics to bring attention to a difficulty seems warranted both nationally and internationally, the temptation to use such information once produced for actual monitoring and even assessment is very great. I believe that if predicted statistics are to be used in such cases, the user must make it very clear that predicted statistics are being used and this use should not become a routine activity. Therefore, publication of databases of annual predicted figures, such as for some MDG indicators, does not seem justified. Reported indicator series should, at an absolute minimum, indicate whether figures are based on crude, corrected, or predicted values. Regional or worldwide aggregates are often based on mixtures of crude, corrected, and predicted data, and the relative proportion of the component national figure based on these three types should be shown to the user. The need for explicit data audit trails {#cesec130} ======================================= Perhaps the most effective method by which to decrease controversy over health statistics and to encourage better primary data collection and development of better analytical methods is a strong commitment to provision of an explicit data audit trail. This method would make primary data, all post-data collection adjustments, models including covariates used for farcasting and forecasting, and necessary documentation available to the public. An explicit data audit trail would allow health statistics to be subject to the scientific principle of replication. A sceptical user should be able to reproduce every figure including all steps along the way. Decision makers, the media, or the public are unlikely to use the explicit data audit trail, but the requirement to publish this trail will over time lead to improved practice. The expectation that peers in the technical community will be able to critique methods, suggest other data sources, and even generate alternative figures will provide a powerful incentive for improved measurement. Few in the international community are opposed to the idea of an explicit data audit trail. There are three difficulties, however, that have restricted the adoption of this approach. First, documentation of all adjustments for known bias and the use of farcasting and forecasting models is time-consuming and expensive. Second, transparency about how some estimates are generated can increase debate about the validity of figures in the short term, which is a concern for some programme managers. Third, the component of the explicit data audit trail that calls for primary data to be accessible to the public or at least to interested analysts might conflict with national privacy laws governing data collection, which is a very important issue. In some countries such as the USA, data are becoming harder to obtain. For example, the National Center for Health Statistics (NCHS) has stopped releasing mortality data for counties because of privacy concerns. Increasing concerns about privacy are in direct conflict with another international trend towards more countries adopting freedom of information laws. In 2002, Mexico passed a sweeping freedom of information act and set up a Federal Institute for Access to Public Information about Mexico, which guarantees unprecedented access to any information held by the government.[@bib39] In India, the Right to Information Act of 2005, seems to provide similar access to information.[@bib40] Various other countries are pursuing similar legislation.[@bib41], [@bib42] Balancing privacy concerns with the need for information to be in the public domain to create a culture of transparency will be a major challenge in the coming decades. Once individual identifiers such as names and addresses have been removed from survey, census, or vital registration data, privacy concerns revolve around the possibility that, with some investigation, individuals could be identified.[@bib43] For example, perhaps only one 84-year-old woman died in Omaha county, USA, so that the analyst could with further investigation find the name of this individual and what she died of. Such a hypothetical possibility must be balanced against the damage to the community by suppression of health information that could improve population health. In the case of death data in the USA, the NCHS position is hard to understand since death certificates for all individuals including names are public records that can be obtained in every state. Clearly, there are important legitimate concerns about data privacy. However, in some cases arguments about data confidentiality are used by institutions to avoid the release of data to other groups. Datasets are often viewed by researchers or organisations as private possessions. Data, of course, are a classic example of a general public good. There is a potential cost that could ultimately be counted in human health of keeping data from the public domain. The principle of an explicit data audit trail and replicability might also be preserved if mechanisms exist to allow restricted access to various datasets with stronger safeguards. Journals such as *The Lancet*, funding agencies, and governments all have an important part to play in transformation of the culture of data from a feudal to an open democratic model. Any efforts that move towards an explicit data audit trail should be encouraged, including intermediate steps such as provision of detailed information on primary data sources and types of adjustments that have been undertaken. Conclusion {#cesec140} ========== Several good practice recommendations for health indicators and their measurement follow from this discussion. These recommendations could lead over time to a more focused effort on production of valid, reliable, and comparable information to serve many information needs. National and worldwide efforts to improve health statistics should focus on a smaller set of priority indicators rather than the thousands that are currently recommended. Priority health indicators should be selected on the basis of public-health importance and the existence of a pragmatic measurement strategy. I believe that focusing attention within and across countries on a restricted set of priority indicators will help show how measurement can foster health improvement. Analyses based on these priority indicators can foster demand from decision makers, the media, civil society, the technical community, and the public for better health measurement. Efforts to strengthen platforms for primary data collection driven by this demand are more likely to gain broad support than are calls for strengthening these platforms that are not linked to specific analytical uses. The measurement strategy should be designed to produce valid, reliable, and comparable information. If the results of measurement are not comparable over time and across places, they will not be useful for monitoring or assessment. Comparability has not been given sufficient attention in the development of measurement strategies. Furthermore, every effort should be made to produce statistics for the true quantity of interest instead of proxies. Proxy indicators should be mapped into the quantity of interest and the uncertainty in that mapping quantified. Crude statistics from primary data collection platforms should be reported as a resource for the analysis and research community, but should be clearly distinguished from corrected statistics for interpretive purposes. The most detailed information possible, including metadata and wherever possible microdata, should be in the public domain. Statistics used for monitoring and assessment should always be corrected for known biases, and the basis for these corrections should be in the public domain. Predicted statistics should in general not be used for actual monitoring of national or local progress, and should never be used for assessing what works and what does not. Predicted statistics have an important and useful role in helping to inform planning, strategic decision making, and research and development prioritisation when corrected statistics are unavailable. Whenever predicted statistics are used, however, efforts should be made to adequately characterise the uncertainty in the predictions from all sources so that they are interpreted with caution. All statistics produced for all purposes should have a well documented, explicit data audit trail, which allows a sceptical scientist to entirely replicate the generation of the corrected or predicted statistics. Due attention should be given to protection of public interest in transparency and accountability that derives from having data in the public domain. The MDG health-related indicators have been used to draw attention to many of the points in this paper. Overall, the set of indicators, the measurement strategies, and the implementation of the MDG health-related indicators is very poor. At least half the indicators are not of high enough public-health importance to warrant major international attention or investments in their assessment. Data availability for developing countries overall is 15%, and even in 2000 when a special effort was made, it reached 44%. Most of these figures are predicted statistics that do not provide a reasonable basis for monitoring progress. The dilemma is that data systems and measurements are so weak that only undernutrition, child mortality, measles immunisation, and attended deliveries have effective measurement strategies in place. WHO, UNICEF, UNAIDS, and other agencies responsible for monitoring the MDGs rely on using predicted statistics for most other indicators so that attention on the MDGs does not fade. Predicted statistics are a reasonable approach for identification of difficulties and for stimulation of policy interest. However, the worldwide community is failing in implementation of viable measurement strategies for real monitoring. I was former Executive Director of the Evidence and Information for Policy Cluster at the World Health Organisation. I have no conflicts of interest that may have influenced this work or the conclusions of the manuscript. [^1]: Official MDG indicators. All others are official supplemental series.

1. Introduction =============== Although hypertension (HTN) is a well-known risk factor for cardiovascular disease (CVD), studies on the effect of each blood pressure (BP) component on CVD have been inconsistent,^\[[@R1],[@R2]\]^ Borghi et al[@R3] demonstrated that, in a European cohort, systolic BP (SBP) was a stronger predictor of coronary heart disease (CHD) and cerebrovascular events compared with diastolic BP (DBP), and this result has been generally consistent in other reports,^\[[@R4],[@R5],[@R6],[@R7]\]^ pointing to the importance of SBP. In contrast, data from the Framingham cohort with a mean age of 61 years, SBP was directly related to, but DBP was inversely associated with, the incidence of CHD risk, which means that pulse pressure (PP: SBP--DBP) is the strongest predictor among the three BP components (SBP, DBP, and PP).^\[[@R8],[@R9],[@R10]\]^ On the other hand, Glynn and colleagues[@R11] described that SBP is more important than DBP, even in a younger population, but they could not show that PP was the most powerful predictor of adverse clinical events in the younger population. Moreover, a few analyses had also focused on the role of mean arterial pressure (MAP) and PP in CVD,^\[[@R6],[@R7]\]^ and several studies using different methodologies have demonstrated the relative importance of all 4 BP components. Therefore, we determined that the effect of all 4 BP components on CV events may need to be investigated case by case, that is, subjects with normal-range BP with prehypertension and with HTN. As a study on the impact of BP components on CV events among prehypertensive individuals was published,[@R12] a study on the effect of each BP component on the risk of CV complications among hypertensive populations is necessary. In addition, the prevalence of HTN and incidence of CV events are different based on sex. A study at Wake Forest Baptist Medical Center of 100 hypertensive subjects with a mean age of 53 years showed 30 to 40 percent higher incidence of vascular disease in women than in men, despite no differences between males and females in terms of BP, heart rate (HR), and body mass index (BMI). The study also found significant differences in the hemodynamic and hormonal mechanisms accounting for the increased BP in women compared to men. Therefore, physicians may need to choose selective BP management considering the age and sex of their patients. Hence, the aim of the current study was to investigate and compare the usefulness of each BP component based on stratification by age and sex for predicting CV events among 22,853 hypertensive patients without chronic kidney disease (CKD) or diabetes mellitus (DM). 2. Materials and methods ======================== 2.1. Health examination sample cohort database ---------------------------------------------- In Korea, the National Health Insurance Service (NHIS) provides general health examinations to several populations: 1. regional insurance subscribers that include a regional household member 40 years or older and other household members, 2. employee subscribers who are 40 years or older and their dependents, and 3. medical aid beneficiaries who are household representatives 19 to 64 years of age and other household members 40 to 64 years of age.[@R13] With the exception of non-office workers, these individuals are asked to undergo biennial health medical examination; non-office workers are required to undergo annual examinations. The health examination sample cohort database included 10% of all participants who underwent a health examination provided by the NHIS during calendar years 2002 and 2003, using simple random sampling, and they were followed until December 31, 2013.[@R14] The number of participants that received health examinations among the general same-aged population in Korea was approximately 40% in 2002 and increased up to 68% in 2013. 2.2. Participants ----------------- We enrolled participants that underwent medical examinations provided by the Korea Medical Insurance Corporation (KMIC) between January 1, 2003 and December 31, 2004. These examinations are referred to as the "baseline". Among the people who received a medical examination during this timeframe, a total of 30,080 people were found to have HTN, we excluded 2230 people that had been diagnosed with current or past CKD and had received chronic dialysis or kidney transplantation at the enrollment point. Another 209 participants were excluded because they had experienced prior CV events, and 4788 people were excluded due to current or past DM. Finally, a total of 22,853 participants with HTN were involved in this study (Fig. [1](#F1){ref-type="fig"}). The study protocol was approved by the Institutional Review Board of Ewha Womans University, College of Medicine (EUMC 2018--01--039), and informed consent was waived because of retrospective study. {#F1} 2.3. Data collection -------------------- Demographic and clinical data that were recorded during the health examination included age, sex, BMI calculated as weight/height^2^ and the Charlson Comorbidities Index (CCI).[@R15] BP was measured at mobile examination centers by trained examiners. SBP and DBP were measured three times, and the average of second and third measurements was used for analysis.^\[[@R16],[@R17]\]^ MAP was calculated as 2/3 DBP + 1/3 SBP, and PP as SBP- DBP. The following laboratory data were measured as part of the health examinations: fasting blood glucose, total cholesterol, hemoglobin, and proteinuria with the dipstick test \[(−), +, 1+, 2+, or over 3+\]. 2.4. Definitions ---------------- 1. HTN was defined on the basis of a prior diagnosis of HTN, prior use of any antihypertensive medications within six months of the defined enrollment time, or as measured BP ≥ 140/90 mmHg.[@R18] 2. DM subjects were defined as those with at least two recorded prescriptions of insulin or oral hypoglycemic agents with a diagnosis code of "E10--14 \[International Statistical Classification of Disease and Related Health Problems (ICD)-10code\]".[@R19] 3. CV endpoints consisted of new onset mortality or hospitalization due to myocardial infarction, angina, or acute coronary syndrome.[@R20] 4. Hospitalization was defined as admission to a hospital for treatment lasting more than 2 days. 5. Smoking status was defined based on guidelines from the Centers for Disease Control and Prevention (<https://www.cdc.gov/nchs/nhis/tobacco/tobacco_glossary.htm>). 1. A current smoker was defined as an adult who had smoked 100 cigarettes in his or her lifetime and who currently smokes cigarettes. 2. A former smoker was defined as an adult who had smoked at least 100 cigarettes in his or her lifetime but who had quit smoking at the time of the interview. 3. A never smoker was defined as an adult who had never smoked or who had smoked less than 100 cigarettes in his or her lifetime. 2.5. Study outcomes ------------------- All study participants were monitored for CV events during 9 years of follow-up, from January 1, 2005 through December 31, 2013. 2.6. Statistical analysis ------------------------- Continuous variables were expressed as mean ± standard deviation and categorical variables as number (percentage). Continuous variables were compared using the independent Student's *t* test, and categorical variables were compared with Pearson chi-square test. Then, we stratified the patients into 3 groups according to age \[40--49, 50--59, and 60 years or older\] and sex \[male and female\], respectively. Among the groups, the crude incidence rates were calculated by dividing the number of subjects with a given event by the person-years of follow-up, which were expressed as cases per 1000 person-years; confidence intervals (CIs) were estimated based on a Poisson distribution. In addition, a Cox proportional hazards analysis was performed to examine the time-to-event association with CV events. We selected adjusted covariates using stepwise selection for analysis of age, sex, total cholesterol level, smoking status, and CCI using a multivariate model. This analysis was conducted using R 3.2.3 software (R Development Core Team, 2013) and SAS 9.4 software (SAS Institute Inc., Cary, NC). The *P* value less than .05 was considered statistically significant. 3. Results ========== 3.1. Baseline characteristics ----------------------------- Among the 22,853 subjects, the mean age was 51.1 ± 8.87 years and 69.2% were male. The mean BMI was 24.4 ± 2.9 kg/m^2^. Out of the total individuals, 106 (0.5%) had cancer as a co-morbidity, congestive heart failure (CHF) was diagnosed in 98 (0.4%), and only four were treated for acute myocardial infarction (AMI). In addition, mean SBP, DBP, PP, and MAP were 140.9 ± 13.9, 89.8 ± 9.0, 51.2 ± 12.6, and 106.8 ± 9.14 mmHg, respectively. At the initial health examination, 5,945 (26%) subjects were current smokers, and the number of non-smokers was 15,996 (70.0%), including past smokers. Laboratory data indicated that mean fasting blood glucose and total cholesterol concentration were 89.8 ± 10.4 and 200.4 ± 36.8 mg/dL, respectively, the mean hemoglobin level was 14.3 ± 1.5 g/dL, and CCI was 0.38 ± 0.70. Additionally, 377 (1.7%) hypertensive patients had proteinuria with a score greater than 1+ \[1+: 264 (1.2%), 2+: 96 (0.4%), and over 3+: 17 (0.1%)\] (Table [1](#T1){ref-type="table"}). Subjects were stratified based on clinical outcomes of CV events. The non-event group included those in which CV events did not occur during the study period, and the event group included those in which CV events occurred during the study period. Study results demonstrated that SBP and PP were significantly elevated in the CV event group (142.8 mmHg SBP and 54.3 mmHg PP, respectively) compared with the CV non-event group (140.8 mmHg SBP and 51.0 mmHg PP), while DBP and MAP were significantly lower in the CV event group (88.5 mmHg in DBP and 106.6 mmHg in MAP) compared to the CV non-event group (89.8 mmHg in DBP and 106.8 mmHg in MAP). Moreover, the hypertensive patients who experienced CV events were significantly older (age 57.0 vs 50.6 years), had a history of previous AMI and CHF, and had a higher CCI (0.62 vs 0.36) compared with the hypertensive patients without CV events (Table [1](#T1){ref-type="table"}). There was a significant difference in smoking status, while there was no significant difference in proteinuria level between the two groups. In the laboratory findings, the average total cholesterol level was significantly higher in the CV event group compared with the CV non-event group (203.5 vs 200.2 mg/dL), whereas the mean hemoglobin level was significantly lower in the CV event group compared to the CV non-event group (14.2 vs 14.3 g/dL) (Table [1](#T1){ref-type="table"}). ###### Baseline characteristics.  3.2. The effects of baseline BP components on CV events among the hypertensive population ----------------------------------------------------------------------------------------- During the median 9 years of follow-up, the incidence rate of CV events was 7.56 (95% CI, 7.18--7.95) per 1000 person-years. When these patients were classified into 2 groups according to sex (male vs female) and then each stratified into 3 groups according to age (40--49, 50--59, and over 60), the crude incidence rates of CV events in ages 40--49, 50--59, and over 60 years in males were 4.43 (95% CI, 3.99--4.89), 8.17 (95% CI, 7.21--9.22), and 2.01 (95% CI, 1.82--2.22), respectively; those in females were 1.13 (95% CI, 1.09--1.17), 6.01 (95% CI, 4.98--7.19), and 1.68 (95% CI, 1.48--1.90) (Table [2](#T2){ref-type="table"}). ###### Incidence rate of cardiovascular events per 1000 person-years.  In univariate Cox proportional regression analyses, a 10 mmHg increase of SBP and PP was significantly associated with the occurrence of more CV events \[10 mmHg increase of SBP: hazard ration (HR) = 1.105, 95% CI (1.067--1.144), *P* \< .001; 10 mmHg increase of PP: HR = 1.202, 95% CI (1.160--1.247), *P* \< .001\]; while 10 mmHg increase of DBP was significantly related to reduced risk of CV events \[10 mmHg increase of DBP: HR = 0.866, 95% CI (0.818--0.916), *P* \< .001\] and 10 mmHg increase of MAP could not observed significant association with the incidence of CV events \[10 mmHg increase of MAP; HR = 0.965, 95% CI (0.912 -1.022), *P* = .223\] (Table [3](#T3){ref-type="table"}). However, multivariate Cox proportional regression analyses revealed that the HR of 10 mmHg increase in PP was 1.063 (95% CI; 1.022--1.106, *P* = .002), but the HR of 10 mmHg increase in DBP was 0.923 (95% CI; 0.873--0.976, *P* = .005) for the risk of CV events after adjusting for age, sex, BMI, total cholesterol concentration, smoking status, and CCI. SBP and MAP were not significantly associated with increased incidence of CV events (Table [3](#T3){ref-type="table"} and Fig. [2](#F2){ref-type="fig"}). ###### Multivariate Cox proportional regression analysis of each blood pressure component for cardiovascular events.  {#F2} 3.3. The different effects of baseline BP components on CV events according to age and sex among the hypertensive population ---------------------------------------------------------------------------------------------------------------------------- Table [4](#T4){ref-type="table"} illustrates that an increase in PP or decrease in DBP was significantly associated with higher occurrence of CV events in males, even after adjusting for age, BMI, total cholesterol concentration, smoking status, and CCI (PP; HR = 1.102, 95% CI; 1.051--1.156, *P* \< .001, and DBP; HR = 0.912, 95% CI; 0.851--0.978, *P* = .010), while an increase in SBP was marginally related to increase in risk of CV events (HR = 1.043, 95% CI; 0.998--1.090, *P* = .059) and an increase in MAP could not observe the association with incidence in risk of CV events (HR = 0.975, 95% CI; 0.910--1.0432, *P* = .461) (Table [4](#T4){ref-type="table"} and Fig. [2](#F2){ref-type="fig"}). In contrast, there was no significant association in females between change of each BP component and incidence of CV events (Table [4](#T4){ref-type="table"} and Fig. [2](#F2){ref-type="fig"}). ###### Multivariate Cox proportional regression analysis of each blood pressure component for cardiovascular events according to sex.  When these patients were stratified according to age and sex, a 10 mmHg increase in SBP or PP was significantly related to increase in risk of CV events among the 40- to 49-year-old male group (SBP; HR = 1.114, 95% CI; 1.031--1.203, *P* = .006, and PP; HR = 1.158, 95% CI; 1.061--1.264, *P* = .001). A 10 mmHg elevation of PP was significantly associated with increased incidence of CV events among the 50- to 59-year-old male group (HR = 1.161, 95% CI; 1.048--1.286, *P* = .004), whereas a decrease of DBP was significantly associated with increased risk of CV events among the 60-year-old and older male group (HR = 0.879, 95% CI; 0.795--0.972, *P* = .012) (Table [5](#T5){ref-type="table"} and Fig. [3](#F3){ref-type="fig"}). However, there was no significant relationship in the female groups between change in BP components and occurrence of CV events, and we could not observe the significant association of MAP with the incidence of CV events, even in the sub-analysis after being stratified by age and sex. (Table [5](#T5){ref-type="table"} and Fig. [3](#F3){ref-type="fig"}) ###### Multivariate Cox proportional regression analysis of each blood pressure component for cardiovascular events according to age group and sex.  {#F3} 4. Discussion ============= Consistent with other studies,^\[[@R4],[@R5],[@R6],[@R7]\]^ we found that, for hypertensive male subjects, the importance of an increase in SBP and PP for risk of CV events was demonstrated in the younger generation; however, a decrease in DBP was significantly associated with increase in the incidence of CV events in older generations. However, there was no significant association between variation in BP components and CV events in hypertensive female subjects irrespective of age stratification and there were no significant associations of MAP in sub-analysis after stratification according to age and sex. The reasons why there was no significant relation between change of BP components and CV events in the hypertensive female populations have not been elucidated. However, it is well-known that there are differences in prevalence of HTN and incidence of CV events between men and women.[@R21] Moreover, women are considered to be protected from most CV events compared to men, and the risk for CV events accelerates after menopause. Therefore, postmenopausal women are at increased risk of CV complications compared to premenopausal women.[@R22] In our study, the crude incidence rates for CV events were lower in female groups compared with male groups, as in other studies (Table [2](#T2){ref-type="table"}),[@R21] which might result in no significant association between variation in BP components and occurrence of CV events in female groups. However, the relative ratio (RR) of incidence rate for CV events between older males and females was greater than that in the younger age groups (RR in 60 years or more: 0.84, RR in age 50 s group: 0.74, and RR in age 40s group: 0.26) when we examined the RR as incidence rate of CV events in the female group incidence rate of CV events in the male group at the same age. This indicates that the occurrence of CV events in hypertensive females after menopause rapidly increased compared to that in hypertensive males of the same age. Moreover, in women aged 65 years or older, the prevalence of HTN was higher than that found in men aged 65 years or older in 2007 (61.8% in women vs 49.3% in men) and 2011 (68.9% in women vs 58.4% in men).[@R22] Nevertheless, we could not find as significant association between change of BP components and risk of CV events even in the 60 years and older, hypertensive female population. The pathophysiological mechanisms for sexual differences in the prevalence of HTN and incidence of CV events have not been established but are not likely as simple as the presence or absence of estrogens, since hormone replacement therapy in older women in the Women\'s Health Initiative or the HERS Trials did not provide primary or secondary prevention. However, our results demonstrate that BP management should be selectively performed according to age and sex to reduce CV events among hypertensive populations. In this study, a 10 mmHg increase of PP was significantly associated with increase in incidence of CV events among the total hypertensive population. Several pathogenic mechanisms for the relationship between PP and CV events have been explained as due to increased arterial stiffness, which causes SBP to increase and DBP to decrease by shifting the wave reflection from diastole to systole.[@R23] SBP elevation has deleterious effects on myocardial oxygen demands, and a reduction of DBP might compromise diastolic perfusion time and coronary perfusion. Finally, widened PP can lead to presence of a clinical or subclinical comorbid condition, such as aortic insufficiency.[@R24] However, in our study, there was no significant association between increase in SBP and risk of CV events, which indicates that diastolic perfusion time and coronary perfusion may be more of an issue for the occurrence of CV events among hypertensive populations compared with myocardial oxygen demands. In Korea, a routine health examination is recommended every other year for those over 40 years of age, and BP is always measured during these examinations.^\[[@R13],[@R14]\]^ However, it is still controversial whether a health examination is beneficial based on cost-effective analysis. Unfortunately, we did not examine serial BP variations or investigate the effect of variability of BP components on the risk of CV events. Thus, it is hard to emphasize the benefit of periodic health examinations based on study results. Nevertheless, this study implies that physicians may need to choose selective BP management based on baseline BP and considering the age and sex of their patients. There were several limitations to our study. First, the health examination sample cohort database included 10% of all participants who underwent a health examination provided by the NHIS from January 1, 2002 through December 31, 2003, using simple random sampling and who were followed until December 31, 2013. Thus, these data do not represent the entire healthy Korean population. Second, the study was a retrospective cohort observational study. Thus, selection bias could not be excluded. In the future, interventional prospective studies will be required to confirm these results. Third, we could not measure serial BP variations individually. Thus, there is a limitation to showing how to manage BP in hypertensive populations. Fourth, there was a relatively small number of women (30.8%) compared to men (69.2%). Thus, future investigation with larger number of women is needed to delineate the effects of BP components in the female hypertensive group. Despite these limitations, the study is the first, to our knowledge, to investigate BP components according to age and sex among hypertensive populations without CKD or DM. This study suggests that BP management in hypertensive populations may need to be performed after full consideration of patient age and sex. However, an interventional, prospective study with larger populations is needed to confirm these results. Author contributions ==================== **Conceptualization:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Dong-Ryeol Ryu. **Data curation:** Eungyu Kang, Seulbi Lee, Eunhee Ha. **Formal analysis:** Eungyu Kang, Seulbi Lee, Eunhee Ha. **Investigation:** Eungyu Kang, Seulbi Lee, Eunhee Ha. **Methodology:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Dong-Ryeol Ryu. **Project administration:** Hyung Jung Oh, Dong-Ryeol Ryu. **Software:** Hyung Jung Oh, Seulbi Lee, Eunhee Ha. **Supervision:** Hyung Jung Oh, Dong-Ryeol Ryu. **Validation:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Dong-Ryeol Ryu. **Visualization:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Dong-Ryeol Ryu. **Writing -- original draft:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Eunhee Ha, Dong-Ryeol Ryu. **Writing -- review & editing:** Hyung Jung Oh, Eungyu Kang, Seulbi Lee, Eunhee Ha, Dong-Ryeol Ryu. Abbreviations: BP = blood pressure, CKD = chronic kidney disease, CV = cardiovascular, DBP = diastolic blood pressure, DM = diabetes mellitus, MAP = mean arterial pressure, PP = pulse pressure, SBP = systolic blood pressure. EK and SL contributed equally to this work. This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2010--0027945). It was also supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI18C0844). The authors have no conflicts of interest to disclose.

Background ========== Lung injury and edema are well-documented consequences of mechanical ventilation with high distending pressures in multiple experimental models \[[@B1]-[@B3]\]. It has been observed that maintaining end-expiratory lung volume, at some level above functional residual capacity, with positive end-expiratory pressure (PEEP) can prevent/reduce this ventilator-induced lung injury (VILI). Multiple animal models employing high distending pressures and/or volumes have demonstrated marked reductions in lung injury when adequate PEEP is applied \[[@B1]-[@B5]\]. In a surfactant-depletion model, lung injury occurs in the absence of large tidal volumes and distending pressures if inadequate or absent PEEP is used \[[@B6]\]. However, applying PEEP above the lower inflection point of the inspiratory pressure-volume curve protected against injury \[[@B6]\]. The protective effect of PEEP has been attributed primarily to prevention of repeated airway collapse and expansion (RACE) \[[@B6],[@B7]\], and to a lesser extent limitation of tidal excursion, and reduced cardiac output \[[@B8]\]. These observations, although not always directly translatable to the clinical management of humans with lung injury, shed light on how management of mechanical ventilatory support might potentially impact patient outcomes. It has recently been observed that outcomes can be improved in ARDS patients if distending pressures are limited by tidal volume reduction \[[@B9],[@B10]\]. Although improved outcomes in ARDS patients ventilated with PEEP set above the lower inflection point of the inspiratory pressure-volume curve has been reported \[[@B10]\], a recent multi-center trial failed to show a survival advantage with a high-PEEP ventilation strategy \[[@B11]\]. Similar outcomes have been observed in two subsequent trials employing either oxygenation-based \[[@B12]\] or plateau pressure-based \[[@B13]\] PEEP protocols. However, another recent study documented significant heterogeneity in how patients with ALI/ARDS respond to higher PEEP levels \[[@B14]\]. They identified two populations of patients: those with a significant recruitable lung volume, and those with negligible recruitable volume. These intra-patient differences may suggest that some patients and not others may benefit from higher PEEP. Additionally, a recent study by Talmor and colleagues \[[@B15]\] examined the effect of higher PEEP based on a trans-pulmonary pressure-based protocol and found a strong trend toward improved survival in the higher PEEP group. The RACE hypothesis has been called into question recently, favoring the tidal movement of fluid and/or foam in the airways as an explanation for the mechanical behavior of the injured lung during positive pressure ventilation \[[@B16]\]. Also, a recent model of saline lavage lung injury found that high tidal volume/low PEEP ventilation resulted in lung injury in the non-dependent lung regions in supine rats, suggesting atelectasis in the dependent lung zones shifts stretch-induced injury to the non-dependent lung and argues against repetitive collapse and expansion as a cause of VILI \[[@B17]\]. The current technological limitations of available imaging modalities preclude accurate real-time imaging of all but the most peripheral alveoli, therefore whether alveoli open and close during mechanical ventilation remains a point of contention. We have recently shown in a rabbit model of VILI that lung injury is greater and more spatially variable with a dorso-caudal gradient in the absence of PEEP\[[@B18]\]. One potential mechanism for this finding is regional repeated airway collapse and expansion in dependent lung areas during tidal breathing promotes subsequent injury. We hypothesize that 1) cyclical airway closure and expansion or RACE occurs in the lungs of anesthetized rabbits mechanically ventilated in the supine posture without PEEP and 2) RACE would occur regionally in the dorso-caudal lung regions, spatially correlating with the previously reported regional distribution of VILI \[[@B18]\]. To test this hypothesis, we measured how relative ventilation regionally redistributes with increasing tidal volume in the lungs of mechanically ventilated rabbits by measuring the distribution of aerosolized fluorescent microspheres. This ventilation redistribution with increasing tidal expansion of the lungs is referred to as sequential ventilation and reflects dynamic regional changes in lung mechanics during inspiration. By measuring regional ventilation redistribution with increasing tidal volume under different PEEP conditions, we could infer whether dynamic regional changes in lung mechanics were related to changes in the lower part of the pressure-volume curve (i.e. cyclical airway closure at FRC followed by expansion and increased ventilation during inspiration) or to changes at the upper part of the pressure volume curve (i.e. alveolar overdistension and regionally reduced compliance resulting in decreased regional ventilation). Methods ======= Animals Preparation ------------------- The University of Washington Animal Care Committee in accordance with National Institutes of Health guidelines approved all methods. New Zealand white rabbits (either sex, 2.4 to 2.8 kg) were sedated with intramuscular ketamine (30 mg/kg) and xylazine (7.5 mg/kg) to allow placement of a 20 ga catheter in each marginal ear vein. A surgical plane of anesthesia was then maintained with a continuous intravenous infusion of ketamine (0.05 mg/kg/hr) and xylazine (0.003 mg/kg/hr) for the remainder of the protocol. A 3.5 mm cuffless endotracheal tube was inserted via tracheotomy to allow positive pressure mechanical ventilation. Arterial catheters were placed in the left carotid (blood gas sampling and arterial pressure measurement) and right femoral (thermistor tipped catheter for thermodilution cardiac output) arteries. A catheter was inserted into the right internal jugular vein for pressure monitoring and administration of thermodilution injectate. Pancuronium bromide (0.15 to 0.2 mg/kg) was administered intravenously, after adequate anesthesia was established, to suppress spontaneous respiratory efforts. A 30-min stabilization period followed the completion of surgical preparation, after which baseline data were collected. During this period, animals were ventilated in pressure control mode (Servo 900 C; Semens-Elema, Stockholm, Sweden) with a 50% inspiratory time and no inspiratory pause. Tidal volume was set at 10 - 12 ml/kg, PEEP = 5 cm H~2~O and a respiratory rate to achieve P~a~CO2 = 35 - 45 mmHg. Physiologic Measurements ------------------------ Data were recorded using Powerlab data acquisition software (AD-Instruments Castle Hill, New South Wales, Australia). Blood pressures (systemic arterial and right ventricular), heart rate, duplicate thermodilution cardiac output (Baxter Edwards SAT-2 Oximeter/Cardiac Output Computer, Irvine, CA), arterial blood gases (Radiometer ABL 5, Copenhagen, Denmark) and ventilatory parameters were measured for each experimental condition after a 20-minute stabilization period. An in-line spirometer (KORR RSS 100; Medical Technologies Research Spirometry System, Salt Lake City, UT) was used to measure airway pressures and tidal volume. Plateau pressure was measured as the pressure achieved at the end of a 5-second end-inspiratory hold maneuver. Sequential Ventilation Measurement ---------------------------------- On completion of surgery, stabilization, and collection of baseline data, each animal (n = 5) was subjected to 4 different experimental conditions combining high (12 ml/kg) or low (6 ml/kg) tidal volume with 8 cm H~2~0 or 0 cm H~2~O PEEP (Table [1](#T1){ref-type="table"}). The rationale for choosing these four conditions is as follows. In the absence of PEEP, if dorso-caudal lung regions are closed at FRC and then subsequently recruited at some point during a tidal inspiration, relative ventilation to these regions will be greater with a larger tidal volume because the regions are open for a greater percentage of inspiration as compared with a smaller tidal volume. Thus, when measuring relative ventilation distribution at these two tidal volumes, there will be greater ventilation to the dorso-caudal regions and correspondingly less ventilation to ventro-cranial regions with the larger tidal volume. In contrast, if PEEP is set sufficiently high as to prevent cyclical airway collapse at FRC, relative regional ventilation distribution should be similar between a smaller tidal volume and a larger tidal volume. ###### Physiological Response to Varying Mechanical Ventilation Strategies Tidal Volume 6 ml/kg 12 ml/kg ---------------------------- ------------- ---------------- ------------- ------------- Heart rate (min^-1^) 214 ± 45 219 ± 26 211 ± 25 230 ± 31 MAP (mmHg) 63 ± 18 47 ± 7 65 ± 9 52 ± 7 Mean RVP (mmHg) 14 ± 2 14 ± 1 13 ± 2 15 ± 1 Cardiac output (L·min^-1^) 0.34 ± 0.02 0.23 ± 0.05^†^ 0.31 ± 0.05 0.25 ± 0.06 Peak P~AW~(cmH~2~O) 8 ± 1 16 ± 2^†^ 13 ± 1\* 30 ± 5\*^†^ Plateau P~AW~(cmH~2~O) 7 ± 1 15 ± 2^†^ 12 ± 2\* 27 ± 2\*^†^ Arterial pH 7.38 ± 0.10 7.38 ± 0.07 7.42 ± 0.04 7.43 ± 0.02 Arterial PO~2~(torr) 69 ± 11 95 ± 6^†^ 69 ± 12 94 ± 11^†^ Arterial PCO~2~(torr) 42 ± 2 40 ± 5 41 ± 2 41 ± 1 A-aDO~2~(torr) 19 ± 21 8 ± 9 25 ± 7 5 ± 8^†^ \* p \< 0.05 compared to other condition with same PEEP but different tidal volume; † p \< 0.05 compared to other condition with different PEEP but same tidal volume. Mixed model used for statistical analysis with post-hoc comparisons using Tukey\'s HSD. Values presented are mean ± SD. MAP - mean arterial pressure, RVP - right ventricular pressure, P~AW~- airway pressure, A-aDO~2~- alveolar-arterial oxygen difference. The order of the four conditions was varied across experiments. Prior to each condition change, two static inflations to 40 cmH~2~0 were performed via the syringe technique and held for 20 seconds each to ensure a standard volume history between conditions. All animals were ventilated with room air throughout all experimental conditions and respiratory rate was adjusted at each condition to achieve P~a~CO~2~of 35 to 45 mmHg. Twenty minutes after each condition was established, physiologic data were recorded. Aerosolized 1-μm diameter fluorescent microspheres were administered over 5 min to measure regional ventilation for each condition as previously described \[[@B19]\]. A total of 4 different fluorescent colors (yellow, orange, orange-red, and red) were used. Each colored microsphere aerosol marked ventilation under one of the four conditions. By using four different aerosols, we can determine spatial ventilation distribution post-mortem for each of the four different ventilation conditions in each animal. At the conclusion of each experiment, a sternotomy was performed, the main pulmonary artery and left atrium were cannulated, and the pulmonary vasculature was flushed with a dextran solution by gravity feed. The lungs were dissected from the chest cavity and dried inflated at 25-cmH~2~O. The dried lungs were fixed in rapid-setting foam, sliced, mapped, and diced into cubes of 1.5-2.0-cm^3^. Each piece was weighed, visually scored for airway and blood content, and soaked for 2 days in 1.5 ml of 2-ethoxyethyl acetate to extract the fluorescent dyes. The fluorescent signals for the four colors were measured in each piece with a fluorimeter (LS50B, Perkin-Elmer), corrected for background and spillover from adjacent signals \[[@B20]\], and converted to weight-normalized, relative ventilation signals as previously described \[[@B19]\]. Data Analysis and Statistics ---------------------------- All values are presented as means ± SD and all statistical analyses were done using JMP (SAS, Cary, NC). Comparisons of physiological parameters among the different ventilator conditions were made using a mixed model in which ventilator condition (the four combinations of tidal volume and PEEP) was the fixed effect and animal number was the random effect to account for correlations from repeated measures within each animal. For a given physiologic parameter there was one value for each animal for each of the four conditions. Post-hoc comparisons were made with Tukey\'s HSD. To assess for ventilation redistribution with change in tidal volume, the base 10 logarithm of the ratio of the relative ventilation with 12 ml/kg tidal volume to ventilation with 6 ml/kg was calculated for each lung piece. For a given piece of lung, if the log ratio was greater than 0, the relative ventilation to that lung piece increased as tidal volume increased; conversely if the log ratio was less than 0, the relative ventilation to that lung piece decreased as tidal volume increased. To evaluate for regional changes in relative ventilation distribution, fluorescent measurements were clustered into five regions based on dome of the diaphragm, major fissure, and a mid-sagittal division as illustrated in Figure [1](#F1){ref-type="fig"}. This clustering correlates with our previously published data on regional lung injury \[[@B18]\]. Because measurements for all four ventilatory conditions were made in each animal and because the log ratio of ventilation with a tidal volume of 12 ml/kg to a tidal volume of 6 ml/kg is not independent among the five regions within any given animal, we could not perform a standard statistical analysis looking at differences between the five regions at different PEEP levels. To quantify the effect of PEEP on ventilation distribution, we calculated the median of the log ratio of ventilation for each region (Figure [1](#F1){ref-type="fig"}) and then the standard deviation of these medians across the five different regions at both PEEP levels. A lower standard deviation represents little difference in median ventilation distribution across the five regions between tidal volumes; whereas, a higher standard deviation represents greater heterogeneity of relative ventilation distribution across the five regions between tidal volumes. A paired t-test was used to compare the log~10~-transformed standard deviation of median log ratios between PEEP levels. {#F1} Results ======= Measurement of sequential ventilation distribution -------------------------------------------------- Five animals were studied using the sequential ventilation experimental protocol. Several of the hemodynamic parameters were similar among the four ventilation conditions, although both mean arterial pressure (MAP) and cardiac output were lower during ventilation with PEEP for both tidal volumes (p = 0.08 and 0.01 for PEEP effect on MAP and cardiac output, respectively, at 6 ml/kg). Airway pressures were higher both with larger tidal volumes and with the addition of PEEP (Table [1](#T1){ref-type="table"}), including statistically significant differences for three out of four comparisons. Arterial oxygenation was statistically higher during ventilation with PEEP (Table [1](#T1){ref-type="table"}) for both tidal volumes; there were no statistically significant differences in arterial pH or PCO~2~among the four conditions. An average of 73 ± 21 lung pieces were obtained from each animal for measurement of regional ventilation across the four conditions. In the absence of PEEP, four out of five animals demonstrated a pattern in which relative ventilation increased to dorsal-caudal lung regions (regions 1 and 2) and decreased to ventral-rostral regions (regions 4 and 5) when tidal volumes were increased from 6 ml/kg to 12 ml/kg (Figure [1A](#F1){ref-type="fig"}). When ventilation distribution was measured in the presence of 8 cmH~2~O PEEP, there was no clear difference in relative ventilation distribution between 6 ml/kg and 12 ml/kg tidal volume ventilation in any animals (Figure [1B](#F1){ref-type="fig"}). A composite graph of all data points across the five animals demonstrates the consistency of this finding (Figure [2](#F2){ref-type="fig"}). The addition of PEEP resulted in a strong trend towards a reduced standard deviation of median log ratios of ventilation with a tidal volume of 12 ml/kg to a tidal volume of 6 ml/kg across the five regions (0.114 ± 0.105 vs. 0.238 ± 0.150, p = 0.08). {#F2} {#F3} In summary, animals ventilated in the supine posture without PEEP demonstrated sequential redistribution of ventilation towards dorsal-caudal lung regions as tidal volume increased suggesting increasing regional compliance in dorsal-caudal lung regions relative to ventral-cranial regions. This sequential redistribution of ventilation was attenuated with the application of PEEP. This finding further suggests that the mechanism for the change in relative regional compliance was recruitment and increased compliance in dorsal-caudal lung, rather than over-distension and decreased compliance in ventral-cranial lung. Discussion ========== In this study, we hypothesized that, in anesthetized, mechanically ventilated, supine rabbits, there are regional areas of lung, which undergo cyclical airway collapse at FRC and expansion during tidal inspiration (RACE) in the absence of PEEP and that this results in spatial redistribution of relative ventilation with increasing tidal volume. We further hypothesized that RACE would spatially correlate with the most regionally severe lung injury observed in a prior study of VILI. We found that relative ventilation increased to dorso-caudal lung regions as tidal volume increased and that this regional ventilation redistribution with increasing tidal volume was ameliorated with the addition of positive end-expiratory pressure. These data support regional cyclical airway closure at FRC and recruitment with tidal inspiration in the absence of PEEP, based on the following assumptions: 1\. A regional increase in relative ventilation with an increase in tidal volume implies that local compliance is increasing relative to other lung regions. 2\. Local lung compliance is roughly constant between volumes associated with either atelectasis (lower inflection point on a pressure-volume curve) or full inflation (upper inflection point on a pressure-volume curve - Figure [3](#F3){ref-type="fig"}). 3\. Regional lung compliance can increase relative to other lung regions either because of local recruitment and improved compliance (Figure [3A](#F3){ref-type="fig"}) or because of over distension and reduced compliance in remote lung regions (Figure [3B](#F3){ref-type="fig"}). 4\. If recruitment of dependent alveoli, which collapse at FRC, is responsible for redistribution of ventilation to dorso-caudal lung with increasing tidal volume, then application of PEEP should reduce this redistribution. However, if over distension of non-dependent alveoli is responsible for ventilation redistribution to dorso-caudal lung, then application of PEEP should augment this redistribution. Although we have not directly visualized RACE, which is not obviously feasible, our data support a spatial distribution of cyclical airway collapse and recruitment, which correlates with our previously reported spatial distribution of lung injury \[[@B18]\]. Because RACE is implicated as one cause of ventilator-induced lung injury \[[@B6],[@B18],[@B21]-[@B23]\], these findings further support regional RACE in uninjured, supine rabbits undergoing mechanical ventilation. All non-invasive measurements of volume change or alveolar density are indirect estimates of regional ventilation subject to erroneous interpretation. Using dynamic measurement of regional ventilation by the novel method of labeled aerosol deposition, we have demonstrated for the first time that regional ventilation redistributes with increasing tidal volume, suggesting dynamic regional changes in lung mechanics. Furthermore, by measuring sequential ventilation at different PEEP levels, we have shown that RACE is the most likely explanation for these dynamic changes in regional lung mechanics. We have previously reported a dramatic dorsal-ventral and cranial-caudal gradient of lung injury severity in a rabbit model of VILI with supine ventilation in the absence of PEEP. The region with the most severe injury was the dorsal-caudal lung. With the application of 8 cmH~2~O of PEEP however, no such gradient is observed, rather a less severe, more homogeneously distributed injury pattern occurs, despite identical end inspiratory pressures \[[@B18]\]. The application of PEEP has been shown to ameliorate VILI in a number of experimental models \[[@B1],[@B3],[@B4],[@B6],[@B24]-[@B26]\]. One potential explanation for the protective effects of PEEP is the prevention of tidal collapse and re-expansion of distal lung units. In the supine posture, lower alveolar volumes at FRC may predispose dependent lung regions to cyclical collapse. Muscedere et. al. \[[@B6]\] demonstrated epithelial injury in distal airways and alveoli, even with low tidal volumes (6 ml/kg), when isolated, unperfused rat lungs were allowed to deflate to volumes below the lower inflection point of the pressure-volume curve during exhalation. Although compelling, these findings must be interpreted in the context of an *ex vivo*, unperfused model which will favor tidal airway collapse and minimize edema formation in comparison with an intact animal model \[[@B16]\]. Distal airways and alveoli are not generally felt to be at risk of tidal collapse and re-expansion *in vivo*in uninjured lungs. In the current study, we examined this during mechanical ventilation in the supine posture by demonstrating sequential distribution of ventilation to the dorsal-caudal lung regions with increasing tidal volume, which was abolished by the application of PEEP. In the supine posture, a reduction was seen in relative ventilation to the dorsal-caudal region. One potential explanation for this is that the FRC is below the closing volume in this region at end-exhalation, resulting in cyclical airway closure. This would result in sequential ventilation with no inspired gas going to this lung region during the initial phase of tidal breathing, followed by recruitment and ventilation in the latter phase of each breath. To test this, we subjected uninjured animals to small (6 ml/kg) and large (12 ml/kg) tidal volume ventilation and measured relative regional ventilation distribution with inhaled fluorescent microspheres. We found a relative increase in ventilation to the dorsal-caudal lung and decreased ventilation to the ventral-cranial lung with larger tidal volumes. Two likely explanations for this observation are 1) alveoli in the dorsal-caudal lung were recruited with increased tidal volume or 2) ventral-cranial regions were over-distended with increased tidal volume, became less compliant, and ventilation was re-distributed to the dorsal-caudal lung. The effect of PEEP on the distribution of ventilation favors the former explanation. The application of PEEP = 8 cm H~2~O eliminated the observed sequential ventilation in the dorsal-caudal lung, consistent with recruitment of alveoli in this region which allowed the same relative ventilation to be delivered independent of tidal volume. There are several limitations to the current study. The tidal volume used to induce VILI in our previous study \[[@B18]\] as well as other studies of VILI were much greater than the tidal volumes used in the measurement of ventilation distribution. The lower tidal volumes were chosen to avoid inducing lung injury during the experiment because aerosol deposition is unlikely to reflect ventilation distribution in the presence of spatially heterogeneous pulmonary edema secondary to differences in particle deposition. We did not measure ventilation distributions in the prone posture to identify whether or not sequential ventilation distribution was diminished. However, we have previously shown in a porcine model that posture change from supine to prone increases dorsal-caudal ventilation consistent with reduced airway collapse in these regions \[[@B19]\]. Additionally, reduced sequential ventilation and airway closure in the prone posture compared with supine posture has been previously shown in dogs with normal lungs by single breath washout test \[[@B27]\]. We have also ventilated animals in the prone posture in an identical fashion to that previously reported in supine animals. With supine ventilation, the most severe injury occurred in the dorsal-caudal lung while less severe injury was seen in the cranial-ventral region. In contrast, prone ventilation produced a more modest and homogeneously distributed injury pattern \[[@B4]\]. These results are similar to that of previous observations in both oleic acid injured \[[@B22]\] and normal dogs \[[@B21]\]. One potential explanation for the protective effects of prone posture is the prevention of tidal collapse and re-expansion of distal lung units. In the supine posture, lower alveolar volumes at FRC may predispose dependent lung regions to cyclical collapse. The more uniform pleural pressure gradient along the dorsal-ventral axis, in the prone posture, results in higher regional FRC in dependent lung zones \[[@B28]\] which may limit tidal collapse and recruitment of airways, and thus reduce VILI. Conclusions =========== We previously reported in a rabbit model of VILI that lung injury is greater and more spatially heterogeneous in the supine posture as compared with the prone posture. One potential mechanism for this finding is regional repeated airway collapse and expansion during tidal breathing. To evaluate whether or not this occurred in the absence of lung injury with supine positioning, we measured regional ventilation at different tidal volumes in the presence and absence of PEEP. We found evidence of sequential ventilation distribution towards dorsal-caudal lung and away from ventral-cranial lung with increasing tidal volume. This sequential ventilation pattern was attenuated with the addition of PEEP compatible with RACE. We speculate that RACE occurs in normal lung with supine ventilation and in the absence of PEEP and that this may contribute to the development of lung injury. List of Abbreviations ===================== PEEP: Positive end expiratory pressure; VILI: Ventilator-induced lung injury; RACE: Repeated airways collapse and expansion; P~a~CO~2~: Partial pressure of arterial carbon dioxide; P~a~O~2~: Partial pressure of arterial oxygen Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= SS and WA designed and performed the experiments, analyzed the data and wrote the manuscript. NP designed the statistical methods and reviewed and edited the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2466/10/25/prepub> Acknowledgements ================ The authors thank Wayne Lamm and Dowon An for assistance in performing these experiments. This work was supported by NIH grants HL71020 and HL004479.

1. Introduction {#sec1-medicina-55-00780} =============== Lymphoepithelioma-like carcinoma (LELC) is a histological type of malignant tumor arising from the uncontrolled mitosis of transformed cells originating in epithelial tissue. It is a common type of poorly differentiated epithelial cells in the nasopharynx \[[@B1-medicina-55-00780],[@B2-medicina-55-00780],[@B3-medicina-55-00780]\]. LELC is seen in salivary glands, lungs, nasopharynx, skin, thymus, stomach, urinary bladder, and uterine cervix \[[@B2-medicina-55-00780],[@B4-medicina-55-00780]\]. The diagnosis is pathomorphological. According to World Health Organization, cervical cancer is the fourth most frequent cancer in women with an estimated 570,000 new cases in 2018 representing 6.6% of all female cancers, and approximately 90% of deaths from cervical cancer occur in low- and middle-income countries \[[@B5-medicina-55-00780]\]. The most common histological type of cervical neoplasia is squamous cell carcinoma (SCC), at around 80% of all cases. A rare subtype of SCC is lymphoepithelioma-like carcinoma. It was reported for the first time by Hamazaki et al. in 1968 \[[@B6-medicina-55-00780]\]. In the literature, LELC is described mainly in case reports \[[@B7-medicina-55-00780]\]. There are significant differences in frequency, mean age, viral status, and outcomes in Asian or Caucasian patients \[[@B7-medicina-55-00780],[@B8-medicina-55-00780]\]. The objective of our study was to analyze the frequency of LELC in hospitalized women with cervical cancer, as well as the clinical characteristics, treatments, and prognosis of LELC. 2. Materials and Methods {#sec2-medicina-55-00780} ======================== A retrospective study of all cases of LELC of the cervix at the Clinic of Oncogynecology, University Hospital, Pleven, Bulgaria between 1 January 2007 and 31 December 2016 was performed. Clinical data were collected from patients' medical records. Patients with clinical stage I who were initially referred to surgery were analyzed. All histological slides were reviewed by an expert, and the diagnosis was reconfirmed. Pathologic and clinical staging were performed according to TNM classification or FIGO. All patients were followed-up until March 2019. The follow-up was done at 3, 6, 9, 12, 15, 18, 21, and 24 months and then annually, including clinical examination, blood tests, and chest X-ray. Annually a whole-body contrast-enhanced CT was performed. We analyzed some clinical characteristics of the patients, calculated the share of LELC from all patients with stage I cervical cancer, and looked at the overall survival (OS) rate, the 5-year survival rate, the correlation between OS and lymph node status, and the correlation between OS and the size of the tumor. Statistical analysis was done by using SPSS for Windows. 3. Results {#sec3-medicina-55-00780} ========== Six hundred and thirty patients with cervical cancer were operated on in our clinic during the study period. Seventeen of the women had LELC, which represented 3.3% of all cases with cervical carcinoma at stage I (all patients who were referred directly to surgery and no neoadjuvant treatment was performed) during the study period. All of the patients had a histological diagnosis before radical surgery (except for one case) from a cervical biopsy that was done due to abnormal genital bleeding. In one patient, the biopsy showed benign pathology but due to persistence of complaints, a laparohysterectomy (LHT) was performed. The mean age of the patients with LELC was 49.6 years (range 32--67). In one patient a simple hysterectomy was performed because of benign histology after dilation and curettage. In all other patients a radical hysterectomy with total pelvic lymph node dissection was performed. In all patients, adjuvant radiotherapy was done. In 14 patients, immunohistochemical staining (IHC) for human papilloma virus (HPV) and Epstein--Barr virus (EBV) was done, and eight of them (47.1%) were positive for any or both viruses and six (35.3%) were negative for both viruses. In three patients (17.6%), the exam was not performed because of the lack of paraffin blocks. Fourteen patients (82.4%) were in the FIGO IB1 stage, and three patients (17.6%) were in the FIGO IB2 stage. The size of the primary tumor was \<2 cm in five patients (29.4%), 2--4 cm in nine patients (52.9%), and \>4 cm in three patients (17.6%). Lymph nodes were metastatic in three patients (17.6%), non-metastatic in 13 patients (76.5%), and unknown in one patient. The overall survival rate was 76.47% for the study period, and the 5-year survival rate of the patients that were followed-up until the 5th year (14 patients) was 69.23%. When comparing the OS between the non-metastatic lymph node group and the metastatic lymph node group, there was a trend of a lower OS in the metastatic lymph node group ([Figure 1](#medicina-55-00780-f001){ref-type="fig"}), which did not reach statistical significance (*p* = 0.087). When studying the correlation between the OS and the size of the tumor ([Figure 2](#medicina-55-00780-f002){ref-type="fig"}), there was no significant difference between the groups (*p* = 0.327). Some clinical and pathoanatomical characteristics of the patients are presented in [Table 1](#medicina-55-00780-t001){ref-type="table"}. 4. Discussion {#sec4-medicina-55-00780} ============= Histological LELC is composed of poorly defined islands of undifferentiated cells in a background intensely infiltrated by lymphocytes. The tumor cells have uniform, vesicular nuclei with prominent nucleoli and moderate amounts of slightly eosinophilic cytoplasm. The cell borders are indistinct, often imparting a syncytial-like appearance to the groups. This typical microscopic appearance and immunohistochemistry for epithelial and lymphoid markers can help in differentiating cervical LELC from the poorly differentiated squamous cell carcinoma and lymphoproliferative lesions. In the female genital tract, LELC has been reported in the vulva, vagina, uterine cervix, and endometrium \[[@B9-medicina-55-00780]\]. When LELC affects the cervix, it is believed to have a better prognosis than the normal SCC of the cervix due to lack of lymph node metastasis \[[@B3-medicina-55-00780],[@B10-medicina-55-00780]\]. Significant differences in the incidence of this type of carcinoma in Asian and Caucasian races have been reported. It represents 0.7% of all primary cervical malignancies among the Western population and is about 5.5% among the Asian population \[[@B3-medicina-55-00780]\]. There is also a difference in the mean age of diagnosis in these patients. In Asian patients it is reported to be between 43 and 50 years (range 30--72) and mean age of 42.3 years in Western patients (range 21--58) \[[@B11-medicina-55-00780]\]. It is assumed that LELC is associated with Epstein--Barr virus (EBV) infection in Asians, whereas Westerns are associated with human papillomavirus (HPV), or viral genesis cannot be proven \[[@B3-medicina-55-00780],[@B12-medicina-55-00780],[@B13-medicina-55-00780]\]. Typically, the diagnosis is made at an early stage and there is no involvement of the lymph nodes. This could be the reason for better prognosis reporting for this disease \[[@B11-medicina-55-00780]\]. In our study, we presented 17 cases with LELC of uterine cervix, which represented 3.3% from all stage I cases with cervical carcinoma during the study period. This rate was four times greater than the literature results. This frequency can be explained by the fact that only those patients who were FIGO I stage and were directly referred to surgery were included in this study. All other cases of cervical cancer were excluded. However, we believe that it is quite possible that the incidence of LELC in Western patients is higher than reported, as it is determined on the basis of case reports and small case series. The mean age in our group was 49.6 (ranging from 32 to 67), which was slightly higher than the published data so far, although Martorell et al. found that the mean age of their patients was 69 years \[[@B11-medicina-55-00780]\]. In our study, only three patients (17.6%) were diagnosed with a tumor larger than 4 cm in diameter, confirming the data from the world literature that LELC is diagnosed early. However, the fact that in nine cases the tumor size was between 2 and 4 cm indicates that the diagnosis was not performed at an early stage. This could be due to the health culture of the population and problems with the coverage of the screening program. All patients had a history of abnormal genital and contact bleeding for at least one year. In two of the patients that died, lymph metastases were observed. The rapid progression of the disease, which could be explained by its possible hematogenic dissemination, was noticeable. In all four patients that died, the tumor was less than 4 cm, in two cases it was less than 2 cm, and in two cases it was between 2 and 4 cm. There was no relationship between the size of the tumor and its prognosis. There was a trend in lower OS in the metastatic lymph node group, which did not reach statistical significance. This could be explained with the small number of patients in the study. 5. Conclusions {#sec5-medicina-55-00780} ============== Lymphoepithelioma-like carcinoma is a rare SCC subtype, but it may be more frequent among western patients than previously thought. Our results do not confirm the data showing low risk of lymph metastasis and good prognosis of LELC, which is why we think that the treatment in these cases has to be more aggressive than is reported in the literature. Due to the low incidence of this disease, a lot is still unknown. Larger studies in the area are needed. Conceptualization, A.Y. and A.K.; Methodology, A.K. and A.Y.; Formal Analysis, A.K. and S.S.; Investigation, A.Y.; Resources, M.V.-S.; Data Curation, S.S. and M.K. (Martin Karamanliev); Writing---Original and Draft Preparation, A.Y. and M.K. (Martin Karamanliev); Writing---Review and Editing, A.Y. and M.K. (Milena Karcheva); Visualization, M.K. (Milena Karcheva); Supervision A.Y. and M.V.-S. This research received no external funding The authors declare no competing interests. {#medicina-55-00780-f001} {#medicina-55-00780-f002} medicina-55-00780-t001_Table 1 ###### Clinical and pathoanatomical characteristics of the patients. Case Age Treatment Clinical Stage Tumor Size (cm) Recurrence Outcome ------ ----- ----------- ---------------- ----------------- ------------------ ---------------------- 1 67 RH + PLND pT1b1pN1Mo b/n 2--4 Unknown Died on 8th month 2 58 RH + PLND pT1b1pNoMo \<2 Liver metastases Died on 88th month 3 42 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on128th month 4 47 RH + PLND pT1b2pN1Mo \>4 No Alive on 128th month 5 48 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on 104th month 6 38 RH + PLND T1b1pNoMo \<2 No Alive on 127th month 7 46 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on 116th month 8 59 TH pT1b2NoMo \>4 No Alive on 103th month 9 49 RH + PLND pT1bpNoMo \<2 No Alive on 102th month 10 59 RH + PLND pT1b1pNoMo b/n 2--4 Bone metastases Died on 18th month 11 40 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on 92th month 12 49 RH + PLND pT1b1pN1Mo b/n 2--4 Unknown Died on 16th month 13 34 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on 68th month 14 66 RH + PLND pT1b2pNoMo \>4 No Alive on 52th month 15 61 RH + PLND pT1b1pNoMo b/n 2--4 No Alive on 44th month 16 48 RH + PLND pT1b1pNoMo \<2 No Alive on 43th month 17 32 RH + PLND T1b1pNoMo \<2 No Alive on 28th month RH---radical hysterectomy; TH---total hysterectomy; PLND---pelvic lymph node dissection.

{#sp1 .238}

Introduction ============ Chromosome instability (CIN), characterized by increased frequency of gain or loss of the whole chromosomes, has long been implicated in tumorigenesis, poor patient prognosis and drug resistance.^[@bib1]^ The most common cause of CIN in cancer cells is a defect in the dynamics of kinetochore-microtubule (k-MT) attachment during mitosis.^[@bib2]^ An evolutionarily conserved spindle assembly checkpoint mechanism has evolved to ensure proper completion of all k-MT attachments before chromosomal segregation.^[@bib3]^ Certain improper k-MT attachments (for example, merotelic attachment with microtubules orientated from both spindle poles) can escape from spindle assembly checkpoint surveillance and persist into the lagging chromosome; these are observed at the spindle midzone during anaphase.^[@bib2]^ Abnormal k-MT attachments occur frequently in early mitosis but are efficiently corrected in normal cells.^[@bib4]^ In cancer cells with CIN, however, stability of k-MT attachments increases, hindering the ability to correct the abnormal k-MT attachment.^[@bib5]^ In addition, abnormal spindle assembly with extra centrosomes significantly increases the frequency of lagging chromosomes during anaphase, indicating that centrosome instability is also a major source of abnormal k-MT attachment.^[@bib6]^ Centrosome hypertrophy is an accepted cause of CIN and is frequently correlated with tumorigenesis.^[@bib7]^ Moreover, centrosome instability is highly linked to certain tumor suppressor protein mutations (for example, those in Brca1).^[@bib8],\ [@bib9]^ Brca1 is a product of a breast and ovarian cancer susceptibility gene and participates in a variety of cellular processes, including DNA damage repair, cell cycle checkpoint response, chromatin remodeling and mitotic progression regulation.^[@bib10]^ The involvement of Brca1 in centrosome regulation was recognized upon analysis of *BRCA1*^−/−^ mice fibroblasts, which contain amplified centrosomes.^[@bib11]^ Brca1 localizes to centrosomes during mitosis, and its ubiquitin ligase activity ensures normal centrosome number and function.^[@bib9],\ [@bib12]^ The direct target of Brca1 in the centrosome is γ-tubulin.^[@bib8]^ Cells that express mutant γ-tubulin that cannot be ubiquitinated by Brca1 are characterized by centrosome amplification; a Brca1 mutant that lacks E3-ligase enzyme activity also loses its effect on centrosome nucleation inhibition.^[@bib12],\ [@bib13],\ [@bib14]^ Chk2 is another centrosome localization tumor suppressor. In response to DNA double-stranded breaks, DNA damage sensor kinase ataxia telangiectasia mutated (ATM) phosphorylates Chk2 at Thr68 residue promoting Chk2 homodimerization and full activation.^[@bib15]^ Phosphorylated Chk2 Thr68 reportedly localizes at centrosomes during mitosis;^[@bib16]^ however, its role in mitotic regulation has not yet been established. Stolz *et al.*^[@bib17]^ recently demonstrated that Chk2 is required for proper and timely spindle formation during normal mitosis; this is critical for accurate attachment of kinetochores to the spindle microtubules and subsequent proper chromosome segregation. Chk2 phosphorylates Ser988 of Brca1, which is important for the function of Brca1 in switching error-prone non-homologous end-joining (NHEJ) to error-free homologous recombination.^[@bib18],\ [@bib19],\ [@bib20]^ Chk2 also phosphorylates Brca1 on Ser988 during normal mitotic progression in the absence of DNA damage. Wild-type Brca1, but not the Ser988 to alanine mutant, can rescue the mitosis defect induced by Chk2 deficiency, suggesting that phosphorylation of Brca1 at this site has an important role in mitotic spindle assembly and chromosome stability maintenance. Moreover, Chk2 deficiency increases CIN and leads to aneuploidy even in chromosomal-stable human colon carcinoma HCT116 cells.^[@bib17]^ How Chk2 is activated in normal mitosis is still an unanswered question. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component in the NHEJ pathway of double-stranded DNA break repair.^[@bib21]^ In response to double-stranded breaks, DNA-PKcs is rapidly phosphorylated on its Thr2609 and Ser2056 clusters by ATM and by itself.^[@bib22],\ [@bib23]^ In addition to its established role in DNA repair, we observed that DNA-PKcs phosphorylated at both Thr2609 and Ser2056 localizes at the mitotic spindle apparatus on centrosomes and kinetochores. Consistent with this localization, DNA-PKcs is important for the stabilization of centrosome and of the spindle structure, as well as in the regulation of mitotic progression.^[@bib24]^ DNA-PKcs regulates mitotic catastrophe in response to irradiation at least partially via the Chk2 pathway;^[@bib25]^ however, whether Chk2 is a target of DNA-PKcs during normal mitotic progression and its biological significance in mitotic spindle assembly has not been clarified. In the present study, we demonstrate that DNA-PKcs regulates Chk2 phosphorylation at the Thr68 site during mitosis and that DNA-PKcs coordinates the spindle assembly and kinetochore/microtubule attachment through the Chk2--Brca1 signaling pathway. We also show that the deficiency of DNA-PKcs leads to chromosomal instability. These results demonstrate that DNA-PKcs mediates Chk2 phosphorylation to regulate microtubule nucleation and the spindle damage response. Results ======= Chk2 is phosphorylated at Thr68 by DNA-PKcs during mitosis ---------------------------------------------------------- Chk2 is the key downstream effector of the ATM signaling pathway for cell cycle checkpoint regulation in the DNA damage response.^[@bib26]^ In addition, Chk2 is activated during normal mitosis, as monitored by Thr68 phosphorylation, and is required for proper mitotic spindle assembly and chromosomal stability.^[@bib27]^ We have reported that DNA-PKcs is also activated and has a critical role in mitosis progression.^[@bib21]^ To test the hypothesis that DNA-PKcs is involved in Chk2 activation during mitosis, human colon cancer HCT116 cells and isogenic DNA-PKcs knockout cells (DNA-PKcs^−/−^) were mitotically synchronized or were treated with ionizing radiation. Western blot analyses revealed significant Chk2 Thr68 phosphorylation in HCT116 cells but little in DNA-PKcs^−/−^ cells ([Figure 1a](#fig1){ref-type="fig"}). In contrast, ionizing radiation-induced Chk2 Thr68 phosphorylation (mediated by the ATM kinase) was observed in DNA-PKcs^−/−^ cells at levels comparable to those in HCT116 cells. Similarly, siRNA-mediated depletion of DNA-PKcs from HeLa cells abolished the increase of Chk2 Thr68 phosphorylation in mitotically synchronized cells but not in irradiated cells ([Figure 1b](#fig1){ref-type="fig"}). Mitosis-dependent Chk2 phosphorylation was also clearly detected in the isogenic and NHEJ-defective Ligase4^−/−^ cells ([Supplementary Figure S1A](#sup1){ref-type="supplementary-material"}), indicating that Chk2 phosphorylation in mitosis is independent of DSB repair regulation. To further examine the role of the kinase activity of DNA-PKcs on Chk2 activation in mitosis, the parental HCT116 cells were synchronized via nocodazole treatment and then they were incubated for 2 h with DNA-PKcs kinase inhibitor Nu7441. As shown in [Figure 2a](#fig2){ref-type="fig"}, Nu7441 effectively abolished Chk2 Thr68 phosphorylation in response to mitosis synchrony but did not affect the levels of Thr68 phosphorylation induced by irradiation. The blocking of mitotic Chk2 phosphorylation was not due to alternation in mitosis synchrony as histone H3 Ser10 phosphorylation was equivalent in sham- and Nu7441-treated HeLa cells. Mitotic Chk2 phosphorylation was also elicited by treatment of cells with the microtubule stabilizer paclitaxel. Paclitaxel-mediated Chk2 Thr68 phosphorylation was abolished by treatment of cells with an siRNA targeted against the gene encoding DNA-PKcs or by Nu7441 treatment ([Supplementary Figures S1B and C](#sup1){ref-type="supplementary-material"}, respectively). In contrast, ATM kinase inhibitor Ku55933 had no effect on mitosis-induced Chk2 phosphorylation but did effectively block IR-induced Chk2 phosphorylation ([Figure 2b](#fig2){ref-type="fig"}). Taken together, these results demonstrate that DNA-PKcs, but not the ATM kinase, activates Chk2 during mitosis. DNA-PKcs is required for mitotic spindle assembly and chromosomal stability --------------------------------------------------------------------------- During the prometaphase to metaphase transition, kinetochores of sister chromatid pairs are captured by microtubules of opposite poles to allow chromosomes to align at the center of the spindle before the sister-chromatid separation at anaphase. Stolz *et al.*^[@bib17]^ reported that Chk2 activation is necessary for proper spindle organization and chromosome segregation during normal mitosis and that Chk2 deficiency resulted in chromosomal numerical instability. Similarly, we observed that there was an increase of chromosomal numerical instability in DNA-PKcs^−/−^ cells as compared with the parental HCT116 cells and Ligase4^−/−^ cells ([Figure 3a](#fig3){ref-type="fig"}). To further explore the involvement of DNA-PKcs in mitotic spindle assembly, HCT116, DNA-PKcs^−/−^ and Ligase4^−/−^ cells were treated with nocodazole and then with proteasome inhibitor MG132 to stall mitosis progress at the metaphase to anaphase transition. Abnormal mitotic spindles were examined via immunofluorescence staining. Cells with misaligned chromosomes were scored ([Figure 3b](#fig3){ref-type="fig"}). DNA-PKcs^−/−^ cells had a higher proportion of chromosomal misalignment during the metaphase to anaphase transition than did the parental HCT116 cells or the Ligase4^−/−^ cells ([Figure 3d](#fig3){ref-type="fig"}). Abnormal mitotic spindles were further examined via analysis of chromosome lagging in anaphase cells, an indication of chromosomal instability or errors during chromosomal segregation. Exponentially growing HCT116, DNA-PKcs^−/−^ and Ligase4^−/−^ cells were scored for chromosome lagging in anaphase ([Figure 3c](#fig3){ref-type="fig"}). Lack of DNA-PKcs, but not of Ligase4, caused a significant increase of lagging chromosomes in HCT116 cells ([Figure 3e](#fig3){ref-type="fig"}), which is consistent with the increase of aneuploidy found in DNA-PKcs^−/−^ cells but not in Ligase4^−/−^ cells ([Figure 3a](#fig3){ref-type="fig"}). Transient knockdown of levels of DNA-PKcs using an siRNA or treatment with DNA-PKcs inhibitor Nu7441 also promoted abnormal spindles and lagging chromosomes in HeLa cells ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}). These results further demonstrated the importance of DNA-PKcs and its kinase activity in mitotic spindle assembly and maintenance of chromosomal stability. The Chk2--Brca1 pathway mediates DNA-PKcs signaling in spindle organization --------------------------------------------------------------------------- To further test the function of DNA-PKcs-dependent Chk2 phosphorylation in spindle assembly and chromosomal stability, wild-type Chk2 and Chk2 mutants with an alanine (to prevent phosphorylation) or aspartic acid (to mimic the phosphorylated state) substitution at Thr68 residue (T68A and T68D, respectively) were expressed in DNA-PKcs^−/−^ cells ([Figure 4a](#fig4){ref-type="fig"}). Overexpression of wild-type Chk2 or of the T68A mutant did not alter the frequencies of chromosomal misalignment and lagging in DNA-PKcs^−/−^ cells as compared with control cells transfected with empty vector, whereas overexpression of the phosphomimetic Chk2 T68D mutant significantly decreased the levels of these abnormalities during mitosis ([Figure 4b](#fig4){ref-type="fig"}). T68D mutant Chk2 was able to restore proper chromosomal alignment and alleviate anaphase lagging in HCT116 cells, whereas vector alone had no effect. Chk2 is known to phosphorylate Brca1 at Ser988 upon DNA damage, which facilitates Brca1 function in DSB repair.^[@bib18],\ [@bib19],\ [@bib20]^ The involvement of Chk2 signaling in mitosis and spindle assembly has also been linked to Brca1 Ser988 phosphorylation.^[@bib17]^ To test whether Brca1 also functions in the same signaling pathway downstream from DNA-PKcs, wild-type Brca1 and non-phosphorylatable or phosphomimetic Brca1 mutants (S988A and S988E, respectively) were overexpressed in DNA-PKcs^−/−^ cells ([Figure 4c](#fig4){ref-type="fig"}). As was observed in Chk2 overexpression experiments, the phosphomimetic Brca1 mutant, but not wild-type Brca1 nor the S988A mutant, was able to rescue mitotic defects in DNA-PKcs^−/−^ cells ([Figure 4d](#fig4){ref-type="fig"}). Taken together, our analyses revealed that Chk2 and Brca1 are the downstream effectors of DNA-PKcs signaling in mitotic regulation. DNA-PKcs inactivation leads to dysfunction of microtubule dynamics ------------------------------------------------------------------ Improper regulation of microtubule dynamics is associated with aberrant mitotic spindle and chromosomal instability.^[@bib28]^ The Chk2--Brca1 signaling axis has also been implicated in regulation of microtubule dynamics in response to spindle damage.^[@bib29],\ [@bib30]^ To further investigate the involvement of DNA-PKcs in microtubule regulation, HCT116 and DNA-PKcs^−/−^ cells were treated with nocodazole in chilled medium to depolymerize microtubules. Microtubule nucleation and regrowth was initiated upon removal of nocodazole-containing medium and addition of prewarmed fresh medium. Microtubule nucleation status was inferred based on the diameter of asters, the radial arrays of microtubules that grow from centrosomes ([Figure 5a](#fig5){ref-type="fig"}). Our analyses revealed that microtubule growth was significantly enhanced in DNA-PKcs^−/−^ relative to HCT116 cells ([Figure 5b](#fig5){ref-type="fig"}). To determine the role of Chk2 and DNA-PKcs in microtubule regulation, HeLa cells stably expressing wild-type or T68A or T68D mutant Chk2 were transfected with siRNA targeting the gene encoding DNA-PKcs. Cells were analyzed for microtubule regrowth as described above. Depletion of DNA-PKcs caused a significant outgrowth of microtubules in cells expressing wild-type Chk2, the T68A mutant and the vector control but not in cells expressing Chk2 T68D ([Figure 5c](#fig5){ref-type="fig"} and [Supplementary Figure S3](#sup1){ref-type="supplementary-material"}). These results indicate that the phosphomimetic Chk2 T68D mutant protein can compensate for the loss of DNA-PKcs and prevent dysregulation of microtubule dynamics in mitosis. Microtubule formation in Brca1-deficient cancer cells is abnormal when cells are treated with an anti-microtubule agent such as paclitaxel.^[@bib31],\ [@bib32]^ The connection of DNA-PKcs and Chk2--Brca1 signaling in regulation of microtubule dynamics prompted us to speculate that DNA-PKcs modulates cellular sensitivity toward paclitaxel. To test the hypothesis, HCT116 and DNA-PKcs^−/−^ cells were treated with paclitaxel and were subjected to a clonogenic survival assay. Our analysis revealed that DNA-PKcs deficiency in HCTT116 cells did confer paclitaxel resistance in clonogenic survival assay ([Figure 6a](#fig6){ref-type="fig"}). In addition, the colony sizes of HCT116 cells were obviously smaller than those of DNA-PKcs^−/−^ cells (data not shown). Similarly, an MTT cell viability assay revealed that DNA-PKcs^−/−^ cells were more resistant to paclitaxel than were the parental HCT116 cells ([Figure 6b](#fig6){ref-type="fig"}). Discussion ========== We have previously reported that DNA-PKcs is autophosphorylated during normal cell cycle progression into mitosis and that activation of DNA-PKcs is necessary for microtubule organization at centrosomes and kinetochores, mitotic spindle organization and chromosomal segregation.^[@bib24]^ In the current study, we report that DNA-PKcs is the upstream regulator of the Chk2--Brca1 signaling pathway, which was known to regulate mitotic spindle formation and maintenance of chromosomal stability. Our results demonstrate that mitotic induction of Chk2 phosphorylation at Thr68 is primarily mediated by DNA-PKcs; inhibition of DNA-PKcs via different approaches eliminated mitotic Chk2 phosphorylation. This is in contrast to ATM-dependent Chk2 phosphorylation under the DNA damage conditions.^[@bib33]^ Chk2 activation has been linked to its downstream effector molecule Brca1 in both DNA damage response and normal mitosis progression, and Chk2-dependent Brca1 phosphorylation at Ser988 is critical for proper assembly of the mitotic spindle and for chromosomal stability.^[@bib17]^ Our analyses revealed that overexpression of phosphomimetic mutants of Chk2 (T68D) or Brca1 (S988E) but not their wild-type counterparts compensated for the loss of DNA-PKcs and decreased chromosomal misalignment and lagging in cells that were deficient in DNA-PKcs ([Figures 4](#fig4){ref-type="fig"} and [5](#fig5){ref-type="fig"}). These results demonstrate that DNA-PKcs, Chk2 and Brca1 are functionally aligned in the pathway that regulates mitotic spindle assembly and guides accurate chromosomal segregation. Our data do not rule out the possibility that, in the absence of DNA-PKcs, a compromised NHEJ mechanism in DSB repair results in aberrant chromosomal structures that hinder chromosomal alignment and segregation in mitosis. However, we did not observe a significant increase in aberrant mitosis in NHEJ-deficient ligase4^−/−^ cells, suggesting that a mechanism independent of both NHEJ and DSB repair but dependent on DNA-PKcs phosphorylation of Chk2 regulates mitosis. The presence of mitotic DNA-PKcs phosphorylation at centrosomes and kinetochores implies that DNA-PKcs is involved in microtubule organization and could modulate k-MT attachment.^[@bib24]^ Accurate chromosome segregation in mitosis relies on the sister kinetochores attached to microtubules emanating from opposite spindle poles. Inaccurate and aberrant k-MT attachments result in CIN, aneuploidy and full-blown carcinogenesis.^[@bib34],\ [@bib35]^ There are three types of k-MT attachment errors in prometaphase: (1) monotelic attachment, in which only one sister kinetochore attaches to microtubule emanating from one pole; (2) syntelic attachment, in which both sister kinetochores bind to microtubules from the same pole; and (3) merotelic attachment, in which a kinetochore is attached to microtubules from both poles. The merotelic attachment is the only attachment error that can escape from the surveillance of the spindle checkpoint, as no kinetochore is left unattached.^[@bib36],\ [@bib37]^ Therefore, cells carrying merotelic kinetochore attachment can progress into anaphase with lagging chromosomes; this is the most common route to gain or loss of a complete chromosome and results in aneuploidy.^[@bib6],\ [@bib35]^ Loss of Brca1 or Chk2 coincides with increased incidents of aneuploidy and merotelic kinetochore attachment.^[@bib27]^ Similarly, there is an increase of misalignment and lagging chromosomes in the absence of DNA-PKcs, and overexpression of phosphomimetic Chk2 T68D or Brca1 S988E partially rescued these aberrances in DNA-PKcs-deficient cells. Thus, it is tempting to speculate that the DNA-PKcs-Chk2--Brca1 pathway triggers correction of merotelic k-MT attachments. Early studies revealed that Brca1 resides at the centrosome throughout all cell cycle phases and suppresses centrosome amplification, microtubule nucleation and aneuploidy.^[@bib30],\ [@bib38]^ Brca1 function in centrosome and microtubule regulation requires its dimerization with Brca1-associated ring domain protein 1 (Bard1) to form an active E3 ubiquitin ligase.^[@bib8]^ A key target of Brca1-dependent ubiquitination is γ-tubulin, which is a component of the γ-tubulin ring complex that anchors microtubule nucleation and growth.^[@bib39]^ The Brca1/Brad1 complex directs γ-tubulin monoubiquitination at lysines 48 and 344 in S/G2 phases, and failure of γ-tubulin monoubiquitination results in a marked amplification of centrosomes and an increase in microtubule nucleation.^[@bib12],\ [@bib14]^ These results thus recapitulate centrosome hyperactivation in Brca1-deficient cells and support a negative regulatory function of Brca1 on microtubule regulation. Such an inhibitory effect on microtubule also requires Chk2-mediated Brca1 phosphorylation at Ser988.^[@bib29]^ Consistent with this model, microtubule regrowth analysis revealed that an enhancement of microtubule nucleation/regrowth occurs in DNA-PKcs-deficient cells and that such dysregulation can be corrected by expression of the phosphomimetic Chk2 T68D mutant ([Figure 5](#fig5){ref-type="fig"} and [Supplementary Figure S3](#sup1){ref-type="supplementary-material"}). It is likely that, in the absence of DNA-PKcs, inadequate activation of the Chk2--Brca1 signaling pathway leads to excessive microtubule nucleation and aberrant microtubule--kinetochore attachment. It has been reported that loss of Brca1 in cancer cells confers resistance to paclitaxel treatment,^[@bib40],\ [@bib41]^ which causes microtubule stabilization and mitotic cell death.^[@bib42]^ Similarly, loss of DNA-PKcs is also associated with paclitaxel resistance, thus further supporting the notion that DNA-PKcs regulates microtubule dynamics via the Chk2--Brca1 pathway. The role of the Chk2--Brca1 pathway in k-MT attachments could be modulated or compensated by the Aurora kinases. Aurora-B kinase is known to have an important role in correcting aberrant microtubule attachment through phosphorylation/activation of the mitotic centromere-associated kinesin (microtubule depolymerizing enzyme MCAK), which depolymerizes microtubules.^[@bib43],\ [@bib44]^ Overexpression of MCAK suppresses lagging chromosomes and aneuploidy owing to the loss of Chk2.^[@bib17]^ However, our result indicates that mitotic activation of DNA-PKcs is independent of Aurora-B regulation (data not shown), suggesting that the DNA-PKcs-Chk2--Brca1 axis acts parallel to the Aurora-B pathway to suppress aberrant microtubule attachment and facilitate chromosomal stability. It has also been reported that Aurora-A kinase phosphorylates Brca1 during mitosis and attenuates its E3 ubiquitin ligase activity.^[@bib45]^ It is possible that DNA-PKcs and Aurora-A are required for a balanced Brca1 activity for optimal regulation of microtubule dynamics and k-MT attachments in mitosis. It is conceivable that the defect in this DNA-PKcs-Chk2--Brca1 signaling pathway will result in chromosomal instability, which not only is a hallmark of cancer cells but also an early event and the driving force for tumorigenesis.^[@bib46]^ The connection of Chk2 and Brca1 to cancer development has been well documented, as they have been recognized as multiorgan cancer susceptibility genes.^[@bib47],\ [@bib48]^ In addition, a growing body of evidence has implicated a role of DNA-PKcs in tumorigenesis.^[@bib49]^ For example, progressive loss of DNA-PKcs expression has been correlated to the advancement in ovarian cancer development.^[@bib50]^ Loss of DNA-PKcs expression has also been found in gastric tumors and correlated to a frameshift mutation of the poly(A)10 tract, as well as microsatellite instability, tumor progression and poor patient survival.^[@bib51],\ [@bib52]^ In addition, reduction in DNA-PKcs activity in peripheral blood lymphocytes has been associated with an increase in chromosomal instability and cancer risk.^[@bib53],\ [@bib54]^ Consistent with these analyses, DNA-PKcs kinase inhibition with small-molecule inhibitor not only attenuated mitotic Chk2 phosphorylation ([Figure 1](#fig1){ref-type="fig"}) but also impaired chromosomal alignment and prolonged mitosis progression.^[@bib24]^ The current study thus provides a molecular mechanism that DNA-PKcs-dependent Chk2--Brca1 signaling pathway is critical for proper chromosomal segregation during mitosis and contributes as a barrier against chromosomal instability and tumorigenesis. In summary, our study suggests that DNA-PKcs is the upstream activator of Chk2 and Brca1. The DNA-PKcs-Chk2--Brca1 pathway has an important role in the maintenance of chromosome stability through the regulation of centrosome homeostasis and proper microtubule attachment to the kinetochore. Our results also highlight the role of DNA-PKcs in the control of microtubule dynamics and the response to microtubule damage. Materials and methods ===================== Cell lines and treatment ------------------------ Human cervical cancer HeLa cells, human colorectal carcinoma HCT116 cells and derivative cells^[@bib55]^ were maintained in α-minimum essential medium supplemented with 10% fetal bovine serum and penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO~2~. For mitosis synchrony, cells were treated with 50 ng/ml nocodazole (Sigma, St Louis, MO, USA) for 16 h; in certain experiments, cells were treated for an additional 2 h with kinase inhibitors before harvest. Cells were treated with ionizing radiation using a Mark-II Cesium-137 irradiator (J L Shepherd and Associates) with or without a 30-min pretreatment with kinase inhibitors. Cells were harvested at 1 h after irradiation. Cell transfection with siRNA oligonucleotides or constructs for expression of Chk2 or Brca1^[@bib18]^ was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\'s instructions. Small inhibitory RNA (siRNA) oligonucleotides against DNA-PKcs were previously described.^[@bib24]^ Immunoblotting and antibodies ----------------------------- Whole-cell lysate preparation and western blotting were performed as previously described.^[@bib22],\ [@bib24]^ For immunofluorescent staining, cells were grown on poly-D-lysine-coated culture slides (BD Pharmingen, San Diego, CA, USA), washed in phosphate-buffered saline (PBS), fixed in PBS containing 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked in PBS containing 5% bovine serum albumin. The cells were incubated with indicated primary antibodies for 2 h at room temperature, washed with PBS and incubated with Alexa-568- and Alexa-488-conjugated secondary antibodies for 1 h (Invitrogen). Cells were then washed with PBS and mounted in Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Images were acquired from a Zeiss AxioImager M2 microscope system equipped with a Plan-Apochromat 63 × /NA 1.40 objective, an AxioCam MRm CCD camera and AxioVision software (Carl Zeiss, Oberkochen, Germany). Anti-Chk2 total (Cell Signaling, Beverly, MA, USA), anti-phosphorylated Chk2 at Thr68 (Cell Signaling), anti-phospho-histone H3 (EMD Millipore, Billerica, MA, USA), anti-β-actin (Sigma) and anti-Brca1 total (Santa Cruz, Dallas, TX, USA) antibodies were purchased from the indicated vendors. Antibodies against total and phosphorylated forms of DNA-PKcs were described previously.^[@bib22]^ Microtubule nucleation assay ---------------------------- Cells were treated with cold medium containing 10 μg/ml nocodazole for 40 min, washed with PBS and added to prewarmed fresh medium without nocodazole to allow microtubule regrowth. Cells were fixed at the indicated time and subjected to immunofluorescent staining as described above. Clonogenic survival and MTT cell proliferation assays ----------------------------------------------------- Exponentially growing cells were trypsinized and counted. HCT116 cells were diluted serially to appropriate concentrations, plated into 60-mm dishes in triplicate and treated with paclitaxel (0.5, 1, 2 and 6 n[M]{.smallcaps}). Cells were fixed at 10--14 days and were stained with 4% formaldehyde in PBS containing 0.05% crystal violet. Colonies containing \>50 cells were counted. For cell proliferation assays, 1 × 10^4^ cells were seeded per well in a 96-well plate. Cells were cultured with paclitaxel for 72 h, and then were analyzed for viability using the previously described MTT assay.^[@bib56]^ This work was supported by the National Institutes of Health (CA166677) and the Cancer Prevention Research Institute of Texas (RP110465-P1). We thank Dr Eric Hendrickson for providing the human HCT116 and derivative cell lines. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on the Oncogenesis website (http://www.nature.com/oncsis). The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. {#fig1} ![DNA-PKcs kinase inhibition attenuates mitotic induction of Chk2 phosphorylation. (**a**) Wild-type HCT116 cells were treated with nocodazole for 16 h, followed by a 2 h incubation with DMSO or 10 μ[M]{.smallcaps} Nu7441 (Nu). (**b**) HeLa cells were subjected to nocodazole (50 ng/ml, 16 h), followed by a 2 h incubation with DMSO, Nu7441, Ku55933 or both Nu7441 and Ku55933 (left panel). HeLa cells were also irradiated (4 Gy, 1 h) with or without pretreatment with Nu7441 and Ku55933 (right panel).](oncsis201349f2){#fig2} ![Loss of DNA-PKcs increases mitosis defects and chromosomal instability. (**a**) Chromosome numbers from individual metaphase spreads of HCT116, DNA-PKcs^−/−^ and Ligase4^−/−^ cells (n⩾100). (**b**) HCT116, DNA-PKcs^−/−^ and Ligase4^−/−^ cells were treated with 50 ng/ml nocodazole for 16 h, and, after release into fresh medium, they were treated with 10 μ[M]{.smallcaps} MG132 for 3 h. Cells were stained for α-tubulin (red), crest (green) and DNA (blue). (**c**) Exponentially growing HCT116, DNA-PKcs^−/−^ and Ligase4^−/−^ cells were subjected to the same immunofluorescent staining protocol. Arrowheads indicate lagging chromosome. (**d**) Percentage of mitotic cells with misaligned chromosomes from three independent analyses. (**e**) Percentages of mitotic cells showed lagging chromosome were counted from three independent analyses. \*\**P*\<0.01.](oncsis201349f3){#fig3} {#fig4} {#fig5} {#fig6}

Background {#Sec1} ========== The global travel and tourism industry has expanded rapidly in recent years. The global number of international tourist arrivals increased from approximately 541 million in 1995 to 1161 million in 2014 \[[@CR1]\]. The ever greater numbers present enormous challenges to the entire global community for epidemic preparedness and control. The increasing complexity of frequent international travel opens an ideal route for local outbreaks of infectious disease to becoming global pandemics. The health and wellbeing of travellers warrants appropriate consultation and treatment in its own right, but in the case of infectious diseases of major public health concern, it is important to address the public health aspects of their illness as patients are also disease carriers promoting the spread of infectious disease on a potentially global scale. The International Health Regulations (IHR) (2005) form one of the few legally binding instruments of the World Health Organisation (WHO). The purpose and the scope of IHR (2005) are "to prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade" \[[@CR2]\]. Most of the existing research in travel medicine and the current guidelines for international health authorities emphasise the role of the air-travel industry in tracking and thus containing the potential international spread of infectious disease. For instance, the WHO guideline on International Travel and Health highlights the role of airlines as well as shipping companies, together with that of tour operators and travel agents in limiting the spread of infectious disease across borders \[[@CR3]\]. Similarly, the Centres for Disease Control and Prevention (CDC) guidelines focus on the airline and cruise industries \[[@CR4]\]. The role of the other important sector in tourism, namely the hotel industry, have been less clearly defined and discussed. This case study will use the example of the Metropole Hotel in Hong Kong in the international spread of Severe Acute Respiratory Syndrome (SARS) in 2003, and the effect of the government mandated quarantine of the Metropark Hotel during the swine flu 2009 in Hong Kong. SARS was caused by the SARS-associated coronavirus, with the primary mode of transmission being direct mucous membrane contact with infectious respiratory droplets \[[@CR5], [@CR6]\]. It had a basic reproduction number of approximately 3, and the spread was mainly to those with close contact in health care and household settings. The first cases were identified in Guangdong province of China in November 2002, and a number of natural reservoirs have been found including civet cats and bats. Incubation period ranged from 3 to 10 days \[[@CR7]\]. The overall case fatality ratio was 9.6% worldwide, with deaths resulting from pneumonia and subsequent respiratory failure. The travel industry contributed to the speed of the spread of this unknown disease at that time, particularly within the Metropole Hotel as it was the first site for global dissemination of the virus \[[@CR8]\]. SARS subsequently caused 774 deaths in 26 countries, with the disease spreading to cover five continents \[[@CR7]\]. This case vividly illustrates how a local outbreak can rapidly evolve into a global pandemic. The H1N1 swine flu pandemic in 2009 was caused by the A(H1N1) pdm09 influenza virus \[[@CR9]\]. This strain containing genes from pig, bird and human influenza viruses has never been reported before 2009, and the outbreak was first identified in March 2009 in Mexico and the United States. It was estimated that between 151,700 and 575,400 deaths resulted globally, with 80% in people younger than 65 years old from respiratory and cardiovascular influenza related complications. The 2009 H1N1 virus infection mortality was estimated to be 0.001--0.011% of the world's population, much lower than the previous 0.03% during the 1968 pandemic and the 1--3% of the 1918 pandemic \[[@CR10]\]. The first imported case in Hong Kong was tested positive for swine influenza on the 1 May 2009 \[[@CR11]\]. This has led to the subsequent quarantine of all guests and staff at the Metropark Hotel for 1 week. Hotels can be a critical component in the evolution of a local outbreak into a global pandemic, and an initial contact point of the import of an impending global pandemic. The aim of this paper is to highlight the role of hotel in the spread of epidemic, and discuss control measures that can be implemented. Methods {#Sec2} ======= This case study focuses on the epidemic outbreaks of SARS in 2003 and H1N1 swine flu in 2009 in Hong Kong. Secondary information was extracted from the literature search and the grey literature looking for published official reports, statements, policy papers, field reports and guidelines for further discussion on the role of the hotel industry on the epidemics. Public health principles of disaster response were used to provide the backbone for discussion. MEDLINE was used to identify research articles published between 1 January 2000 and 31 December 2017 in the English language, to answer these specific questions: 1. what factors in the hotel setting contributed to international spread of SARS; 2. the decision making and implementation of hotel quarantine during swine flu, and the impact of the hotel quarantine. Any study design regarding aspects on timelines of sequence of events, environmental sampling and contamination, evaluation of hotel related interventions, modelling of interventions were included. Studies not meeting the above inclusion criteria or answering the research questions was excluded. The search for grey literature involved the searching and browsing the websites of relevant organisations including the World Health Organisation, US CDC, EU European Centre for Disease Prevention and Control, Hong Kong Centre for Health Protection and Department of Health, Food and Health Bureau of Hong Kong SAR government, SARS expert committee, Hong Kong Government information page. MEDLINE (Ovid SP) search was performed using these terms: (Disease outbreaks \[mesh\] OR Epidemics \[mesh\] OR Pandemics \[mesh\] OR Severe Acute Respiratory Syndrome \[mesh\] OR Influenza A Virus, H1N1 Subtype \[mesh\]) AND Hotel \[keyword\]. Results {#Sec3} ======= The search identified 34 records from MEDLINE of which five were relevant (references \[[@CR6], [@CR12]--[@CR15]\]), and an additional 13 records were identified through other sources and the grey literature. SARS 2003 {#Sec4} --------- A medical professor from Guangzhou in China arrived in Hong Kong on 21 February 2003 and checked into a room on the ninth floor of the Metropole Hotel in Kowloon \[[@CR16]\]. During his stay, he infected at least seven other guests and visitors staying on the ninth floor of the hotel including three visitors from Singapore, one visitor from Vietnam, two visitors from Canada and a local individual \[[@CR17]\]. On 8 March 2003, the Singapore Ministry of Health reported to the Hong Kong Department of Health that three patients who presented with pneumonia were admitted to hospital after returning to Singapore from Hong Kong. They had all stayed in the Metropole Hotel. During the conversation, laboratory investigations were pending and there was not sufficient evidence to suggest that their illnesses had been related to the Metropole Hotel \[[@CR16]\]. On 12 March 2003, the WHO issued a global alert about unusual cases of an acute respiratory syndrome. On 14 March 2003, the index case for the significant outbreak at the Prince of Wales Hospital was identified. It was not until 19 March 2003, after multiple enquiries, the patient revealed that he had visited the Metropole Hotel around that period as a visitor but not a guest \[[@CR16]\]. On the same day, the Hong Kong Department of Health reported the chain of transmission of the outbreak at the Metropole Hotel \[[@CR16]\]. The Metropole Hotel exemplified the potential international spread of infectious diseases. The index cases in the Hong Kong, Toronto, Singapore and Hanoi outbreaks were all associated with the hotel. SARS patients in Ireland and United States had also visited the Metropole Hotel around the same time when there were other sick guests present in the hotel \[[@CR12]--[@CR14], [@CR18]\]. Subsequently there were hospital outbreaks when these index cases returned and were treated at their home countries. Table [1](#Tab1){ref-type="table"} showed the timeline of the 2003 SARS action of the Department of Health.Table 1Hong Kong Health Authority's action to hotel industry during SARS 2003DateAction by the Health Authority21 February 2003A medical professor from Guangzhou in China arrived in Hong Kong and checked into a room on the ninth floor of the Metropole Hotel in Kowloon. He was later found to be the source of SARS outbreak in Hotel M and beyond.14 March 2003The Secretary for Health, Welfare and Food sets up and chairs the 1st meeting of Task Force on SARS.19 March 2003Department of Health (DH) announced the chain of transmission of at least 7 SARS cases were related to the Hotel M, the index case of the epidemic in Hong Kong.19 and 20 March 2003DH's Kowloon Regional Office conducted site investigation at Hotel M. DH ensured hotel management conducted proper cleansing and disinfection.22 March 2003DH inspected hotel environment inspected and found satisfactory and informed hotel management that they could resume business on 9/F.28 May and 6 June 2003DH met with tourism and hotel industries, Tourism Commission and Tourism Board on health awareness programme for visitors.Note: Materials extracted from references \[[@CR16], [@CR35]\] Little was known about the new disease SARS when the outbreak began at hotel and hospitals in February and early March. The WHO did not issue its first emergency travel advisory naming the illness as SARS until 15 March 2003 \[[@CR16]\]. There was local media coverage of an outbreak of atypical pneumonia in Guangzhou on 10 February 2003, and the health authority in Hong Kong had made contact with the Guangzhou and Guangdong authorities. However, accurate information about the atypical pneumonia outbreak in Guangdong Province was not available to Hong Kong or the international community at the time. Case investigation and contact tracing conducted by the Department of Health on 24 February 2003 on the index case revealed that the Guangzhou professor and his wife had stayed at the Metropole Hotel. However, no contact tracing was conducted in the hotel at that stage since the Department of Health had not received any other reports of severe community-acquired pneumonia related to the hotel \[[@CR16]\]. There were no environmental factors or triggers identified that warranted further action. However, environmental sampling on the carpet outside the room in which the index case resided, and elevator area showed a hot zone which tested positive for the SARS virus by polymerase chain reaction (PCR) 3 months after the index case stayed at the hotel \[[@CR19]\]. Another study investigating German guests staying at the hotel also suggested the possibility of environmental contamination as a source of infection \[[@CR15]\]. It is not known how long the infectious virus persists in the surroundings of a SARS patient. The established practice was for contact tracing to be conducted on close contacts (friends or family), but not on the basis of a shared location \[[@CR16]\]. A number of researchers estimated the basic reproduction number of SARS by fitting models to the initial growth of the epidemics in a number of countries \[[@CR19]\]. Modelling of SARS epidemiology in Hong Kong and China showed rapid public health measures such as contact tracing for confirmed and suspected cases, and quarantining, monitoring, and restricting the travel of contacts had an effective reduction in reproduction number \[[@CR20]--[@CR23]\]. Swine flu 2009 {#Sec5} -------------- A 25-year-old male from Mexico arrived in Hong Kong on 30 April 2009, and stayed at the Metropark Hotel. He attended hospital on the same evening where he was admitted to an isolation ward. He subsequently developed a fever and was confirmed to have swine flu on 1 May 2009 \[[@CR11]\]. The Hong Kong Special Administrative Region (HKSAR) government raised the response level to 'Emergency' on the same day under the *Emergency Preparedness Plan for Influenza Pandemic*. An 'Emergency Response Level Steering Committee on Human Swine Influenza (Flu A H1N1) Pandemic' was also established on the 1st May to formulate the overall disease control strategy \[[@CR11]\]. Under the *Prevention and Control of Disease Ordinance*, the Director of Health ordered that all guests and staff at the Metropark Hotel should be quarantined on the evening of 1 May 2009 \[[@CR11]\]. The quarantine was led by the Department of Health in collaboration with other government departments. Quarantined persons were provided with oseltamivir (Tamiflu) and other medical treatment. The Social Welfare Department provided daily necessities and emotional support to the quarantined. A help desk was set up at the hotel involving the Department of Health, the Home Affairs Department, the Social Welfare Department, the Immigration Department, the Civil Aid Service, the Auxiliary Medical Service and the Police. The quarantine ended 1 week later, which covered the incubation period of influenza of 1 to 7 days. For those persons who had completed the quarantine period without showing symptoms of being infected, they were issued with Certificates of Conclusion of Quarantine. At the same time, the Centre for Health Protection (CHP), other government departments and relevant agencies conducted contact tracing starting on the 1st May 2019. Close and selected social contacts were prescribed chemoprophylaxis and put under medical surveillance. The hotel and nearby streets, as well as other public places, were cleansed and disinfected. Hygiene guidelines had been issued to all licensed hotels/guesthouses and rented rooms to encourage enhanced cleansing and improvement of hygiene. All industrial associations had been informed of the situation and reminded the need to take precautionary measures. The Occupational Safety and Health Council had organised public seminars to raise public awareness of preparedness for influenza in the workplace \[[@CR11]\]. Table [2](#Tab2){ref-type="table"} showed the timeline of the 2009 swine flu action of the Department of Health.Table 2Hong Kong Health Authority's action to hotel industry during Swine flu 2009DateAction by the Health Authority30 April 2009A 25-year-old male from Mexico (index patient) arrived in Hong Kong and stayed at the Metropark Hotel.1 to 7 May 2009The Director of Health ordered that the Metropark Hotel should be isolated. All guests and staff were quarantined. Quarantined persons were provided with oseltamivir (Tamiflu) and other medical treatment.1 May 2009Department of Health conducted contact tracing in the disease containment phase1 May 2009Cleansing and disinfection of the hotel lobby and common areas as well as bathrooms in individual guest rooms have been arranged by Food and Environmental Hygiene Department (FEHD).Early May 2009The Metropark Hotel as well as 8 other hotels/hostels in which passengers of flight MU505 (flight of the index patient) had stayed have been disinfected.Early May 2009Hygiene guidelines have been issued to all owners' corporations, owners' committee, mutual assistance committees and the Hong Kong Association of Property Management Companies, licensed hotels/guesthouses and bed space apartments to encourage enhanced cleansing and improvement of hygiene.Note: Materials taken from references \[[@CR11], [@CR36]\] Characteristics of hotels involved {#Sec6} ---------------------------------- Both the Metropole Hotel at Kowloon (now renamed Metropark Hotel Kowloon) and the Metropark Hotel at Wan Chai (now renamed Kew Green Hotel) were four stars hotels situated at busy part of the city. Both hotels were managed by the same management group -- the China Travel Service (Holdings) Hong Kong Limited. The two hotels were no different in terms of access to public health facilities and general standard of care. The difference in the timing of public health actions by the health authority was likely contributed by experience of SARS preceding swine flu. Government initiatives on epidemic preparedness with the hotel industry after SARS and swine flu {#Sec7} ------------------------------------------------------------------------------------------------ After the SARS outbreak in Hong Kong the health authority established the Guidelines for Hotels in Preventing Severe Acute Respiratory Syndrome (SARS) \[[@CR24]\] and Guidelines on Infection Control & Prevention in the Hotel Industry \[[@CR25]\]. The guidelines provided practical information for hotel staff members on how measures to prevent communicable diseases should be done. It offered comprehensive information on ways to implement infection control measures, in particular the maintenance of good hygiene on hotel premises \[[@CR25]\]. The CHP organised Infection Control Seminars for the Hotel Industry on a regular basis. For instance, in response to the Ebola virus outbreak in West Africa in 2014--2016, the CHP provided advice for the local hotel industry on receiving guests with a travel history or residence in an Ebola virus disease affected area. The guideline stressed the importance of enquiring about the travel history of guests and outlined procedures on handling these guests who may feel unwell. The guideline reiterated the need to keep a record of staff and residents who had stayed in the hotel, with their personal and contact details, for possible future public health actions and contact tracing \[[@CR26]\]. Ideally, hotels should be setting their own standards of hygiene measures and providing training to staff before an outbreak occurs. Further roles and responsibilities included contingency arrangement, plan of acquisition of protective equipment, disease reporting and surveillance mechanism during outbreak period \[[@CR27]\]. From our online and database internet search, however, there is little mention of collaboration between the government and the hotel industry. No documentation was found on setting up of task forces or committees, or of invitation to hotel representatives to the working group advising on infection control guidelines in Hong Kong. Discussion {#Sec8} ========== SARS served as the classic example of how tourism and international travel can present challenges to the global health system. The spread of the illness within a single hotel and the subsequent international air travel of the victims contributed to and accelerated the speed of the spread of SARS across the globe. The experience from SARS in Hong Kong had a profound impact on the public health reform especially on the infectious disease surveillance and epidemic response \[[@CR16]\]. These included strengthening the surveillance and the isolation and treatment of individuals with the disease according to case definitions, the establishment of communication channels between hotel and the government system, and the development of guidelines and response plans that allows the implementation of stringent infection control measures when necessary. The establishment of contingency plans and command structures including the 'Emergency Response Level Steering Committee on Influenza Pandemic' allowed a clear structural framework and key lines of responsibility \[[@CR28]\]. However, collaboration with the private sector and the hotel industry were found to be limited and focused around infection control measures. According to subsequently published literature, application of appropriate measures had likely reduced the number of people who were infected, requiring medical care and died during the influenza pandemic \[[@CR29]\]. It has been shown that case isolation or household quarantine could have a significant impact at reducing attack rates in the community, and chemoprophylaxis can greatly reduce disease transmission during the pandemic \[[@CR30], [@CR31]\]. However, the quarantine of guests at the Metropark Hotel in 2009 inevitably stirs up much discussion and controversy among the media and the public health community on the balance between the need to protect the public health and the need to safeguard civil liberties. The decision of quarantine created enormous tension between the government, guests and the hotel management. The decision of the need for quarantine and the scale of the quarantine needs to be scientifically justified. The negative effects overall of such a policy on the tourism attractiveness of a destination cannot be neglected. The quarantine at the Metropark Hotel during swine flu also highlighted the extensive assistance needed for the quarantined persons, and the cooperation necessary in the possible future need for a hotel quarantine. Pre-established partnerships and coordination between the government and the hotel industry is key in epidemic preparedness and response. Effects of epidemics on the hotel industry {#Sec9} ------------------------------------------ Studies have shown that the psychological impacts of SARS and the government restrictions on travel, had a great impact on the travel industry far beyond the region of SARS hit areas \[[@CR32]\]. For the hotel industry in Hong Kong, the number of hotel guests dropped dramatically to a level that was never experienced before \[[@CR33]\]. In order for hotels to sustain their business, the Hong Kong hotel industry adopted an industry-wide recovery effort and empathized on mutual support \[[@CR26]\]. Previous papers called for a better preparedness of the hotel industry for future crises and epidemics \[[@CR32], [@CR33]\]. Collaborations with the hotel industry to mitigate the impact of epidemics {#Sec10} -------------------------------------------------------------------------- Hotels are often the first point of contact for tourists arriving at a host country. Hotels could provide an additional line of defence beyond entry border screening, and they could offer another layer of protection against illnesses that border screening processes may have missed, for example in the situation where travel occurs during the incubation period of an infectious disease. In view of this, the capacity of hotels in the detection of potential illness and the launching of an initial response should be fully recognised and utilised. The WHO pandemic influenza risk management recommended involving civil society and the private business sector in pandemic preparedness planning and national committees \[[@CR34]\]. The case of hotel industry collaboration with the health sector in Hong Kong has the potential to provide a positive example of effective disaster risk reduction coordination. Conclusions {#Sec11} =========== The epidemic preparedness and infection control measures mounted against SARS and H1N1 swine flu demonstrated a role that needed to be filled by the hotel industry. During SARS, late recognition of the environmental contamination of hotel facilities and the failure of timely intervention on the hotel guests with close contact contributed to the spread of the disease internationally. While the appropriateness and best method of quarantine in future pandemic influenza warrants further research, the 2009 swine flu hotel quarantine exposed gaps in the partnership with hotel industry. Health authorities in Hong Kong had since provided guidelines mostly in the area of disinfection and hygiene, and focused on educating hotel workers on basic hygiene to prevent the spread of infectious diseases. The potential to establish traveller screening, timely reporting and isolation for the infected guests during epidemics could be explored. The capacity of the hotel industry in controlling infections should be recognised not only in Hong Kong but also in other parts of the world. Not applicable. Funding {#FPar1} ======= No funding available for this study. Availability of data and materials {#FPar2} ================================== Not applicable. Conceptualization, KH and CM; Data curation, CM and MY; Formal analysis, KH and CG; Supervision, EC and CG; Writing -- original draft, KH and CM; Writing -- review & editing, MY, EC and CG. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== Not applicable. Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mangroves are intertidal ecosystems that have a pantropical distribution. The distributional range of species inhabiting these ecosystems is typically restricted to either the Indo-West Pacific (IWP) region or the Atlantic-East Pacific (AEP) region ([@bib12]). How this pattern of distribution formed is one of the main biogeographic questions in mangrove research. Phylogenetic studies have detected significant levels of divergence in several tree species across the IWP and AEP (*Rhizophora* L. in [@bib3] and [@bib11]; and *Hibiscus* L. in [@bib9]). However, the divergence history, at a global scale, of many other mangrove plants remains to be clarified. *Acrostichum aureum* L. (common name "mangrove fern"; Pteridaceae) is of particular interest because this species is the only mangrove plant that is distributed pantropically (i.e., in both the IWP and AEP regions). This species also differs from other mangrove plants in that it has wind-dispersed spores, while most other mangrove plants have sea-dispersed seeds, fruits, or propagules. This different dispersal system might have enabled this species to achieve its relatively wide distribution compared to other mangrove plants. To address this question, it is important to perform population genetic studies to analyze the genetic structure and demographic history of the species using highly polymorphic microsatellite markers. Therefore, we developed novel microsatellite markers for *A. aureum* using next-generation sequencing. We tested the markers on samples from across the pantropical distribution of the species to check their levels of polymorphism and to determine their usefulness as markers for future studies. METHODS AND RESULTS =================== One sample of *A. aureum* was collected from Sabah (Malaysia) ([Appendix 1](#app1){ref-type="app"}), and total DNA extracted using a DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). We then performed shotgun sequencing, using one-third of a run on a Roche 454 Genome Sequencer Junior (Roche Applied Science, Penzberg, Germany). The GS Junior Titanium Sequencing Kit (Roche Applied Science) and Multiplex Identifier (MID) adaptors (see [@bib6]) were used following the manufacturer's protocol. The run generated a total of 81,415 reads with an average length of 490 bp. The program QDD version 2.1 ([@bib7]) was used to identify di- to hexanucleotide motif microsatellites with at least five repeats. Sequence similarity and establishment contigs were detected following the procedure in [@bib10]. A total of 1452 perfect microsatellite sequences were obtained and 48 primer pairs designed using the following criteria: (1) PCR product size of 80--300 bp; (2) flanking region containing at least five repetitions of any di- to hexanucleotide motifs; and (3) primers with length 18--27 bp, annealing temperature 57--63°C, and GC content 20--80%. Forty-eight primer pairs with at least 12 repeats of various fragment sizes appropriate for multiplex PCR were selected. The 5′-tailed primer method ([@bib8]) was used to label and visualize the PCR amplicons of the selected primers. The 19-bp U19 sequence (GGTTTTCCCAGTCACGACG) was added to the 5′-tail of forward primers, and the GTTT PIG-tail was added to the 5′ end of the reverse primer. This PIG-tail facilitates the addition of adenosine by *Taq* polymerase, thereby reducing stuttering ([@bib1]). PCR amplification tests of each primer pair were performed in individual PCR reactions using two individuals, collected from Sabah (Malaysia) and Pará (Brazil), using the standard protocol of QIAGEN Type-it Microsatellite PCR Kit (QIAGEN), with a final volume of 5.0 μL and 1.0 μM of each primer. The PCR thermal conditions were as follows: initial denaturation at 95°C for 5 min; 30--32 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 90 s, extension at 72°C for 30 s; and final extension at 60°C for 30 min. The PCR products were electrophoretically separated on 1.5% agarose gels stained with ethidium bromide. Thirty loci exhibited clear PCR amplification. Twenty-four individuals sampled from Sabah (Malaysia) were used to assess the quality of amplification and polymorphism of these 30 loci. Loci were amplified using QIAGEN Type-it Microsatellite PCR Kits (QIAGEN) in three tubes, each with 5.0-μL mixtures containing 0.5 μL of 1--10 ng of genomic DNA, 2.5 μL of multiplex PCR master mix buffer, 1.2 μL of primer mix (with the concentration of each primer pair adjusted from 1.0 μM), and 0.8 μL of U19 fluorescent dye--labeled primer (6-FAM, VIC, NED, or PET; 1.0 μM). We used the same PCR protocol as described above. Twenty-seven of the 30 loci showed clear fragment patterns using one singleplex and 11 multiplex PCR sets (two to three primer pairs per multiplex; [Table 1](#tbl1){ref-type="table"}). Samples from two more populations (16 individuals each from Piti \[Guam\] and Pará \[Brazil\]; [Appendix 1](#app1){ref-type="app"}) were then included to check the genetic diversity of these loci. Cross-species amplification of these loci was also assessed by testing in the other two species in the genus *Acrostichum* L.: four individuals of *A. danaeifolium* Langsd. & Fisch. collected in Pará (Brazil) and Colima (Mexico), and four individuals of *A. speciosum* Willd. from Sungei Buloh (Singapore) ([Appendix 1](#app1){ref-type="app"}). ###### Characteristics of 27 microsatellite markers developed for *Acrostichum aureum*.^[a](#tblfn1){ref-type="table-fn"},[b](#tblfn2){ref-type="table-fn"}^ Locus Primer sequences (5′--3′) Repeat motif Allele size range (bp) Fluorescent dye Multiplex DDBJ accession no. ------- -------------------------------------------------- -------------- ------------------------ ----------------- ----------- -------------------- AA07 F: GGTTTTCCCAGTCACGACAATGGGCTACTCAAATGGG (GA)~17~ 194--246 FAM Set 1 LC065390 R: GTTTGTGTTCCTTGTATGTCGATCAAT AA08 F: GGTTTTCCCAGTCACGACGAAGAGGTGGGACAAGCAAG (AG)~16~ 120--150 VIC Set 4 LC065391 R: GTTTGTGGTTGAGAGTGGGTTGA AA09 F: GGTTTTCCCAGTCACGACGTGCGATGGCTACTTCTCCT (AG)~15~ 144--170 FAM Set 1 LC065392 R: GTTTCCCTTTCTCCACACTCC AA10 F: GGTTTTCCCAGTCACGACAGCCTTGCAACCTGCTCTAC (AC)~15~ 197--259 VIC Set 4 LC065393 R: GTTTCCATCATGGCCAGCTTTACT AA11 F: GGTTTTCCCAGTCACGACCCGTAGGCTCTGATACCAA (AC)~15~ 129--159 NED Set 8 LC065394 R: GTTTCTCCCATGTCAAACACTCCA AA12 F: GGTTTTCCCAGTCACGACGCCAGCCTAGACACCTCTTG (TG)~15~ 123--159 VIC Set 5 LC065395 R: GTTTGCATGCATAAGAAGACCAACC AA14 F: GGTTTTCCCAGTCACGACAGGTCAAGCACAAGCTCAA (AG)~14~ 169--177 PET Set 10 LC065396 R: GTTTACACCTGCACACTGGTGAGT AA15 F: GGTTTTCCCAGTCACGACAGTTCTTGTCTTGGGTGAGCA (TG)~14~ 269--281 PET Set 10 LC065397 R: GTTTGGAGTAAGCTTGGTGCATATC AA16 F: GGTTTTCCCAGTCACGACGGTGCAAGGAGATGCCATAG (GA)~14~ 114--134 NED Set 9 LC065398 R: GTTTAGTCAGGGTCGTTCAAGCTG AA17 F: GGTTTTCCCAGTCACGACGGGTGTGAGGGATTTGAGAA (AG)~14~ 118--182 VIC Set 6 LC065399 R: GTTTATCGTTGGAGATGATGGAGG AA23 F: GGTTTTCCCAGTCACGACGAGAGGAGAGAAGCAAATAGGG (GA)~12~ 285--293 NED Set 7 LC065400 R: GTTTGGAGTCTTGGTAGACGGG AA24 F: GGTTTTCCCAGTCACGTTGAGCCAATGAAATGCT (TG)~11~ 267--269 FAM Set 2 LC065401 R: GTTTAGGAAGAGAAAGCGAGGGAG AA27 F: GGTTTTCCCAGTCACGACGTTGTCCTCTACTTGAGCTCCC (CA)~15~ 140--152 NED Set 7 LC065402 R: GTTTCACACAAGAGAGCATGTTTGTA AA28 F: GGTTTTCCCAGTCACGACGTCTCCTGAAGGGAGTGGTGA (GA)~15~ 84--128 VIC Set 6 LC065403 R: GTTTGAGTTCCACACCATGCCAG AA29 F: GGTTTTCCCAGTCACGACGAAAGATGCAAAGAAAGGGAGA (AC)~15~ 103--135 FAM Set 3 LC065404 R: GTTTGAAGATGAGAAGTGTGGTCG AA30 F: GGTTTTCCCAGTCACGACGTCTTCAAGTGTCTTGGGTTTGA (AC)~14~ 104--124 FAM Set 2 LC065405 R: GTTTATTCATGAGGAGCATGACCTA AA33 F: GGTTTTCCCAGTCACGACGCGCACCTTGTCCAAGTAAGC (AT)~13~ 160--172 FAM Set 2 LC065406 R: GTTTGGAATAGGTAATGGAGTAGACTTGA AA34 F: GGTTTTCCCAGTCACGACGTCTTCAATCCTCTCTATAAACTAGCG (CA)~13~ 188--216 PET Set 12 LC065407 R: GTTTCTCACAAGGGAGGCTATCCA AA35 F: GGTTTTCCCAGTCACGACGATGAAGCCAAGATCCCAAA (GA)~13~ 352--376 FAM Set 1 LC065408 R: GTTTGCCACCACACCTTCTCTGAT AA37 F: GGTTTTCCCAGTCACGACGTTCCGATCCTTGTTGGTAGC (AG)~13~ 173--219 VIC Set 5 LC065409 R: GTTTAAGTGGACGGCGTAATCAAG AA38 F: GGTTTTCCCAGTCACGACGCAATGGCGAATAGCGAAGC (TG)~13~ 205--223 NED Set 9 LC065410 R: GTTTGTCACCCAAGACTCCCTCT AA40 F: GGTTTTCCCAGTCACGACGTTGCAGGTTAGAGCTCCCAT (TC)~13~ 145--163 PET Set 11 LC065411 R: GTTTAGTGTCCACCAACCATCCA AA41 F: GGTTTTCCCAGTCACGACGTTGATGCAAATCAACCCTTT (CT)~13~ 167--199 NED Set 8 LC065412 R: GTTTCATGATCCTTACCTTGCCC AA42 F: GGTTTTCCCAGTCACGACGAAGGATTGATGCAACCAAGG (AC)~13~ 145--165 PET Set 12 LC065413 R: GTTTCCAATGTGAGCCATCAAGG AA43 F: GGTTTTCCCAGTCACGACGTTGGATGGACCTTCTTCGTC (CA)~13~ 313--315 VIC Set 4 LC065414 R: GTTTGATGCTCTGATCCCTCCTT AA46 F: GGTTTTCCCAGTCACGACGGGAGTGTGACAAGGTGTAAGA (CT)~12~ 176--224 FAM Set 3 LC065415 R: GTTTGACCGAGGCCAAGAATAAGG AA48 F: GGTTTTCCCAGTCACGACGTTCTACACGTGGTGGGAGGT (AG)~12~ 114--136 PET Set 10 LC065416 R: GTTTCAAGGCTTCATATGAGGTGAG *Note*: DDBJ = DNA Data Bank of Japan. All values are based on 56 samples representing Sabah (Malaysia), Piti (Guam), and Pará (Brazil) populations ([Appendix 1](#app1){ref-type="app"}). Annealing temperature for all loci was 57°C. The amplified products were loaded into an ABI3500 automatic sequencer (Applied Biosystems, Waltham, Massachusetts, USA) with GeneScan 600 LIZ Size Standard (Applied Biosystems), and their sizes and genotypes were determined using GeneMarker ([@bib5]). Expected heterozygosity (*H*~e~) and fixation index (*F*~IS~) were calculated to evaluate genetic diversity of the three populations using FSTAT version 2.9.3.2 ([@bib4]; hereafter, FSTAT). The significance of deviations of *F*~IS~ from zero, as evidenced by deviation from Hardy--Weinberg equilibrium, and genotypic disequilibrium for all locus pairs, were tested by randomization using FSTAT. The obtained *P* values (with a 0.05 significance threshold) were adjusted based on a sequential Bonferroni correction. The presence of null alleles and their bias on genetic diversity among the three populations (*F*~ST~) ([@bib13]) were evaluated using FreeNA ([@bib2]). In the Sabah population, the number of alleles detected and *H*~e~ ranged from one to 15 and 0.000 to 0.893, respectively, and 26 of the 27 loci were polymorphic ([Table 2](#tbl2){ref-type="table"}). A significant deviation in *F*~IS~ was found in only one locus (AA16). Although null alleles were detected and their frequencies estimated at each locus ([Table 4](#tbl4){ref-type="table"}), the *F*~ST~ value after the null allele correction was 0.619, the same as the original value without correction (= 0.619), suggesting that biases, due to null alleles, in genetic structure analysis would be limited. Although 19 of the 27 loci were amplified in samples from the other two populations, most were fixed for different alleles among populations. Seven and six loci were amplified in *A. danaeifolium* and *A. speciosum*, respectively ([Table 3](#tbl3){ref-type="table"}). ###### Genetic variation of the 27 newly developed microsatellite markers in three *Acrostichum aureum* populations.[^a^](#tblfn3){ref-type="table-fn"} Locus Sabah, Malaysia (*N* = 24) Piti, Guam (*N* = 16) Pará, Brazil (*N* = 16) ------- ---------------------------- ----------------------- ------------------------- ----------------------------------------- ----- ------- ------- ------------------------------------------ ----- ------- ------- ------------------------------------------ AA07 12 0.875 0.893 0.052 1 0.000 0.000 NA 1 0.000 0.000 NA AA08 10 0.870 0.883 0.037 1 0.000 0.000 NA 2 0.133 0.391 0.678 AA09 9 0.563 0.758 0.288 --- --- --- --- 1 0.000 0.000 NA AA10 13 0.647 0.870 0.285 2 1.000 0.500 −1.000[\*](#tblfn7){ref-type="table-fn"} 2 1.000 0.500 −1.000[\*](#tblfn7){ref-type="table-fn"} AA11 6 0.542 0.682 0.226 --- --- --- --- --- --- --- --- AA12 8 0.625 0.752 0.200 1 0.000 0.000 NA 1 0.000 0.000 NA AA14 4 0.375 0.541 0.326 1 0.000 0.000 NA 1 0.000 0.000 NA AA15 6 0.583 0.523 −0.095 2 0.000 0.305 1 2 0.000 0.219 1.000 AA16 8 0.333 0.782 0.590[\*](#tblfn7){ref-type="table-fn"} --- --- --- --- --- --- --- --- AA17 10 0.824 0.804 0.007 1 0.000 0.000 NA 1 0.000 0.000 NA AA23 5 0.762 0.667 −0.119 1 0.000 0.000 NA 1 0.000 0.000 NA AA24 1 0.000 0.000 NA 1 0.000 0.000 NA --- --- --- --- AA27 8 0.773 0.744 −0.016 1 0.000 0.000 NA 1 0.000 0.000 NA AA28 15 0.722 0.773 0.094 1 0.000 0.000 NA 1 0.000 0.000 NA AA29 9 0.765 0.827 0.105 1 0.000 0.000 NA 1 0.000 0.000 NA AA30 6 0.583 0.681 0.164 1 0.000 0.000 NA 1 0.000 0.000 NA AA33 5 0.375 0.490 0.255 1 0.000 0.000 NA --- --- --- --- AA34 8 0.714 0.659 −0.060 1 0.000 0.000 NA 3 0.188 0.174 −0.047 AA35 10 0.875 0.855 −0.002 1 0.000 0.000 NA 2 0.188 0.170 −0.071 AA37 9 0.688 0.732 0.093 2 0.000 0.305 1.000[\*](#tblfn7){ref-type="table-fn"} 2 0.063 0.061 0.000 AA38 9 0.682 0.830 0.201 1 0.000 0.000 NA 1 0.000 0.000 NA AA40 10 0.833 0.852 0.044 1 0.000 0.000 NA 2 0.067 0.064 0.000 AA41 9 0.792 0.813 0.048 3 0.250 0.584 0.593 1 0.000 0.000 NA AA42 10 0.857 0.862 0.030 3 0.250 0.498 0.522 1 0.000 0.000 NA AA43 2 0.063 0.170 0.651 --- --- --- --- --- --- --- --- AA46 13 0.941 0.796 −0.153 1 0.000 0.000 NA --- --- --- --- AA48 9 0.583 0.745 0.237 1 0.000 0.000 NA 1 0.000 0.000 NA *Note*: --- = not amplified; *A* = number of detected alleles; *F*~IS~ = fixation index; *H*~e~ = expected heterozygosity; *N* = number of individuals genotyped; NA = not applicable. Voucher and locality information are provided in [Appendix 1](#app1){ref-type="app"}. Significant deviation from Hardy--Weinberg equilibrium (*P* \< 0.05). ###### Fragment sizes detected in cross-amplification tests of the 27 newly developed *Acrostichum aureum* microsatellite markers in two closely related species.[^a^](#tblfn4){ref-type="table-fn"} Locus *A. danaeifolium* *A. speciosum* ------- ------------------------ -------------------------- ----------------------------------- Pará, Brazil (*N* = 4) Colima, Mexico (*N* = 4) Sungei Buloh, Singapore (*N* = 4) AA07 --- --- 188 AA08 --- --- --- AA09 134--2154 134--2158 154--2158 AA10 --- --- --- AA11 --- --- --- AA12 --- --- 120--2132 AA14 --- --- --- AA15 --- --- --- AA16 --- --- --- AA17 --- --- --- AA23 --- --- --- AA24 --- --- --- AA27 --- --- 144 AA28 --- --- --- AA29 --- --- 110--2112 AA30 --- --- --- AA33 --- --- --- AA34 213 --- --- AA35 316 --- --- AA37 --- --- --- AA38 273 --- --- AA40 --- --- --- AA41 207--219 207--2217 --- AA42 175 --- --- AA43 344--2348 344--2348 --- AA46 --- --- 217--2221 AA48 --- --- --- *Note*: --- = not amplified. Voucher and locality information are provided in [Appendix 1](#app1){ref-type="app"}. ###### Null allele frequencies at each locus estimated by FreeNA software in three *Acrostichum aureum* populations.[^a^](#tblfn5){ref-type="table-fn"} Locus Sabah, Malaysia (*N* = 24) Piti, Guam (*N* = 16) Pará, Brazil (*N* = 16) ------- ---------------------------- ----------------------- ------------------------- AA07 0.000 0.001 0.001 AA08 0.000 0.001 0.200 AA09 0.122 NA 0.001 AA10 0.114 0.000 0.000 AA11 0.111 NA NA AA12 0.086 0.001 0.001 AA14 0.111 0.001 0.001 AA15 0.000 0.263 0.224 AA16 0.247 NA NA AA17 0.000 0.001 0.001 AA23 0.000 0.001 0.001 AA24 0.001 0.001 NA AA27 0.000 0.001 0.001 AA28 0.000 0.001 0.001 AA29 0.053 0.001 0.001 AA30 0.048 0.001 0.001 AA33 0.090 0.001 NA AA34 0.000 0.001 0.000 AA35 0.000 0.001 0.000 AA37 0.065 0.263 0.000 AA38 0.054 0.001 0.001 AA40 0.000 0.001 0.000 AA41 0.000 0.211 0.001 AA42 0.000 0.175 0.001 AA43 0.138 NA NA AA46 0.000 0.001 NA AA48 0.098 0.001 0.001 *Note*: NA = not applicable. Voucher and locality information are provided in [Appendix 1](#app1){ref-type="app"}. CONCLUSIONS =========== The 26 polymorphic microsatellite markers developed in this study will be useful to evaluate the genetic structure and to infer the past demographic history of *A. aureum* to study how this mangrove fern achieved the widest distributional range of all mangrove plants. Cross-species amplification also suggested that some markers could be used to evaluate genetic diversity in other species in the same genus. ###### Voucher information for *Acrostichum* species used in this study. Species Voucher specimen accession no.[^a^](#tblfn6){ref-type="table-fn"} Collection locality Geographic coordinates *N* ------------------------------------ ------------------------------------------------------------------- ----------------------------------- -------------------------- ----- *A. aureum* L. TK 11072403 (348--371) (URO) Klias, Sabah, Malaysia 5.426454°, 115.559861° 24 *A. aureum* TK 13122001 (1--16) (URO) Piti, Guam 13.440381°, 144.678365° 16 *A. aureum* GMM 14112102 (207--222) (UEC) Perimirim, Pará, Brazil −0.973032°, −46.591348° 16 *A. danaeifolium* Langsd. & Fisch. GMM 14112201 (231--234) (UEC) Capanema, Pará, Brazil −1.299979°, −47.099699° 4 *A. danaeifolium* TK 14071804 (50--53) (URO) Ciudad de Armería, Colima, Mexico 18.912410°, −104.036289° 4 *A. speciosum* Willd. TK 091112003 (61--63) (URO) Sungei Buloh, Singapore 1.449007°, 103.730684° 4 *Note*: *N* = number of individuals sampled. Collectors and herbaria: GMM = Gustavo Maruyama Mori; TK = Tadashi Kajita; UEC = Universidade Estadual de Campinas herbarium; URO = Herbarium, Faculty of Education, University of the Ryukyus. [^1]: The authors thank all those who provided support during field collections and Dr. Leanne Faulks for critical reading of this paper. This work was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI 22405005 and 25290080 to T.K.), JSPS Invitation Program for East Asian Young Researchers (JENESYS) Program 2009 and 2011 to Chiba University (coordinated by T.K.), the Ministry of Education, Culture, Sports, Science, and Technology TOBITATE! Young Ambassador Program 2014 (to T. Yamakawa), and the Chiba University International Exchange Program to Support Sending Graduate Students Abroad (FY2014 to T. Yamakawa). [^2]: These authors contributed equally to this work.

Background ========== Peripheral T-cell lymphomas (PTCLs) represent an heterogeneous group of non-Hodgkin\'s lymphomas (NHL), characterized by poor outcome, accounting approximately for 10%-15% of all non-Hodgkin lymphomas in the western countries, and with an higher prevalence in Asia \[[@B1],[@B2]\]. Peripheral T-cell lymphomas derive from lymphocytes at the post-thymic stage of maturation. According to recent WHO (World Health Organization) classification more than 20 biologically and clinically distinct entities of Peripheral T-cell lymphomas have been described, such as Peripheral T-cell lymphomas Not Otherwise Specified (NOS), angioimmunoblastic T-cell lymphoma (AITL), natural killer/T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL) and anaplastic large-cell lymphoma (ALCL), the most common ones \[[@B3]\]. Cutaneous lymphomas represent a distinct entity of T lymphomas according to WHO, because same of those show even an indolent course \[[@B4]\]. Unlike other non-Hodgkin\'s lymphomas, only two subtypes of Peripheral T-cell lymphomas are characterized by disease-defining genetic abnormalities, such as the t(2;5)(p23;q35) in anaplastic large-cell lymphoma and DNA integration of human T-lymphotropic virus 1 (HTLV1) in adult T-cell leukemia/lymphoma \[[@B5],[@B6]\]. Peripheral T-cell lymphomas- Not Otherwise Specified account approximatively for 60-70% of T-cell lymphomas and it cannot be furtherly classified on the basis of morphology, phenotype, and conventional molecular studies, representing often a diagnosis of exclusion with respect to other T cell lymphomas histotypes \[[@B7]\]. *Cyclin D1* is well-established human oncogene, frequently deregulated in cancer, playing a specific role in cancer phenotype characterization and disease progression \[[@B8]\]. Cyclin D1 over-expression is often due to chromosomal aberration. Among lymphomas, translocation (11;14)(q13;q32) is typically observed in mantle cell lymphoma. Thus the *cyclin D1* gene at chromosome 11q13 is juxtaposed to *IgH* gene on chromosome 14q32, resulting in overexpression of cyclin D1 \[[@B9],[@B10]\]. Moreover *cyclin D1* amplification and gain copies with consequent protein over-expression have been frequently described in multiple myeloma, T cutaneous lymphomas and in solid cancer, such as oral squamous cell carcinoma, lung cancer, melanoma, breast cancer \[[@B11],[@B12]\]. *Cyclin D1* gene abnormality has also described in cutaneous lymphoma where the *cyclin D1* gene copy gain is an infrequent event and it seems associated to malignant phenotype \[[@B12]\]. Here we report a case of Peripheral T-cell lymphomas Not Otherwise Specified with *cyclin D1* gene copy gain associated with protein overexpression. Case presentation ================= A 74 year old man was admitted to Hematology Unit of Moscati Hospital, Avellino, because of multiple superficial adenopathies, splenomegaly and bilateral lower limbs lymphedema. Laboratory data revealed elevated LDH levels, hyperuricemia and positivity for hepatitis B antibodies. Peripheral blood counts were normal, since leucocitosi was not found. Parametres are as follows: white blood cells count (WBC) 8500/mmc (Neutrophilis 73.5%; Leukocytes 20.8% Monocytes 5,5%); red blood cells count (RBC) 3.970000/mmc Haemoglobin (Hb) 13,4 g/dl; hematocrit **(**HCT) 38.3%; mean corpuscular volume (MCV) 96,4 Platelet count (PLT) 129.000/mmc. On the basis of this clinical presentation and histological findings was excluded a diagnosis of lymphoma with a leukemic presentation. CT (computed tomography) scan showed multiple deep and superficial lymph-nodes enlargement in the neck, thorax and abdomen. Focal hypoechoic lesions were detectable in the spleen. A latero-cervical/submandibular nodal biopsy was performed for diagnosis purpose. The specimen was fixed in 10% neutral buffered formalin and paraffin embedded. Five microns thick sections were stained with hematoxylin and eosin for histological examination (Figure[1](#F1){ref-type="fig"}). {#F1} Further sections were utilized for immunohistochemical study, performed with Ventana automatic stainer . Antibodies against CD2, CD3, CD4, CD8, CD5, CD43, bcl2, bcl6, CD10, CD56, CD57, CD1a, CD34, CD99, CD30, ALK1, CD23, CD20, CD79a, BSAP/Pax5, MIB1 and cyclin D1 were tested. The histological examination showed an effacement of the normal lymphoid parenchyma, because of the diffuse relatively monotonous proliferation of atypical, small-medium size cells with rounded or irregularly cleaved nuclei, finely dispersed chromatin and inconspicuous nucleoli. The proliferation showed a predominantly paracortical pattern of growth, entrapping residual follicles. Occasional larger cells were interspersed. The mitotic activity was high. Neoplastic cells show immunohistochemical positivity for T cell markers (CD2, CD3, CD5, CD43, CD4) and bcl2 (Figure[1](#F1){ref-type="fig"}); B cell markers (CD20, CD79a and BSAP/Pax5) were expressed in residual follicles, in which a CD23 positive dendritc cells meshwork was occasionally observed (Figure[1](#F1){ref-type="fig"}). The atypical cells were unreactive to CD10, CD8, CD56, CD57, CD1a, CD34, CD99 and ALK1. Only rare larger cells stained with CD30. The proliferation marker MIB1 was positive in almost 80% of cells. Unexpectedly cyclin D1 antibody was expressed by a cospicous part of the neoplastic T cells (Figure[2](#F2){ref-type="fig"}). {#F2} A peripheral T cell lymphoma, unspecified, was diagnosed, according to the morphology and immunophenotype, with unusual cyclin D1 expression. Moreover molecular analysis of IgH and TCR rearrangement was done. In particular detection of B clonality was investigated by identification of VDJ segments amplification of the hypervariable region of immunoglobulin heavy chain (IgH) using multiple primers complementary to conserved regions in the involved gene (*Nanogen-Master Diagnòstica)*; the detection of T clonality was investigated by identification of VJ segments amplification of TCRgamma gene using primers complementary flanking regions of the V and the J segments (Nanogen-*Master Diagnòstica*). In electrophoresis study, clonal rearrangement of TCR gamma gene is shown by the presence of a single strong sharp band within the expected size range from clonal control ( Figure[3](#F3){ref-type="fig"}). Our PCR analysis definitively demonstrated neoplastic T cell proliferation, being clonally rearranged for TCRgamma gene, in particular our sample presents VJ-B rearrangement as shown from band approximately for 215bp (Figure[3](#F3){ref-type="fig"}). {#F3} Although morphological and immunoprofile excluded a mantle cell lymphoma, chromosomal translocation (11; 14) (q13; q32) involving cyclin D1/IGH genes has been searched. FISH analysis for the detection of *cyclin D1* status was performed using Vysis LSI IGH/CCND1 XT Dual Color Dual Fusion Probes (Vyses). This probe set uses the dual-color, dual fusion strategy and consists of a mixture of locus-specific fluorophore-labelled DNA probes containing sequences homologous to the IGH regions (Spectrum Green) and *cyclin D1* breakpoint region (Spectrum Orange). The *cyclin D1* contig is composed of three segments covering a region approximately of 942Kb locus 11q13 where are present different genes including *cyclin D1.* Green fluorescent spots represent *Igh* and red spots stand for *cyclin D1*. In a normal tissue we have two split signals of both colors while in a traslocated sample we have two or one fused signals (yellow). The cytogenetic analyses revealed a copy gain of the *cyclin D1* without evidence of translocation because the sample shows two split signals of both colours (Figure[3](#F3){ref-type="fig"}). Bone marrow biopsy showed a huge CD3+ T cell lymphoma infiltration (Figure[4](#F4){ref-type="fig"}). {#F4} The patient was placed on GEMOX chemotherapy regimen (gemcitabine and oxalyplatin). Only one cycle of therapy was administered because of hematological and systemic toxicity. The patient died of disease two months after the diagnosis. In this short report we show overexpression of cyclin D1 in a peripheral T-cell lymphoma. Worldwide, Peripheral T-cell lymphomas represent approximately 12% of all non-Hodgkin\'s lymphomas \[[@B13]\]. Although Peripheral T-cell lymphomas Not Otherwise Specified represent most of the T-cell lymphomas, the genetic features are only poorly characterized \[[@B14]\]. Gene profiling studies performed on small series of Peripheral T-cell lymphomas showed frequent aberrations, particularly over-expression of critical genes involved in a *proliferation signature*, also significantly associated with shorter survival. This *proliferation signature* included genes commonly involved in cell cycle progression, such as CCNA, CCNB, TOP2A, and PCNA \[[@B15]\]. Cyclins play a central role in cell cycle regulation and are involved in the pathogenesis of specific hematologic malignancies. D-cyclins (D1, D2, and D3) are structurally and functionally similar proteins that bind and activate cyclin-dependent kinases 4 and 6 during the G1 phase of the cell cycle as the cell prepares to initiate DNA synthesis \[[@B16]\]**.** In mammalian cells, deregulation of these proteins leads to significantly increased cell proliferation and turnover \[[@B17]\]. In the current literature, T-cell lymphoma subtypes could be characterized by overexpression of cyclin D2, D3, in particular when proliferation rate is greater than 50% \[[@B8]\]. In addition increased Cyclin D1 expression has been observed in 9 of 23 Mycosis Fungoides (39%), 7 of 10 C- primary cutaneous CD30+ anaplastic large-cell lymphoma (70%), and 6 of 30 Sezary Syndrome (20%) \[[@B12]\]. On the contrary cyclin D1 overexpression, to the best of our knowledge, has not been hitherto described in nodal Peripheral T-cell lymphomas, Not Otherwise Specified \[[@B17]\]. Cyclin D1 overexpression is described as a driving molecular event in various types of cancer, including mantle cell lymphoma (MCL), plasmacellular dyscrasia, a subset of cutaneous T cell lymphomas, ,non-small cell lung cancer, and carcinomas of breast, head and neck, and esophagus \[[@B12],[@B18]-[@B22]\]. In various studies, cyclin D1 immunohistochemical expression in several tumors seems to be related to other proliferation markers such as Ki-67, PCNA and other cell-cycle regulatory proteins such as CDK4, p21, E2F1 proapoptotic protein p53, and inversely correlated with expression of tumor suppressor pRb protein, and bcl-2 \[[@B23]-[@B26]\]. In the literature, there are conflicting reports about prognostic impact of cyclin D1 expression and clinical outcome of different cancers. Cyclin D1 overexpression is responsible for the cell cycle deregulation playing a significant role for a greater aggressiveness, tumour extension, regional lymph node metastases and advanced clinical stage in many cancer types, such as oral cancer, breast cancer and lung cancer \[[@B27]-[@B29]\]. Cyclin D1 may be a prognostic indicator for survival \[[@B30],[@B31]\]. Many studies confirm that cyclin D1 over expression is indicative of poor outcome in B cell lymphoma patients and as it might be used also as poor prognostic index as in our case \[[@B32]\]. Aberrant expression of Cyclin D1 can be due to chromosomal translocations, single nucleotide polymorphism and gene amplification or copy gains. Chromosomal translocation is a common genetic mechanism for the pathogenesis of B-cell lymphomas \[[@B18]\]. Indeed more than 90% of mantle cell lymphoma is characterized by t(11; 14) (q13; q32) \[[@B9]\]. In addition translocation involving *Cyclin D1*, i.e. t(11; 14) (q23; q32), is also observed in 15-25% of non-IgM MGUS (monoclonal gammopathy of undetermined significance) \[[@B33]\]. As a consequence of this translocation, *cyclin D1* is constitutively expressed under the control of an active *Ig* locus in B cells presenting traslocation. Elevated expression of cyclin D1 has also been demonstrated in other lymphoproliferative disorders as hairy cell leukemia, plasma cell dyscrasias, rare cases of B-cell chronic lymphocytic leukemia/ small lymphocytic lymphoma and epithelial malignancies. Copy number change at locus 11q13 *cyclin D1* has been described in melanomas and it is strictly related to prognosis. \[[@B34]\] Gene amplifications of *cyclin D1* with consequent overexpression has been reported in several tumor types such as head and neck cancer, pituitary tumors, esophageal squamous cell carcinoma, and breast cancer. \[[@B20],[@B35]-[@B37]\] In addition, also small genetic changes such as single nucleotide polymorphisms, producing specific *cyclin D1* splice variant, have been described as responsible of cyclin D1 overexpression \[[@B38]\]. Further cyclin D1 G/A870 polymorphism has been implicated as a modulator of cancer risk and/or poor prognosis in human disease. \[[@B39]\] Conclusion ========== In this paper we described a case of peripheral T cell lymphoma with atypical expression of cyclin D1. The monomorphic Cyclin D1 high proliferation observed in this case led to a diagnosis with other lymphomas, in particular with mantle cell lymphoma. In addition, we showed molecular alteration never described in the literature for this type of lymphoma which affects cyclin D1 expression through gene gain of function. This abnormality produces a dysregulation of the cell cycle and it could have contribute to a more aggressive behavior of this lymphoma. Since we consider the difficult that the pathologists encounter in the diagnosis of T cell lymphomas and we underline the importance of molecular biology integration tests as a diagnostic tool to escape the pitfall. Consent ------- Written informed consent was obtained from our patient for the treatment of biological material for diagnostic and researching pourpose. A copy of the written consent is available by the Hospital "S. G. Moscati" AV, Italy. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= LP, Fro and RF have been directly involved in Diagnosis and interpretation of patient\'s examinations. RFr and LP were responsible for the conception and design of the Case Report. LP and FRo were responsible for provision of case report biological sample. LR is responsible for the technical part concerning the processing of the biological material. GA, RFr have been involved in PCR and FISH analysis. ADC, AA and LP have been involved in clinic-patological elaboration. The manuscript was prepared by GA under the supervision of RFr and LP. All authors read and approved the final manuscript.

Background {#Sec1} ========== Colorectal surgery has traditionally been associated with significant morbidity and prolonged hospital stay \[[@CR1]--[@CR4]\]. Overall complication rates have been reported to be 26--35 % \[[@CR1], [@CR3], [@CR4]\]. Infectious complications, in particular, represent a major cause of morbidity and mortality after colorectal surgery \[[@CR4], [@CR5]\]. Albumin is considered a negative acute-phase protein because its concentration decreases during injury and sepsis. The rate of loss of albumin to the tissue spaces (measured as transcapillary escape rate) rises by more than 300 % in patients with septic shock \[[@CR6], [@CR7]\]. Hypoalbuminemia is a risk factor for mortality and postoperative complications \[[@CR8]--[@CR13]\]. Therefore, nutritional control has been an important focus of perioperative management \[[@CR14]\]. The magnitude of the systemic inflammatory response during the perioperative period, as indicated by the acute-phase proteins---C-reactive protein (CRP) in particular---may help to identify the risk of a postoperative infectious complication \[[@CR4], [@CR15]--[@CR21]\]. The correlation between serum albumin and CRP with gastrointestinal cancer has been reported \[[@CR22], [@CR23]\]. However, it is unclear whether antecedent CRP could be used to predict future hypoalbuminemia in the perioperative period of colorectal surgery. The primary endpoint of this study was to reveal whether antecedent CRP could be used to predict future hypoalbuminemia in the perioperative period of colorectal surgery. The secondary endpoint was to clarify the relationship between CRP on postoperative day (POD) 3 and postoperative infectious complications. Methods {#Sec2} ======= Study design {#Sec3} ------------ This retrospective study included patients who had been admitted for elective open colorectal surgery from July 2011 to March 2013 at the Izumi Regional Medical Center. The following patient data were collected from medical charts: sex, age, albumin administration in the postoperative period, body mass index (BMI), type of surgery, tumor site, American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging, depth of tumor invasion, lymph node involvement, and postoperative oral intake. The tumors were staged according to the TNM criteria \[[@CR24]\]. The following laboratory data were determined preoperatively and on PODs 3 and 7: serum albumin, CRP, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (γ-GTP), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), serum creatinine (Scr), blood urea nitrogen (BUN), hemoglobin (Hb), and white blood cell (WBC) count. Serum levels of albumin (normal range 4.0--5.0 g/dL) and CRP (normal range 0--0.3 mg/dL) were measured using the bromocresol green dye-binding method and turbidimetric assay with an autoanalyzer (Hitachi 7180; Hitachi, Tokyo, Japan). Patients underwent mechanical bowel preparation with 2 L of polyethylene glycol electrolyte solution (Niflec; Ajinomoto Pharma, Tokyo, Japan). Prophylactic cefmetazole was administered from the day of the surgery (3 g/day) to POD 2 (2 g/day on POD 1 and POD 2). The study protocol was approved by the ethics committee of Izumi Regional Medical Center (approval number 20130812-1). Examination of factors affecting perioperative serum albumin with colorectal surgery {#Sec4} ------------------------------------------------------------------------------------ Preoperative hypoalbuminemia is a risk factor for postoperative complications \[[@CR8], [@CR11], [@CR12]\]. Platt et al. provided data WBC, CRP, and albumin concentrations on preoperative and PODs 1--7 in 454 patients undergoing surgery for colorectal cancer, of whom 104 developed infectious complications. Results demonstrated that CRP measurements on POD 3 could accurately predict infectious complications, including anastomotic leak, after resection for colorectal cancer \[[@CR4]\]. The average time to development of an infectious complication, including an anastomotic leak, was between 6 and 8 days postoperatively \[[@CR4]\]. These results demonstrated the utility of factors affecting serum albumin on the preoperative day and PODs 3 and 7. Moyes et al. reported that preoperative elevated modified Glasgow Prognostic Score predicts postoperative infectious complications in patients undergoing potentially curative resection for colorectal cancer \[[@CR25]\]. Therefore, the independent variables with a possible effect on serum albumin were chosen by referring to this report \[[@CR25]\]. The dependent variable was serum albumin and the independent variables were CRP, sex (male, 1; female, 0), age, albumin administration on the postoperative day (yes, 1; no, 0), tumor site (rectum, 1; colon, 0), AJCC TNM cancer stage (I, 1; II, 2; III, 3; IV, 4), depth of tumor invasion (T1, 1; T2, 2; T3, 3; T4, 4), lymph node involvement (N0, 0; N1, 1; N2, 2), BMI, postoperative oral intake (bad, 1; good, 0), AST, ALT, γ-GTP, LDH, ALP, Scr, BUN, Hb, and WBC count. Postoperative oral intake was used as an independent variable only on POD 7. Postoperative albumin administration was used as an independent variable on PODs 3 and 7. Correlations between antecedent CRP and future serum albumin {#Sec5} ------------------------------------------------------------ We examined correlations between preoperative CRP and serum albumin on POD 3, between preoperative CRP and serum albumin on POD 7 and between CRP on POD 3 and serum albumin on POD 7. Relationships between antecedent CRP and future hypoalbuminemia {#Sec6} --------------------------------------------------------------- By receiver operating characteristic (ROC) analysis \[[@CR4]\], we examined relationships between preoperative CRP and hypoalbuminemia on POD 3, between preoperative CRP and hypoalbuminemia on POD 7 and between CRP on POD 3 and hypoalbuminemia on POD 7. Hypoalbuminemia was defined as serum albumin ≤3.0 g/dL \[[@CR14], [@CR26]\]. Relationship between CRP on POD 3 and postoperative infectious complications {#Sec7} ---------------------------------------------------------------------------- Patients were assessed for the following infectious complications: wound infection, intra-abdominal abscess, anastomotic leak, pneumonia and septicemia \[[@CR25]\]. The criteria used to define infectious complications were taken from the methods reported by Moyes et al. \[[@CR25]\]: (1) wound infection was defined as the presence of pus, either discharged spontaneously or requiring drainage. Wound infection included a subgroup of patients who developed perineal infection after abdominoperineal resection of the rectum. (2) Intra-abdominal abscess was verified by either surgical drainage or by ultrasonographically guided aspiration of pus. (3) Anastomotic leakage was defined as radiologically verified fistula to bowel anastomosis or diagnosed by repeat laparotomy. (4) Pneumonia was defined as a positive chest radiograph and requirement for antibiotic treatment. (5) Septicemia was defined by clinical symptoms combined with a positive blood culture. To reveal the relationship between CRP on POD 3 and postoperative infectious complications, the diagnostic accuracy of CRP was assessed by ROC analysis \[[@CR4]\]. Statistical analysis {#Sec8} -------------------- Multiple regression analysis with stepwise variable selection was used to examine the factors affecting preoperative day and PODs 3 and 7 serum albumin with significance level of entering a selection at *p* \< 0.05 and of keeping a selection at *p* \< 0.10 \[[@CR27]\]. The significance level for keeping an independent variable in the final model was set at 0.01. The relationships between antecedent CRP and future hypoalbuminemia were examined by ROC analysis \[[@CR4]\]. The relationships between CRP on POD 3 and postoperative infectious complications were performed using ROC analysis \[[@CR4]\]. The area under the ROC curve (AUC) results were considered excellent for AUC values between 0.9 and 1, good for AUC values between 0.8 and 0.9, fair for AUC values between 0.7 and 0.8, poor for AUC values between 0.6 and 0.7 and failed for AUC values between 0.5 and 0.6 \[[@CR28]\]. Statistical analysis was performed using Excel 2010 (Microsoft Corp., Redmond, WA, USA) with the add-in software Ekuseru-Toukei 2012 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Additionally, EZR (Saitama Medical Center, Jichi Medical University, Japan), which is a graphical user interface for R \[[@CR29]\] (The R Foundation for Statistical Computing, Vienna, Austria) was used for ROC analysis only. Results {#Sec9} ======= Patient characteristics {#Sec10} ----------------------- Patient characteristics are presented in Table [1](#Tab1){ref-type="table"}. Three-quarters of patients were older than 65 years of age. Laboratory values revealed no severe perioperative liver or kidney dysfunction.Table 1Characteristics of 37 patients who underwent colorectal surgeryPatients characteristics (n = 37)ValuesSex, no (%) Male/female19 (51):18 (49)Age (years)77 (38--86)BMI (kg/m^2^)22 (15.8--31)Tumor site, no. (%) Colon/rectum25 (68):12 (32)Type of surgery, no. (%) Colectomy25 (68) Anterior resection9 (24) Abdominoperineal resection of rectum2 (5) Hartmann procedure1 (3)TNM staging, no. (%) Stage I3 (8) Stage II14 (38) Stage III17 (46) Stage IV3 (8)Laboratory values in preoperative period Serum albumin (g/dL)4.1 (2.5--5.0) CRP (mg/dL)0.31 (0.03--16.67) AST (IU/L)21 (10--37) ALT (IU/L)17 (6--46) γ-GTP (IU/L)22 (8--123) LDH (IU/L)186 (124--500) ALP (IU/L)257 (122--679) Serum creatinine (mg/dL)0.64 (0.42--1.42) BUN (mg/dL)13.5 (5.8--40.8) WBC (/μL)6100 (2400--16500) Hemoglobin (g/dL)10.7 (5.4--16.6)Quantitative variables are expressed as medians (minimum--maximum). Qualitative variables are expressed as absolute numbers (percentages)*ALP* alkaline phosphatase, *ALT* alanine transaminase, *AST* aspartate transaminase, *BMI* body mass index, *BUN* blood urea nitrogen, *CRP* C-reactive protein, *γ-GTP* gamma glutamyl transpeptidase, *LDH* lactate dehydrogenase, *WBC* white blood cell Postoperative infectious complications {#Sec11} -------------------------------------- Postoperative complications are presented in Table [2](#Tab2){ref-type="table"}. Twelve (32 %) patients experienced postoperative complications, and nine (24 %) experienced only infectious complications. The most serious infectious complication was anastomotic leak. The median time to development of an infectious complication was 5 postoperative days.Table 2Postoperative complications after colorectal surgeryPostoperative complicationsNumberPercentageInfectious complications924Anastomotic leak13Wound infection25Intra-abdominal abscess25Pneumonia4^a^10Ileus25Cardiac complications13All complications1232Mortality00^a^2 patients with ileus Factors affecting perioperative serum albumin with colorectal surgery {#Sec12} --------------------------------------------------------------------- In the preoperative period, CRP and BUN were effective variables. CRP was significant (*p* \< 0.01), and the partial correlation coefficient was −0.497 (Table [3](#Tab3){ref-type="table"}).Table 3Variables identified as predicting serum albumin in the perioperative period of colorectal surgeryPointVariableRegression coefficientStandard errorStandardized regression coefficientPartial correlation coefficient*P*Preoperative periodConstant4.6760.254\<0.001CRP−0.0780.026−0.468−0.4970.005BUN−0.0370.015−0.393−0.4340.016POD 3Constant2.3790.376\<0.001Hb0.1190.0350.6170.5320.002CRP−0.0220.008−0.416−0.4390.012Albumin administration0.3050.1330.3740.3870.029Lymph node involvement0.1820.0850.3110.3660.040Scr−0.5290.279−0.262−0.3270.068Tumor site−0.2640.141−0.332−0.3240.070POD 7Constant1.9120.388\<0.001Hb0.1070.0320.5010.5060.002CRP−0.0240.008−0.429−0.4570.007γGTP−0.0060.002−0.399−0.4240.012Depth of tumor invasion0.1620.0850.2680.3190.066*BUN* blood urea nitrogen, *CRP* C-reactive protein, *γ-GTP* gamma glutamyl transpeptidase, *Hb* hemoglobin, *POD* postoperative day On POD 3, Hb, CRP, albumin administration on the postoperative day, lymph node involvement, SCr, and tumor site were effective variables. Hb was significant (*p* \< 0.01), and the partial correlation coefficient was 0.532 (Table [3](#Tab3){ref-type="table"}). On POD 7, Hb, CRP, γ-GTP, and depth of tumor invasion were effective variables. Hb and CRP were significant (*p* \< 0.01), and partial correlation coefficients were 0.506 and −0.457, respectively (Table [3](#Tab3){ref-type="table"}). Correlations between antecedent CRP and future serum albumin {#Sec13} ------------------------------------------------------------ Significant correlations were observed between preoperative CRP and serum albumin on POD 3 (*p* = 0.023), between preoperative CRP and serum albumin on POD 7 (*p* = 0.023) and between CRP on POD 3 and serum albumin on POD 7 (*p* \< 0.001) (Table [4](#Tab4){ref-type="table"}).Table 4Correlations between antecedent CRP and future serum albumin in the perioperative period of colorectal surgeryVariableCorrelation coefficient*P*CRP in preoperative period and serum albumin on POD 3−0.37420.0225CRP in preoperative period and serum albumin on POD 7−0.37230.0233CRP on POD 3 and serum albumin on POD 7−0.54470.0005*CRP* C-reactive protein, *POD* postoperative day Relationship between antecedent CRP and future hypoalbuminemia {#Sec14} -------------------------------------------------------------- The AUC of CRP in the preoperative period to the development of hypoalbuminemia on POD 3 was 0.579 (95 % CI 0.392--0.766) with an optimal threshold of 0.86 mg/dL, sensitivity of 36.4 % and specificity of 93.3 % (Fig. [1](#Fig1){ref-type="fig"}), and the diagnostic accuracy resulted as failed. The AUC of CRP in preoperative period to the development of hypoalbuminemia on POD 7 was 0.683 (95 % CI 0.481--0.886) with an optimal threshold of 0.94 mg/dL, sensitivity of 50 % and specificity of 92 % (Fig. [1](#Fig1){ref-type="fig"}) and the diagnostic accuracy was poor. The AUC of CRP on POD 3 to development of hypoalbuminemia on POD 7 was 0.833 (95 % CI 0.679--0.987) with an optimal threshold of 12.43 mg/dL, sensitivity of 75 % and specificity of 80 % (Fig. [1](#Fig1){ref-type="fig"}), and the diagnostic accuracy was good.Fig. 1Diagnostic accuracy of antecedent CRP with regard to development of future hypoalbuminemia. **a** AUC of CRP in preoperative period to development of POD 3 hypoalbuminemia was 0.579 (95 % CI 0.392--0.766) with an optimal threshold of 0.86 mg/dL, sensitivity 36.4 % and specificity 93.3 %. **b** AUC of CRP in preoperative period to development of hypoalbuminemia on POD 7 was 0.683 (95 % CI 0.481--0.886) with an optimal threshold of 0.94 mg/dL, sensitivity 50 % and specificity 92 %. **c** AUC of CRP on POD 3 to development of hypoalbuminemia on POD 7 was 0.833 (95 % CI 0.679--0.987) with an optimal threshold of 12.43 mg/dL, sensitivity 75 % and specificity 80 %. *AUC* the area under the receiver operating characteristic curve, *CRP* C-reactive protein, *POD* postoperative day Relationships between CRP on POD 3 and postoperative infectious complications {#Sec15} ----------------------------------------------------------------------------- The AUC of CRP on POD 3 was 0.96 (95 % CI 0.902--1) with an optimal threshold of 13.8 mg/dL, sensitivity of 100 % and specificity 88 % (Fig. [2](#Fig2){ref-type="fig"}), and the diagnostic accuracy was excellent.Fig. 2Diagnostic accuracy of CRP on POD 3 with regard to development of infective complications after colorectal surgery. AUC of CRP on POD 3 was 0.96 (95 % CI 0.902--1) with an optimal threshold of 13.8 mg/dL, sensitivity 100 % and specificity 88 %. *AUC* the area under the receiver operating characteristic curve, *CRP* C-reactive protein, *POD* postoperative day Discussion {#Sec16} ========== In the present study, we examined whether antecedent CRP could be used to predict future hypoalbuminemia in the perioperative period of colorectal surgery. The main finding is that CRP on POD 3 may be of use in predicting the development of hypoalbuminemia on POD 7 (Fig. [1](#Fig1){ref-type="fig"}). Three-quarters of patients were older than 65 years of age in the present study (Table [1](#Tab1){ref-type="table"}). We searched for similar studies that evaluated infectious complications of colorectal surgery and found that 67 % of the patients in the study by Moyes et al. \[[@CR25]\] were over 65 years of age, which is similar to the 67 % in Platt's report \[[@CR4]\], suggesting that the population in the present study is similar to the population in previous reports. Twelve (32 %) patients experienced postoperative complications, and nine (24 %) experienced only infectious complications in the present study (Table [2](#Tab2){ref-type="table"}). Overall complication rates have been reported to be 26--35 % in colorectal surgery \[[@CR1], [@CR3], [@CR4]\]. Infectious complication rates have been reported to be 15--42 % in colorectal surgery \[[@CR4], [@CR25], [@CR30], [@CR31]\]. Therefore, the rates of all complications and infectious complications in the present study are similar to those in previous reports. The correlation between serum albumin and CRP with gastrointestinal cancer has been reported previously \[[@CR22], [@CR23]\]. In present study, correlations were observed between serum albumin and CRP preoperatively (*p* \< 0.01) and between serum albumin on POD 3 and CRP on POD 3 (*p* = 0.012) and between serum albumin on POD 7 and CRP on POD 7 (*p* \< 0.01) (Table [3](#Tab3){ref-type="table"}) in stepwise multiple regression analysis. These findings suggest that CRP has the greatest association with serum albumin, and concur with the results of other related reports. Hypoalbuminemia is a risk factor for mortality and postoperative complications \[[@CR8]--[@CR13]\]. Therefore, the identification of a predictor of hypoalbuminemia may be clinically significant. In present study, significant correlations were observed between CRP in preoperative period and serum albumin on POD 3 (*p* = 0.023), between CRP in the preoperative period and serum albumin on POD 7 (*p* = 0.023) and between CRP on POD 3 and serum albumin on POD 7 (*p* \< 0.001) (Table [4](#Tab4){ref-type="table"}). Additionally, the AUC of CRP on POD 3 to the development of hypoalbuminemia on POD 7 was 0.833 (95 % CI 0.679--0.987) with an optimal threshold of 12.43 mg/dL, sensitivity 75 % and specificity 80 % (Fig. [1](#Fig1){ref-type="fig"}), suggesting that CRP on POD 3 could be useful in predicting the development of hypoalbuminemia on POD 7. Therefore, CRP on POD 3 may be valuable for the indicator of early nutritional intervention. We consider that hypoalbuminemia resulted from increased CRP, which can be explained by the following: inflammatory cytokines decrease the synthesis of constitutive proteins, such as serum albumin, and increase its degradation \[[@CR7]\]. They also promote capillary permeability and leakage of serum albumin into the extravascular space \[[@CR7]\]. Because CRP is affected by increased interleukin-6 during acute inflammation, a decrease in serum albumin occurs with increased CRP \[[@CR32]\]. The clinical utility of postoperative CRP has been reported \[[@CR4], [@CR33]\]. In particular, a large study (n = 454) by Platt et al. showed that CRP was a predictor of postoperative infectious complications after curative resection in patients with colorectal cancer and that postoperative measurement of CRP on POD 3 was clinically useful in predicting surgical site infectious complications, including anastomotic leak \[[@CR4]\]. In that study, the AUC of CRP on POD 3 was 0.8 (*p* \< 0.001) and the optimal cutoff value was 17 mg/dL, and the AUC of serum albumin on POD 3 was 0.68 (*p* \< 0.001) and the optimal cutoff value was 2.5 g/dL. The diagnostic accuracy for postoperative infectious complications of CRP on POD 3 was better than that of serum albumin on POD 3 \[[@CR4]\]. In the present study, the AUC of CRP on POD 3 with regard to development of infective complications after colorectal surgery was 0.96 (95 % CI 0.902--1) with an optimal threshold of 13.8 mg/dL, sensitivity 100 % and specificity 88 % (Fig. [2](#Fig2){ref-type="fig"}), suggesting that CRP on POD 3 could be useful to predict postoperative infective complications. Therefore, these results are consistent with those reported by Platt et al. A limitation of this study is that retrospective data collection relied only on evaluation of clinical progress notes, laboratory test results, and other documentation. However, three-quarters of patients in this study were older than 65 years of age. Therefore, we believe our results apply to the elderly, in whom serum albumin is likely decreased. Prospective studies are needed to confirm whether our findings can be adapted to all colorectal surgery patients. Conclusions {#Sec17} =========== The present study revealed that CRP has the greatest association with serum albumin in the preoperative period and on PODs 3 and 7 and that antecedent CRP was associated with future serum albumin. Additionally, CRP on POD 3 could be useful in predicting hypoalbuminemia on POD 7. This result suggests that CRP on POD 3 may be valuable as an indicator of early nutritional intervention. AJCC : American Joint Committee on Cancer ALP : alkaline phosphatase ALT : alanine aminotransferase AST : aspartate aminotransferase AUC : the area under the receiver operating characteristic curve BMI : body mass index BUN : blood urea nitrogen CRP : C-reactive protein γ-GTP : gamma-glutamyl transpeptidase Hb : hemoglobin LDH : lactate dehydrogenase POD : postoperative day ROC : receiver operating characteristic Scr : serum creatinine TNM : tumor-node-metastasis WBC : white blood cell AS, KI, and TI designed the study. AS, SO, YaI, SN, NH, and KI performed research. AS, YoI, KI, and TI analyzed the data. AS, SO, KI, YK, YoI, and TI drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements {#FPar1} ================ We thank the Department of Gastroenterology for its contribution to the study. Competing interests {#FPar2} =================== The authors declare that they have no competing interests.

Post-hoc secondary analysis of data from our recent Edinburgh and Lothians Viral Intervention Study (ELVIS) pilot randomised controlled trial (RCT) indicates that hypertonic saline nasal irrigation and gargling (HSNIG) reduced the duration of coronavirus upper respiratory tract infection (URTI) by an average of two-and-a-half days. As such, it may offer a potentially safe, effective and scalable intervention in those with Coronavirus Disease-19 (COVID-19) following infection with the betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) \[[@R1]\]. ELVIS was undertaken in 66 adults with URTI. Results have been reported in detail elsewhere \[[@R2]\]. Briefly, volunteers with URTI were within 48 hours of symptom onset randomised to intervention (n = 32) or control (n = 34) arms. The intervention arm made hypertonic saline at home and performed HSNIG as many times as needed (maximum of 12 times/day). Control arm participants dealt with their URTI as they normally did. Nose swabs collected at recruitment and first thing in the morning on four consecutive days were sent to the laboratory for testing. Both arms kept a diary (which included the Wisconsin Upper Respiratory Symptom Survey-21 questionnaire) for a maximum of 14 days or until they were well for two consecutive days. Follow-up data were available for 92% of individuals (intervention arm: n = 30; control arm: n = 31). HSNIG reduced the duration of URTI by 1.9 days (*P* = 0.01), over-the-counter medication use by 36% (*P* = 0.004), transmission within household contacts by 35% (*P* = 0.006) and viral shedding by ≥0.5 log~10~/d (*P* = 0.04) in the intervention arm when compared to controls \[[@R2]\]. We also recently reported that epithelial cells mount an antiviral effect by producing hypochlorous acid (HOCl) from chloride ions \[[@R3]\]. HOCl is the active ingredient in bleach. Epithelial cells have this innate antiviral immune mechanism to clear viral infections. Since bleach is effective against all virus types \[[@R4]\], we tested to see if a range of DNA, RNA, enveloped and non-enveloped viruses were inhibited in the presence of chloride ions supplied via salt (NaCl). All the viruses we tested were inhibited in the presence of NaCl. The human viruses we tested were: DNA/enveloped: herpes simplex virus; RNA/enveloped: human coronavirus 229E (HCoV-229E), respiratory syncytial virus, influenza A virus; and RNA/non-enveloped: coxsackievirus B3 \[[@R3]\]. In COVID-19, high titres of SARS-CoV-2 are detectable in the upper respiratory tract of asymptomatic and symptomatic individuals \[[@R5]\]. The titres are higher in the nose than the throat suggesting measures that control the infection and viral shedding will help reduce transmission \[[@R5]\]. In the context of the COVID-19 pandemic, we have undertaken a post-hoc re-analysis of the ELVIS data with a focus on those infected with coronaviruses. Coronaviruses were the second most common cause of URTI (after rhinoviruses). Fifteen individuals were infected by a coronavirus: 7 in the intervention arm, 8 in the control arm. In the intervention arm, four participants were infected by an alphacoronavirus (HCoV 229E = 3, HCoV NL63 = 1) and three by a betacoronavirus (HCoV HKU1 = 3). In the control arm, two were infected by an alphacoronavirus (HCoV NL63 = 2) and six by a betacoronavirus (HCoV OC43 = 1, HCoV HKU1 = 5). An individual in the control arm with HCoV HKU1 had dual infection with rhinovirus. {#Fa} The duration of illness was lower in the intervention arm compared to the control arm in the subset of patients infected with coronavirus (mean days (SD): 5.6 (1.4) vs 8.1 (2.9)). Using a two-sample *t* test, this was difference of -2.6 days (95% confidence interval (CI) = -5.2, 0.05; *P* = 0.054). The difference in the duration of blocked nose was -3.1 days (95% CI = -6.0, -0.2; *P* = 0.04), cough -3.3 days (95% CI = -5.9, -0.7; *P* = 0.02) and hoarseness of voice -2.9 days (95% CI = -5.6, -0.3; *P* = 0.03) in favour of HSNIG ([**Table 1**](#T1){ref-type="table"}). The severity of symptoms in individuals in the two arms can be seen in [**Figure 1**](#F1){ref-type="fig"}. ###### Number of days for self reported symptom improvement in the control and intervention arms infected by a coronavirus Variable label Treatment N Mean SD Difference in mean (intervention -- control) (95% CI for difference) *P*-value ------------------ -------------- --- ------ ----- ---------------------------------------------------------------------- ----------- Blocked nose Intervention 7 4.0 2.2 -3.1 (-6.0, -0.2) 0.0362 Blocked nose Control 8 7.1 2.9 Chest congestion Intervention 7 1.9 1.2 -0.8 (-2.7, 1.2) 0.4056 Chest congestion Control 8 2.6 2.1 Cough Intervention 7 2.7 1.3 -3.3 (-5.9, -0.7) 0.0179 Cough Control 8 6.0 3.0 Head congestion Intervention 7 3.4 1.9 -1.9 (-5.0, 1.1) 0.1931 Head congestion Control 8 5.4 3.3 Hoarseness Intervention 7 2.4 1.6 -2.9 (-5.6, -0.3) 0.0325 Hoarseness Control 8 5.4 2.9 Scratchy throat Intervention 7 2.6 1.0 -2.1 (-5.1, 1.0) 0.1712 Scratchy throat Control 8 4.6 3.6 Sneezing Intervention 7 3.9 1.7 -1.0 (-3.8, 1.8) 0.4469 Sneezing Control 8 4.9 3.0 Sore throat Intervention 7 3.6 1.9 -1.1 (-4.4, 2.3) 0.5139 Sore throat Control 8 4.6 3.7 Runny nose Intervention 7 4.4 1.3 -1.6 (-4.1, 0.9) 0.1999 Runny nose Control 8 6.0 2.8 Feeling tired Intervention 7 3.6 1.8 -2.1 (-5.1, 1.0) 0.1671 Feeling tired Control 8 5.6 3.3 SD -- standard deviation, CI -- confidence interval {#F1} The individual in the control arm with a co-existing rhinovirus infection could have affected the results. Excluding this individual, the duration of illness in the control arm was a mean of 7.3 days (SD = 1.8). The impact on the intervention control comparison was to reduce the size of the difference to -1.7 days (95% CI = -3.6, 0.2; *P* = 0.07). In the absence of a suitable antiviral agent or a vaccine, we need a safe and effective intervention that can be globally implemented. Our *in-vitro* data gives the evidence that NaCl has an antiviral effect that works across viral types. The findings from this *post-hoc* analysis of ELVIS need to be interpreted with caution. These data do however suggest that HSNIG may have a role to play in reducing symptoms and duration of illness in COVID-19. **Funding:** The study was funded by Edinburgh and Lothians Health Foundation. The funder reviewed the grant application, but had no role in the study design, collection, analysis, interpretation of data, writing of the report or and in the decision to submit the paper for publication. **Authorship contributions:** SR conceived the ELVIS trial and was PI on this leading it together with AS. SR, AS and CG planned this post-hoc subgroup analysis. CG was the trial statistician and undertook the secondary analysis. JD managed the virological testing, LM supported with project management expertise. SR and AS jointly drafted the manuscript, which was contributed to by LM and CG. All authors approved the final version of the manuscript. **Competing interests:** The authors have completed the ICMJE uniform disclosure form (available upon request from the corresponding author) and declare no conflicts of interest.

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Background {#Sec1} ========== Breastfeeding is associated with a variety of positive health outcomes in children, such as: lower incidence of diarrhea; influenza; ear infections; and respiratory tract infections \[[@CR1]\]. It has been suggested that breast-fed infants have advanced immune system development compared to formula-fed infants \[[@CR2], [@CR3]\]. The microbiota acquired in early life has been reported to be important for mucosal immune response and tolerance, suggesting linkage to mucosal inflammation, autoimmunity, and allergy disorders \[[@CR2], [@CR3]\]. However, the underlying mechanisms remain to be fully elucidated. Complementary to human infant studies, formula-fed piglets compared to sow-fed piglets differed significantly in: diarrhea frequency; levels of mucosal IgG and IL-2; lactic acid bacteria/E.coli ratio \[[@CR4]\]; and counts of colonic *Clostridium cluster IV and Bacteroides vulgatus* \[[@CR5]\], all of which are consisent with microbiota differences found between human infants fed breast milk or formula. Comparison of infants' microbiota with piglet microbiota indicates that predominant phyla observed in breast-fed and formula-fed infants are *Bacteroides* and *Fimicutes,* which are also the abundant phyla in sow-fed and formula-fed piglets. However, *Actinobacteria* is also predominantly observed in breast-fed and formula-fed infants but very small percent was observed in sow-fed piglets and none in formula-fed piglets \[[@CR6]\]. Furthermore, previous report on metagenome profile comparison between porcine fecal and human fecal samples showed 70% functional similarity \[[@CR7]\]. Given the similarity of the infant and piglet intestine \[[@CR8]\], the neonatal piglet is arguably the most useful model to explore the interaction between infant diets and gut microbiota to understand the mechanisms underlying health complications in infants. Although diet-associated alterations in the composition of the infant gut microbiota are well documented, the functional effect on gastrointestinal tract physiology and immune response remains to be fully elucidated. One potential mechanism is an interaction of the microbiota with the host gastrointestinal tract and beyond through "xeno-metabolites" produced through microbial metabolism. For example, lower levels of SCFAs (short-chain fatty acids) were correlated with *Bacteroides, Prevotella, Clostridium clusters XIVa and IV, Bifidobacterium spp*. and *Lactobacillus* from colons of formula fed piglets \[[@CR9]\]. Interestingly, Yano et al. reported that gut microbiota regulate host sertotonin biosythesis \[[@CR10]\]. Several exogenous factors including specific dietary components and microbiota alter serotonin release \[[@CR10], [@CR11]\]. However, it is not currently known whether the serotonergic pathway is altered by the microbiota in infants and, thus, may be a potential pathway influencing gut immune system development and function. Previously, we demonstrated that neonatal pigs fed formula differed from sow-fed piglets in small intestine morphology and had decreased lymphoid follicle size and germinal centers in Peyer's patch and ileum tissue \[[@CR12]\]. Herein, we explore the effects of formula on the colon microbiota and the impact on colon morphology and immune response, as well as metabolites involved in the serotonin pathway and serotonin metabolism in the colon. Methods {#Sec2} ======= Animal housing and feeding {#Sec3} -------------------------- Thirty-six Yorkshire/Duroc crossbreed piglets (2 days old, *n* = 6/group/gender) were used in this study. The animals were maintained by the ethical guidelines established and approved by Institutional Animal Care and Use Committee (IACUC) of University of Arkansas Medical Sciences. The Animal feeding and diet composition details and body weights are published in our previous paper \[[@CR12]\]. In brief, piglets were randomized into three groups, i.e., group 1-sow fed; group 2-soy-based formula (soy) (Enfamil Prosobee Lipil powder; Mead Johnson Nutritionals, Evansville, IN) fed; group 3-cow's milk-based formula (milk) fed (Similac Advance powder; Ross Products, Abbott Laboratories, Columbus, OH). Piglets were fed formula milk (1.047 MJ/kg/day) from small bowls on a fixed schedule (i.e., 1st week every 2 h, 2nd week every 4 h, and 3rd week every 6 h) until sacrifice on a postnatal day (PND) 21. These piglets were anesthetized with isoflurane, blood was collected and then sacrificed by exsanguination. All tissues and contents were flash frozen in liquid nitrogen and stored at −80 °C until use, while a portion of tissues was fixed in formalin for histomorphometric analyses. Microbiota analysis using 16sRNA amplicon sequencing {#Sec4} ---------------------------------------------------- Distal colon contents were subjected to DNA isolation using Qiagen DNA isolation kit \[[@CR13]\]. Amplicons were generated by PCR of variable region 4 (V4) of bacterial 16S rRNA genes. Multiplex sequencing was carried out with an illumina platform. QIIME software was used for clustering of V4 rRNA reads at 97% nucleotide sequence. PiCRUST (phylogenetic investigation of communities by reconstruction of unobserved states) was used to generate a profile of putative functions (via metagenome prediction) from the 16srRNA OTU data \[[@CR14]\]. OTUs were picked from a demultiplexed fasta file containing the sequences for all distal colon contents samples using the closed-reference protocol where we searched sequences against the GG reference OTUs at 97% percent identity. These OTUs were normalized by the predicted 16S copy number. BIOM table containing the predicted metagenome for each sample was attained. We used STAMP (statistical analysis of metagenomics profiles) \[[@CR15], [@CR16]\] software to determine statistical significance of functional metagenomics prediction data \[[@CR15], [@CR16]\]. The significant differences in functional category relative abundances among sow, soy, and milk formula fed samples at level-3 profile were determined by ANOVA followed by Tukey-Kramer post-hoc test with effect size Eta-squared to test. Colon morphology and immune response {#Sec5} ------------------------------------ Colon samples were embedded in paraffin after dehydration. Six-micrometer-thick sections were stained with hematoxylin and eosin and used for histomorphometric analysis. The digital images were captured using Aperio microscope scanner (Aperio CS) and were used to measure the crypt height, crypt density (crypt number per mm length of tissue), full thickness of membrane, thickness of granular muscularis, and colon circumference in the sections of distal colon using ImageScope (Version 12.1.0.5029, Aperio Technologies) and Image J (Image J 150b, NIH, USA). Gene expression was measured by real time PCR (Additional file [1](#MOESM1){ref-type="media"}: Table S2). Western Blot {#Sec6} ------------ Frozen colon tissue (\~100 mg) was homogenized in cell lysis buffer (500 μl) (Cat EPX-99999-000 eBioscience, San Diego, USA) containing 0.1% proteinase inhibitor cocktail (Sigma, St. Louis, MO) and 1% NP40, in ceramic bead tubes using Fast Prep-24^TM^ 5G machine (M.P. Biomedical LLC, California, USA) at a speed 6.0 ms for 30 s (twice). Samples were centrifuged at 12,000 g for 15 min at 4 °C, supernatants were collected and protein concentration was determined using Bio-Rad protein estimation kit (BioRad). 100 μg of protein was subjected to Western Blot analyses using 8% acrylamide gel. Membranes were probed with anti-pig primary antibodies raised in rabbit overnight at 4 °C and subsequently incubated with 1:10,000 dilution of goat-anti-rabbit HRP for 1 h at room temperature (BioRad Laboratories Inc., California). We used 1:1000 dilution of anti-VE-cadherin, anti-catenin, anti-HSP 27 (Abcam, Cambridge, MA) as primary antibodies. Detection was performed using a chemiluminescence system (super signal west chemiluminescent substrate, Thermo Scientific). Image Quant software (Image Quant TL 8.1 Version) was used for densitometric analysis. Anti-rabbit vinculin (Abcam ab73412) that cross reacts with pigs was used as a housekeeping protein for normalization of a target protein. Intestinal alkaline phosphatases activity measurement {#Sec7} ----------------------------------------------------- The supernatants of distal colon contents, duodenum contents, and serum were used to measure intestinal alkaline phosphatases (ALP) using commercial kit (Anaspec CA, AS72146). This kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λmax = 405 nm) when dephosphorylated by ALP. In brief, 40 μl of sample, 10 μl of assay buffer and 50 μl of 5mM pNPP solution were mixed and incubated in the dark at room temperature for 30 to 60 min. The reactions were stopped by adding 50 μl Stop Solution and plates were read at 405 nm. The serial diluted pNP (5μ Mol) and pNPP (mM), ALP enzymes (2ng) were used for preparation of substrate, product and enzyme standard curves respectively. Absorbance readings were taken immediately and every 5 min for 60 min to obtain enzyme kinetics. Alkaline phosphatase activity in the test samples was calculated as enzyme activity (moles substrate/min) = (V × vol)/(e x l). V is reaction velocity (OD 405/min), vol is reaction volume in liters, e is the extinction coefficient of pNPP, 1.78 × 10^4^ M^−1^cm^−1^and l is the path length of light through the sample in cm (for 100 ml sample, *l* = 0.5 cm). This equation determines enzyme activity in terms of moles of substrate conversion per minute. Immunohistochemistry and Immunofluorescence {#Sec8} ------------------------------------------- To see the expression of HSP27, immunohistochemistry were carried out with 6 μm thick sections of distal colon. Anti-rabbit HSP27 (1:1000 dilution), ABC kit, DAB kit, and Texas Red labeled - anti-rabbit IgG (Vector Laboratories, Inc, Burlingame, CA) were used to perform immunohistochemistry. Olympus microscope (BX50) with 3CCD color camera (DCX-976MD) were used to capture the images. To see the expression of 5HT producing enterochromaffin cells in distal colon, 6 μm thick sections of distal colon were deparaffinized and rehydrated. Citra Plus or Dako Target Retrieval (pH6) solution was used for antigen retrieval. After blocking for 1hour (hr) with 5% donkey serum in PBS, tissue sections were incubated for 3hr at room temp with a cocktail of 1:2000 rabbit anti-5-HT antibody (ImmunoStar, 20080) and 1:250 mouse anti-chromogranin A (Abcam, ab199014), prepared in PBS with 5% donkey serum. Tissue sections were then incubated with secondary cocktail containing 1:100 donkey anti-rabbit Alexa 488 (Jackson Immuno, 711-545-152) and 1:100 donkey anti-mouse Alexa 594 (Jackson Immuno, 715-585-150) in 1x PBS for 1 h at room temp. The sections were mounted with prolong gold containing DAPI. The images were acquired using fluorescence microscope (Olympus BX51TRF). The green and red fluorescent cells indicated 5-HT and CgA expressing cells, respectively. We counted the number of 5-HT expressing EC cells (5HT+/CgA+) (yellow color) and a total number of EC cells (CgA+) cells. Serotonin ELISA {#Sec9} --------------- Frozen distal colon tissues (\~80 mg/400 ul) or distal colon contents (\~50 mg/300 ul) or duodenum contents (\~50 mg/300 ul) were homogenized in ceramic bead tubes containing cell lysis buffer (Cat EPX-99999-000 eBioscience, San Diego, USA) with 0.1% proteinase inhibitor cocktail (Sigma, St. Louis, MO), 1% NP40 and 0.1% ascorbic acid using Fast Prep-24^TM^ 5G machine (M.P. Biomedical LLC, California, USA) at speed 6.0 ms for 30 s (twice). Samples were centrifuged at 12,000 g for 15 min at 4 °C, supernatants were collected, and total protein concentration was determined using Bio-Rad protein estimation kit (BioRad). Serotonin levels were detected in clear supernatant of distal colon tissues, distal colon contents, duodenum contents, diet samples, serum and urine by ELISA according to the manufacturer instructions (Eagle Biosciences SEU39-K01). The plate was read at 450 nm (reference wavelength 600 nm) using a microplate spectrophotometer (Benchmark plus, BioRad). The data were normalized with total protein and tissue weight. Serotonin metabolites measurements {#Sec10} ---------------------------------- The presence and relative abundance of serotonin metabolites in serum, urine, and colon contents of neonatal piglets was determined using an ultra-high-performance liquid chromatography (UHPLC) system coupled to a high-resolution accurate mass spectrometer (HRAM). All instrumentation, columns, and software used are products of Thermo Fisher Scientific (Waltham, MA). All solvents used were of optimal grade and purchased from Fisher Scientific (Pittsburgh, PA). Analytical HPLC grade compounds were obtained from Sigma-Aldrich (St. Louis, MO). Metabolites in serum (100 μl) were extracted in methanol (2:1); colon contents (\~50 mg) were homogenized in 50% aqueous methanol and extracted in acetonitrile (2:1). All extracts were dried under a nitrogen stream and reconstituted in 5% aqueous methanol spiked with an internal standard (Lorazepam, 500 ng/ml). Urine samples were first analyzed for creatinine using a commercially available assay (\#STA-378, Cell Biolabs, San Diego, CA). Urines were then normalized to the lowest creatinine concentration (0.036 mg/ml) by dilution in water before analysis. For serum and colon content metabolites, chromatographic separation was performed on an Ultimate 3000 UHPLC system fitted with a Hypersil GOLD C18 reversed-phase column (100 × 2.1 mm, 1.9 μ). Mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was set at 0.4 ml/min with an elution gradient as follows: 0 to 1% B from 0.0 to 2.0 min; 1 to 20% B from 2.0 to 6.5 min; 20--95% B from 6.5 to 11.5 min; 95 to 99% B from 11.5 to 13.5 min; 99--1% B from 13.5--16.5 min; hold at 1% B until 20.0 min. Sample injection volume was 5 μl. Urine metabolites were separated using a Hypersil GOLD (50 × 2.1 mm, 1.9 μ) column using the following elution gradient: 0 to 20% B from 0.0 to 4.0 min; 20--100% B from 4.0 to 9.0 min; 100 to 0% B from 9.0 to 12.0 min. Sample injection volume was 5 μl. Detection was carried out on a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with data acquisition executed using Xcalibur 4.0 software. All samples were analyzed by positive electrospray ionization (ESI+) Full-MS scan mode. Nitrogen as sheath, auxiliary, and sweep gas was set at 50, 13, and 3 units, respectively. Other conditions included: resolution, 70,000 FWHM; AGC target, 3e6 ions; maximum injection time, 200 ms; scan range, 50--750 m/z; spray voltage, 3.50 kV; and capillary temperature, 320 °C. All data was then processed using TraceFinder 3.3 software. Raw files were imported and screened against a compound database generated from ESI+ Full-MS scans of standard mixtures of 5-hydroxytryptophan (5HTP), 5-hydroxyindoleacetic acid (5HIAA) and tryptophan. Metabolites were identified by exact mass (±3 ppm) and a retention time (±15 s) as shown in Additional file [1](#MOESM1){ref-type="media"}: Table A. Following identification, all samples within each biological matrix were pooled together. These pooled samples were then analyzed by ESI+ Full-MS with PRM (parallel reaction monitoring) targeting the five compounds of interest. MS2 spectra was generated at three fixed collision energies-10, 30, and 45%. Raw files were imported and individual MS2 spectra were matched to an in-house MS2 database created in Library Manager 2.0 with a match score \>80% to the compound of interest (Additional file [1](#MOESM1){ref-type="media"}: Table S6). Tryptamine and its metabolite measurements {#Sec11} ------------------------------------------ Tryptamine levels were evaluated as previously described by Sangwan et al. (1998) \[[@CR17], [@CR18]\]. A homogenate of distal colon content or duodenum contents (\~50 mg) was prepared in assay buffer containing 0.1 M sodium phosphate (pH 7.5), 5 mM beta-mercaptoethanol, 5 mM thiourea and 1 mM ethylenediaminetetraacetic acid followed by centrifugation for 30 min at 10,000 g. The clear supernatant of distal colon contents (25 μl) or duodenum contents (25μl) or serum (75 μl) or urine (75 μl) or diet samples were diluted up to 250 μl with assay buffer and used for tryptamine analyses. Standard curve was generated by serial dilution of 1 mg/ml tryptamine (Sigma 193747) in assay buffer. The fluorescence intensity of samples was determined using a fluorescence spectrometer (Molecular Device Spectra Max Gemini XPS) with excitation and emission wavelengths at 280 and 360 nm, respectively. Indole acetic acid (IAA) concentration was measured by using Salkowski method \[[@CR19], [@CR20]\]. 20 μl of the supernatant was diluted with 80 μl of phosphate buffer and 440 μl of reagent R1 was added. Reagent R1 (Salkowski reagent) contains 12 g of FeCl~3~ per liter of 7.9 M H~2~SO~4~. Red color formation was quantified as the absorbance at a wavelength of 540 nm in a microplate spectrophotometer (Benchmark plus, BioRad). Standard curve was generated by serial dilutions of a 5mM IAA stock solution in phosphate buffer. Statistical Analysis {#Sec12} -------------------- Microbiota OTU reads were imported into R version 3.2.1 and all statistical analysis were performed using the vegan and phyloseq packages unless specifically noted. OTU richness was measured by Chao1 and OTU diversity was measured by several diversity indices (Shannon, Simpson, Inverse Simpson, and Fisher). Group differences in α-diversity (richness and diversity) were assessed by ANOVA using the stats package. Between-specimen diversity (β-diversity) was assessed by calculating a matrix of dissimilarities using the Bray-Curtis method and then visualized using non-metric multidimensional scaling (NMDS). Group differences in β-diversity was assessed using permutational multivariate analysis of variance (PERMANOVA) with 999 permutations. Group differences among genus level OTUs were assessed by pairwise comparisons on read counts using Negative Binomial Wald Tests from the DESeq2 package. OTU relative abundance is given as median % relative abundance when described in text. All statistical tests used on 16S-rRNA gene sequencing data were considered significant at *α* ≤ 0.05. All tests were corrected for multiple comparisons using the false discovery rate (FDR) correction by Benjamini and Hochberg. Histomorphometry, protein expression, serotonin, and tryptamine measurements were analyzed by one/two way ANOVA followed by post hoc test (Tukey's) using GraphPad software. Data are presented as mean ± SEM and are considered significant at *p* \< 0.05. *n* = 12/group until/unless indicated. Associations among selected variables were assessed with Spearman's correlations. Results {#Sec13} ======= Formula alters microbiota diversity and relative abundance in piglet colon {#Sec14} -------------------------------------------------------------------------- Sample richness as evidenced by Chao1 index was significantly lower in formula groups' at all taxonomical levels except at the order level. Sample diversity (Shannon index) inferred significance only at the phylum level (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Non-metric multidimensional scaling (NMDS) plots of β-diversity revealed no dietary difference at the phylum level; however, a significant diet effect was observed at the genus level with sow-fed animals discriminated from soy- or milk-fed piglets (Fig. [1a](#Fig1){ref-type="fig"}). At the phylum level a total of 16 phyla were found in sow-fed, but not in soy- or milk-fed piglets, (Fig. [1b](#Fig1){ref-type="fig"}) suggesting a reduction of microbial diversity in formula-fed piglets. *Bacteroidetes* represented the most abundant phyla in all groups (52.9, 65.1, and 66.1% in sow-, soy- or milk-fed piglets, respectively), followed by *Firmicutes* (23.9, 22.6, 20.5%) and then *Proteobacteria* (9.3, 10.9, 10.8%), respectively (Fig. [1b](#Fig1){ref-type="fig"}). Detailed description of relative abundances for class, order, and family levels are discussed in supplemental results (Additional file [1](#MOESM1){ref-type="media"}: Figure S1A-1C). At the genus level, 50 total OTUs were differentially impacted by diet (Fig. [2](#Fig2){ref-type="fig"}). Relative to sow feeding, 40 OTUs were significantly altered with milk-formula feeding and 33 with soy formula feeding. Of these, 24 OTUs were differentially altered in both formula groups compared to sow-fed piglets. In addition, 18 OTUs were altered significantly in milk-fed piglets relative to soy-fed. *Streptococcus, Blautia, Citrobacter, Butrycimonas, Parabacteroides, Lactococcus* were all identified genera that were increased with soy or milk formula feeding relative to sow feeding. *Butrycimonas* and *Parabacteroides* were greater in formula-fed animals by at least a factor of five (*Butrycimonas:* 0.09, 1.95, and 3.63% in sow, soy, and milk, respectively; *Parabacteroides* 1.57, 8.52, 8.47% in sow, soy, and milk, respectively). In addition, relative to sow-fed piglets, *Biliophila, Ruminococcus, Clostridium* were greater with soy formula feeding while *Anaerotruncus, Akkermansia, Enterococcus, Acinetobacter, Christensenella, Holdemania* were greater with milk feeding. Almost half of significant OTUs were decreased in formula-fed piglets relative to those who were sow-fed (Fig. [2](#Fig2){ref-type="fig"}; 20 OTUs). Twelve genera, including *Treponema*, *Catenibacterium*, *RFN20*, *YRC22*, and 8 unassigned OTUs, were present only in sow-fed piglets (i.e., completely absent in soy and milk-fed piglets (Fig. [2](#Fig2){ref-type="fig"}). Altogether, changes in microbial composition and diversity indicate that neonatal diet shapes the colon microbiota.Fig. 1Formula diets alter the colon microbiota relative to sow diet in porcine neonates. **a** NMDA plot of microbial diversity displays the separation of sow, soy, and milk from each other at the phylum and the genus level. **b** *Stacked bar chart* shows sow, soy, and milk groups microbial relative abundance in the distal colon at the phylum level Fig. 2**a** Heat-map displays relative abundances of the genus that had a significant diet effect. Genera are ordered by hierarchical clustering of Bray-Curtis dissimilarities. Significant differences are expressed as *circles* in panel **b**. *Closed black circles* represent genus that were greater in the column numerator relative to the denominator. *White circles* represent genus that were lower in the column numerator relative to the denominator (\**p* \< 0.05) Formula impact on colon morphology and immune response piglets {#Sec15} -------------------------------------------------------------- No differences were observed in gross colon-morphology among the diet groups (Additional file [1](#MOESM1){ref-type="media"}: Figure S2A). A significant increase in colon length (Additional file [1](#MOESM1){ref-type="media"}: Figure S1B) and circumference (Additional file [1](#MOESM1){ref-type="media"}: Figure S1E) and a decrease in crypt depth/tissue thickness ratio (Additional file [1](#MOESM1){ref-type="media"}: Figure S1C) were observed in soy-fed relative to sow-fed groups. No significant differences were observed in colon crypt counts (Additional file [1](#MOESM1){ref-type="media"}: Figure S2D). To determine the membrane integrity, several membrane proteins e.g. VE-Cadherin, β-catenin, HSP-27, and alkaline phosphatase were measured by western-blot and cytokine gene expression was measured by real time PCR. No significant difference in expression of cadherin proteins was observed among the groups (data not shown). Interestingly, HSP-27 and β-catenin showed a trend for an increased protein expression in soy relative to sow-fed piglets (Additional file [1](#MOESM1){ref-type="media"}: Figure S2F-H). We confirmed HSP-27 expression by Immuno-staining (Additional file [1](#MOESM1){ref-type="media"}: Figure S2H). We observed increased alkaline phosphatase activity in distal colon contents especially in milk group (Fig. [3a](#Fig3){ref-type="fig"}) with no difference in serum. Furthermore, in soy or milk group upregulation of BMP4, CCL21, CSF3, CCL25, and VEGFA and down regulation of TNFSF10 and CXCL11 was observed relative to sow-fed group, suggesting that formula diet impacts the immune system (Fig. [3b](#Fig3){ref-type="fig"}, Additional file [1](#MOESM1){ref-type="media"}: Table S2).Fig. 3Formula diets significantly alters tissue immune response relative to sow diet in porcine neonates. **a** *Bar graph* shows intestine alkaline phosphatase activity in distal colon (DC) contents and serum (analyzed by two-way ANOVA followed by tukey's post hoc test). **b** *Bar graph* shows the expression of different cytokines and chemokines in distal colon of sow-, soy-, and milk-fed piglets (analyzed by multiple *t* test). (\**p* \< 0.05, \*\*\*\* *p*\<0.0001  for formula diet in comparison to sow fed, ^\#^ *p* \<0.05 for milk diet in comparison to soy diet) Predictive functional profiling of neonatal diet driven microbiota {#Sec16} ------------------------------------------------------------------ As microbiota shift did not appear to impact the colon morphology or colon membrane protein expression, we assessed the functional significance of the microbiota using PICRUSt. The analyses predicted the functional composition of a metagenome using marker gene data and a database of reference genomes. A significant difference was observed in 43 pathways among the diet groups based on cellular process, environmental, genetic information processing, metabolism, and human diseases (Additional file [1](#MOESM1){ref-type="media"}: Figure S3). Our analyses predicted significant higher apoptosis, and bacterial invasion of epithelial cells in soy- or milk-fed groups in comparison to the sow-fed among the cellular and environmental pathways predicted (Additional file [1](#MOESM1){ref-type="media"}: Figure S3, level 1). Correlation analyses revealed a significant positive association of apoptosis with *Bacteroidetes* across diets (Fig. [4a](#Fig4){ref-type="fig"}). Moreover, the milk-fed group demonstrated a positive correlation to apoptosis with *Megasphaera,* and unassigned genus from *Firmicutes* and Synergistetes phyla*.* In the sow-fed group *Butyricimonas* was positively correlated with bacterial invasion of epithelial cells while in the soy fed *Enterococcus, Lactococcus* genera and unassigned genera from *Proteobacteria* and *Actinobacteria* phyla were positively correlated. In the milk group, *Acinetobacter, Citrobacter* genera and unassigned genera from *Bacteroidetes* and *Proteobacteria* phyla were positively correlated with bacterial invasion of epithelial cells.Fig. 4Correlation matrix of microbiota to tryptophan and cellular processes. **a** Significant correlations among genera and tryptophan metabolism, apoptosis, and bacterial invasion of epithelial cells are shown. The significant Spearman correlation coefficients are displayed as *ellipses. Let* and *right directions* of ellipses indicate positive and negative correlations, respectively. **b** The *bar graph* shows the prediction analyses of tryptophan metabolism. (\*\**p* \< 0.05 for milk diet in comparison to sow-fed, ^\#^ *p* \< 0.05 for milk diet in comparison to soy diet) Impact of diet on tryptophan metabolism {#Sec17} --------------------------------------- Correlation analyses (Fig. [4a](#Fig4){ref-type="fig"}) indicates that *Akkermansia, Butyricimonas, Clostridium, Holdemania, and Bilophila* were positively correlated with tryptophan metabolism in the soy-fed, while only *Bilophila* genus correlated with tryptophan metabolism in the milk group. However, unassigned genus from phyla *Tenericutes, Proteobacteria, Bacteroides,* and *Lentisphaerae* in addition to *RFN20* genus from *Firmicutes* phylum were positively correlated with tryptophan metabolism in the sow-fed group but not in formula-fed groups. In addition, *Butyricimonas* was negatively correlated with tryptophan metabolism in the sow-fed group while unassigned genus from *Proteobacteria* and *Bacteriodetes* phyla were negatively correlated with tryptophan metabolism in the soy group. Among the metabolism pathways predicted, we explored mainly the tryptophan metabolism (Additional file [1](#MOESM1){ref-type="media"}: Figure S3, Fig. [4b](#Fig4){ref-type="fig"}). Formula suppresses the host serotonin level in colon of piglets {#Sec18} --------------------------------------------------------------- Tryptophan is a precursor for both host serotonin and bacterial tryptamine biosynthesis. In the host, enterochromaffin (EC) cells synthesize \>90% of serotonin in the gastrointestinal tract \[[@CR21]\], where tryptophan is converted into 5-Hydroxytryptophan (5-HTP) and serotonin (5-HT) by the enzymes tryptophan hydroxylase (TPH) and aromatic acid decarboxylase (AADC). Serotonin catabolizes into 5-hydroxyindole aldehyde (5-HIA) and 5-hydroxyindole acetic acid (5-HIAA) in the presence of MAO (monoamine oxidase) and aldehyde dehydrogenase \[[@CR22]\] (Fig. [5a](#Fig5){ref-type="fig"}). To understand the impact of formula diet on serotonin synthesis, we determined EC cell and serotonin producing EC cell numbers by immunofluorescence staining. Both EC cells and 5HT- were significantly reduced in the distal colon of soy- and milk-fed piglets relative to sow, while the ratio of 5-HT/CgA reached significance only in milk-fed group in comparison to sow (Fig. [5b](#Fig5){ref-type="fig"}). To determine if EC cell number is truly changed because both EC cells and L cells stain positive for CgA \[[@CR23]\], we measured EC cell and serotonin transporter (VMAT~1~) expression by real time PCR (Tph1, Lmx1a, NKX2). No difference was observed in EC cell gene expression (Tph1, Lmx1a, NKX2) in all diet groups, however serotonin transporter (VMAT~1~) expression was decreased in both formula groups relative to sow (Fig. [5c](#Fig5){ref-type="fig"}). These data suggest that VMAT~1~ expression is possibly dependent on the amount of serotonin available and EC cell number is not affected by neonatal diet. Interestingly, distal colon tissue serotonin concentrations measured by ELISA were decreased, but no differences were found in the distal colon content (Fig. [5d](#Fig5){ref-type="fig"}). Diet groups did not differ in serum and urine serotonin levels (Additional file [1](#MOESM1){ref-type="media"}: Figure S4A) either. To determine serotonin regulation, 5-HTP and 5HIAA levels were measured. No significant differences in 5-HTP and 5HIAA levels were observed among diet groups in distal colon contents (Additional file [1](#MOESM1){ref-type="media"}: Figure S4B-D). In addition, increased tryptophan and 5HTP levels were noted in serum and urine of soy and milk groups (Additional file [1](#MOESM1){ref-type="media"}: Figure S4B-D), suggesting a diet effect in these piglets. To determine the diet contribution to serotonin levels observed, we measured serotonin in diet samples and noted that soy and milk groups have significantly more serotonin than sow diet (Fig. [4d](#Fig4){ref-type="fig"}). However, the amount of serotonin observed in three diet groups is very minimal (0.05--0.20 pg/mg) relative to distal colon tissue and contents (50--100 pg/mg; *p* \< 0.005) (Fig. [5d](#Fig5){ref-type="fig"}). Altogether these findings suggest that formula diets have impact on serotonin levels observed in colon.Fig. 5Formula diets alter the serotonin level in porcine neonates fed with soy or milk formula relative to sow diet. **a** *Cartoon* displays the tryptophan metabolism to serotonin and bacterial tryptamine and the enzymes involved in the pathway (**b**) The digital images show the immunostaining of EC cells in distal colon sections with 5-HT (*green*), CgA (*red*) and 5-HT/CgA (*yellow*). The *bar graph* displays the decrease in a number of EC cells in soy- and milk-fed groups relative to the sow-fed group. (**c**) Relative EC cell gene expression in distal colon tissue. (**d**) Serotonin level in diets (*n* = 3--4/diet group), distal colon tissue (normalized with tissue weight), distal colon contents of soy and milk groups in comparison to the sow-fed (*n* = 12/group). Data were analyzed by two-way ANOVA followed by post hoc test (Tukey's). (\**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001, \*\*\*\**p* \< 0.0001 for formula diet in comparison to sow-fed, ^\$^ *p* \< 0.005 comparison of serotonin levels in diets versus contents or tissue independent of treatment group). EC = Enterochormaffin cells, CgA = Chromogranin A Formula promotes microbial tryptamine production {#Sec19} ------------------------------------------------ Tryptamine is produced by specific bacteria (*Clostridium, Ruminococcus*) (Fig. [5a](#Fig5){ref-type="fig"}) that utilize tryptophan. To understand if tryptophan is converted to tryptamine, distal colon contents and tissue tryptamine levels were measured. We observed signficantly more tryptamine in colon contents of formula-fed groups relative to the sow group, with no differences in distal colon tissue among diet groups (Fig. [6a](#Fig6){ref-type="fig"}). Furthermore, the level of tryptamine observed in distal colon contents (Fig. [6a](#Fig6){ref-type="fig"}) was significantly higher than in tissue in all diet groups (*p* \< 0.0001 contents vs tissue). In addition, amount of tryptamine observed in the diet is significantly lower than distal colon contents independent of diet groups (*p* \< 0.0001) (Fig. [6a](#Fig6){ref-type="fig"}). To determine if the small intestinal tract makes any contribution to tryptamine levels observed in distal colon contents, we measured tryptamine in duodenal contents (Additional file [1](#MOESM1){ref-type="media"}: Figure S4E). The levels of tryptamine in duodenum contents are significantly lower than in distal colon contents (*p* \< 0.0001 duodenum vs distal colon) in all diet groups. In addition, tryptamine metabolizes into indole acetic acid (IAA) in the presence of MAO enzyme (Fig. [6a](#Fig6){ref-type="fig"}) \[[@CR24]\]. Thus, we measured IAA levels in duodenum and distal colon contents, and noted significantly more IAA in distal colon contents of milk-fed group relative to the sow-fed group, but not in duodenum contents (Fig. [6b](#Fig6){ref-type="fig"}). Furthermore, in serum no significant differences were observed among groups in IAA levels, while in urine lower levels of IAA were observed in soy, and milk-fed relative to sow-fed piglets (Additional file [1](#MOESM1){ref-type="media"}: Figure S4F).Fig. 6Formula diet enhances the bacterial tryptamine biosynthesis relative to sow diet in porcine neonates. **a** The *bar graph* shows tryptamine levels in diets (*n* = 3-4/diet group), distal colon contents and distal colon tissue (**b**) The *bar graph* displays indole acetic acid (IAA) in distal colon (DC) (and duodenum (DD) contents (*n* ~sow~ = 7, *n* ~soy~ = 9, *n* ~milk~ = 11). The data were analyzed by two-way ANOVA followed by post hoc test (Tukey's) (\**p* \< 0.05, \*\**p* \< 0.01, for formula diet in comparison to sow-fed; ^\#^ *p* \< 0.05, for milk diet in comparison to soy-fed, ^\$^ *p* \< 0.0001 comparison of tryptamine levels in diets versus contents, and in tissue versus contents independent of treatment group). Discussion {#Sec20} ========== The gut microbiota plays an important role in health and development of infants and diet may be the most important environmental factor on gut bacterial composition. It is well known that the microbiota of breast-fed and formula-fed infants differ significantly \[[@CR3]\]. Very little information about the relationship of diet, microbiota and immune system development and function is available for infants. We have used a pig model to study these effects. Previous reports showed that cecum from the sow-fed group was enriched with *Prevotella, Oscillibacter, and Clostridium* \[[@CR21]\], while the formula group was enriched with *Bacteroides, Parabacteroides, and Alistipes* \[[@CR25]\]. However, our study found that relative to sow-fed piglets, those fed with formula showed increased *Streptococcus, Blautia, Citrobacter, Butrycimonas, Parabacteroides, Lactococcus*. In addition, in milk-fed piglets, *Anaerotruncus, Akkermansia, Enterococcus, Acinetobacter, Christensenella,* and *Holdemania* were increased, while in soy-fed group *Biliophila, Ruminococcus, Clostridium* were increased relative to those who were sow-fed. Furthermore, pooled ascending colon contents from day 9 and day 17 old sow-fed or formula-fed piglets were assessed for microbiota. Data showed *Bifidobacterium* higher counts in sow-fed while, formula-fed had higher counts of *Clostridium cluster IV, XIVa* and *Bacteroides vulgatus* and our study samples are collected on day 21. The differences observed in our study to reported studies could be explained by the specific location of the contents collected, differences in housing environment, and possibly the time point samples were collected. Collectively, these data suggest that bacterial diversity is much greater in colon than in cecum, and that the microbiota profiles differ based on the location of the gut. Thus, understanding microbiota differences at each location in the gastrointestinal tract and its impact on the gut will inform the physiology of the gut in detail. Furthermore, formula feeding reduced microbial richness and diversity relative to sow feeding in the distal colon. It is possible that the higher relative abundance of genera (e.g., *Streptococcus, Blautia, Citrobacter, Butrycimonas, Parabacteroides, Lactococcus),* are known to produce substances such as bacteriocins, enzymes, lactic acid, and fatty acids, prevent the colonization of certain commensal microorganisms \[[@CR26]\]. This may account for the lower diversity observed in formula-fed piglets. Bacterial diversity can impact the host immune system, for example, in the primate model \[[@CR27]\], dam-reared rhesus macaques had a distinct colonic microbiota compared to those bottle-fed, and disparate immune systems were observed even after weaning from neonatal diet, suggesting that initial microbial colonization impacts immune system. Previously in small intestine we observed a significant morphological changes in formula-fed piglets along with an increased expression of cytokines and decreased expression of anti-inflammatory molecule IL-10 (mRNA and protein). Interestingly no major tissue morphological changes or membrane protein expression were noted in the colon with formula feeding. Thus, it is possible that tissue defense mechanisms protect the colon. For instance, alkaline phosphatase-detoxifes LPS, ameliorates intestinal inflammation, and regulates gut microbial communities and their translocation across the gut barrier \[[@CR28]\]. Interestingly, alkaline phosphatase activity is increased approximately threefold in distal colon contents of milk-fed piglets relative to the sow-fed group. Furthermore, dairy products contain a high concentration of alkaline phosphatase \[[@CR29]\] that may account for the increased alkaline phosphatase activity observed in the colon contents. The presence of more HSP-27 in the soy fed piglets may be an indication of another defense mechanism in this group. In addition, increased BMP4, (known to cause intestine barrier dysfunction), and CCL25, (a chemokine known to regulate the trafficking of gut-specific memory/effector T cells into gut mucosa) expression in soy- and milk-fed piglets was noted. Of relevance to these results are findings that an exclusively breastfed infants (at 3 months of age), but not exclusively formula-fed infants, showed down-regulation of genes such as KLRF1, BPL1, ALOX5, IL-1α, and AOC3 that prime mucosal inflammatory responses \[[@CR30]\]. Overall, data suggests that formula diet impacts immune response relative to breastfeeding. The microbiota fuctional prediction analyses showed several pathways that differed significantly between the diet groups. We observed more genera in milk-fed piglets (3 vs 1) correlating with apoptosis relative to the sow-fed group, suggesting the colon microbiota may increase the exfoliation of epithelial cells from gut mucosa and possibly reduce EC cell number as we observed here. In the sow-fed group, only *Butyricimonas* was positively correlated with bacterial invasion of epithelial cells, while in soy and milk groups four genera were positvely correlated, suggesting a higher chance of epithelial cell invasion in formula groups. It is well accepted that gut microbiota-programming of host epithelial cell transcription is region-specific \[[@CR31]\]. Thus, the specific set of microbiota present in the colon could have diverse physiological effects on the host. However, it should be noted that the pathways are based on prediction analyses and future studies will be needed to confirm the impact on the specific pathways. Based on microbiota prediction analyses, we focused on tryptophan metabolism and interestingly, several genera were correlated to tryptophan metabolism. Consistent with our data Poroyko et al. reported that formula group cecal microbiota was enriched with sequences for aromatic amino acid degradation. Thus, to understand tryptophan fate in the distal colon, we looked at the serotonin pathway and observed significantly lower levels of serotonin in colon tissue of formula-fed piglets. However, no differences were observed in serum serotonin levels, suggesting that the small intestine (and other tissue) contributed to the serum serotonin levels. Furthermore, serotonin precursors-tryptophan and 5HTP were unaffected in distal colon contents, but increased in serum and urine of soy and milk groups. It is possible that 5HTP from the colon is transported into serum more readily in soy and milk groups and excreted into urine and thus lower levels of serotonin were observed in these groups. Moreover, 5HIAA, a catabolic product of serotonin level was not altered in distal colon contents and urine and undetectable in serum, ruling out the possibility of low serotonin due to increased catabolism of serotonin into 5HIAA in formula-fed piglets. We also noted significantly increased levels of tryptamine in formula group relative to sow in colon contents, but not in tissue. However, to understand if proximal gut regions contribute to tryptamine levels present in the colon, tryptamine was measured from duodenum contents. These analyses showed lower level of tryptamine in duodenum contents in all groups. In addition, in serum and urine tryptamine levels did not differ among the diet groups. Moreover, the amount of tryptamine observed in the diet or distal colon tissues is minimal in comparison to distal colon contents, suggesting that diet or tissue possibly do not contribute to high levels of tryptamine observed and highlighting the fact that tryptamine is possibly produced by distal colon microbiota. Furthermore, *Clostridium, Ruminococcus, Bilophila, Butyrricimonas, and Blautia* genera are positively correlated with tryptophan metabolism. Interestingly, *Clostridium sporogenes and Ruminococcus gnavus* are known to consume tryptophan to produce tryptamine \[[@CR32]\]. However, mammals convert tryptophan to tryptamine \[[@CR33]\] and tryptamine produced by host cells could be readily transported to the blood and be released into the lumen. Thus, levels of tryptamine observed are possibly due to microbiota or host. Future studies are required to determine if tryptamine is derived from host or bacterial species under these conditions. It is known that serotonin receptors contribute to the immune system regulation \[[@CR34]\]. Interestingly, the serotonergic receptor-mediated signaling pathway is not only activated by endogenous agonist serotonin, but also by an exogenous tryptamine. The relative affinity of serotonin and tryptamine for serotonin receptors could cause imbalance of local and systemic serotoninergic system and thereby affect the immune system and other physiological functions. Most recently, using the Ussing-chamber model it was reported that tryptamine causes ion release in intestinal epithelial cells, suggesting tryptamine might affect the food particles transit or gastric motility \[[@CR32]\]. In addition, an early study conducted in 1950 showed that tryptamine injection into skin flap or gastronemius muscle of cat resulted in histamine release \[[@CR35]\] and it is well known that increased histamine levels are reported in allergic conditions \[[@CR36]\]. Previous reports have indicated that tryptamine might play a role in mood and appetite \[[@CR37], [@CR38]\] as it can cross the blood brain barrier unlike serotonin (5-HT). Interestingly tryptamine could also promote 5-HT release from enterochromaffin cells, and possibly have impact locally on the GI tract \[[@CR37]\]. However, future studies are needed to determine the programming effect of the microbiota and the tryptophan metabolites observed during neonatal period and how that impacts host immune system and allergies. In addition, how tryptamine may differ from serotonin in the activation of cellular signaling pathways and the kyneurine pathway (another tryptophan pathway) and how this activation impacts in these piglets needs further investigation. Conclusions {#Sec21} =========== In summary, we studied the microbiota composition in distal colon and focused on aromatic amino acid metabolism (tryptophan). We demonstrated that relative to sow feeding, formula feeding: (1) alter colon microbiota richness and diversity; (2) affect colon cytokine response (3) reduce host serotonin levels; and (4) increase tryptamine production. In conclusion, these data suggest that the formula-associated colon microbiota possibly impacts sertonin biosynthesis and favors the bacterially-derived typtamine production, and thereby altering the colon's immune response. If recapitulates in human infants, these early changes in GI and immune system development due to formula feeding could have long-term health consequences. Limitations {#Sec22} ----------- Many factors other than diet can contribute to the development of gut microbiota composition such as housing environment, maternal proximity, pens, and mother's diet. In this study the sow-fed piglets were housed at the farm until day 21 and formula-fed were brought to the animal facility on day 2. There is a need to use more controlled environment to understand microbiota changes due to the dietary differences, which could be achieved by using human breast milk-fed piglet model. Thus, future studies will focus on human breast milk-fed piglet model. Additional file {#Sec23} =============== Additional file 1:Formula diet alters tryptophan metabolism. (ZIP 709 kb) 5-HIA : 5-hydroxyindole aldehyde 5-HIAA : 5-Hydroxyindole acetic acid 5-HT : 5-Hydroxy tryptamine/serotonin 5-HTP : 5-Hydroxytryptophan AADC : Aromatic acid decarboxylase ADH : Aldehyde dehydrogenase ALOX5 : Arachidonate 5-lipoxygenase AOC3 : Amine oxidase copper containing 3 BMP4 : Bone morphogenetic protein 4 BPL1 : Biotin protein ligase 1 CCL25 : C-C motif chemokine ligand 25 CgA : Chromogrannin A DC : Distal colon DD : Duodenum EC : Enterochormaffin cells GI : Gastro-intestine HSP-27 : Heat shock protein 27 IACUC : Institutional Animal Care and Use Committee IL-1α : Interleukin-1α KLRF1 : Killer cell lectin-like recetor F1 LPS : Lipopolysaccharide MAO : Monoamine oxidase TPH : Tryptophan hydroxylase **Electronic supplementary material** The online version of this article (doi:10.1186/s40168-017-0297-z) contains supplementary material, which is available to authorized users. We thank our animal core personnel Matt Ferguson, Bobby Fay and Trae Pittman for vivarium help with the piglets and the ACH genomics core for microbiota DNA sequencing. Funding {#FPar1} ======= The research work was supported by USDA-ARS Project 6026-51000-010-05S. Availability of data and materials {#FPar2} ================================== Microbiota data are available at [https://qiita.ucsd.edu](https://qiita.ucsd.edu/), EBI accession \# ERP023002. LY and TMB cotributed to the study concept and design. : MKS, AKB, KEM, KS, and SVC contributed to the data acquisition. MKS and LY contributed to the analyses and interpretation of the data. MKS and LY contributed to the drafting of the manuscript. : MKS, BDP, and LY contributed to the statistical analysis. All authors approved the final version of the manuscript. Ethics approval and consent to participate {#FPar3} ========================================== The study was approved by IACUC of UAMS, Little Rock (Protocol number 3471). Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Anecdotal reports have demonstrated an association between chemotherapy and accelerated aneurysmal growth.[@bib1], [@bib2] However, such a report has not been noted related to transarterial chemoembolization (TACE). Localized TACE therapy requires significantly lower doses of the chemotherapeutic agent, theoretically reducing the systemic effects. We report the first clearly documented case of significant, rapid aneurysmal dilatation after TACE. The patient consented to publication. Case report {#sec1} =========== A 64-year-old Caucasian man was referred to our clinic for evaluation of aortoiliac aneurysmal disease identified during workup for a liver transplant. The patient\'s medical history was significant for hepatocellular carcinoma (HCC) secondary to hepatitis C, portal hypertension, chronic obstructive pulmonary disease, and a resolved hepatitis B infection. He received treatment with ribavirin and interferon for hepatitis C, resulting in an undetectable viral load for 11 months leading up to his presentation. Of note, he had no prior or family history of aneurysmal diseases or vasculitides. On initial magnetic resonance imaging during the HCC workup, a noninflammatory infrarenal aneurysm measuring 3.9 cm was identified in November 2013, with a right common iliac artery (RCIA) aneurysm measuring 3.3 cm and a left common iliac artery (LCIA) measuring 2.3 cm ([Fig 1](#fig1){ref-type="fig"}).Fig 1Magnetic resonance imaging of the abdomen and pelvis, November 2013. Three rounds of TACE were performed over the course of 5 months while the patient was being evaluated for liver transplantation. During round one (March 2014), doxorubicin (50 mg) with QuadraSphere Microspheres Merit Medical Systems Inc, South Jordan, Utah) was delivered through the right hepatic artery. Follow-up magnetic resonance imaging in April 2014 demonstrated rapid growth of the aneurysms, with the infrarenal aorta measuring 4.1 cm, the RCIA measuring 3.6 cm, and the LCIA measuring 2.8 cm ([Fig 2](#fig2){ref-type="fig"}).Fig 2Magnetic resonance imaging abdomen and pelvis, April 2014. During round two of TACE, doxorubicin (50 mg), lipiodol (1 mL), and LC Beads (AngioDynamics, Queensbury, NY) were administered to the right hepatic lobe. Computed tomography angiography imaging in June 2014 revealed progressive aneurysmal dilatation, with measurements of the infrarenal aorta at 4.8 cm, RCIA at 4.3 cm, and the LCIA at 3.3 cm ([Fig 3](#fig3){ref-type="fig"}).Fig 3Computed tomography angiography of the abdomen and pelvis, June 2014. The patient was offered endovascular repair of the aneurysm given the significant growth, the size of the aneurysm, and the transplant committee\'s decision to exclude the patient for liver transplantation secondary to the aneurysms. After his third TACE procedure using lipiodol (10 mL) as the sole agent, the patient elected to undergo an endovascular repair. Off-label techniques, as previously described, were used to perform successful exclusion of the aneurysm, with bilateral hypogastric artery preservation, and he was discharged home on postoperative day 2 without incident.[@bib3], [@bib4] Discussion {#sec2} ========== This is the first documented case of accelerated CIA aneurysm growth after TACE treatment. TACE is an accepted treatment for unresectable HCC.[@bib5], [@bib6], [@bib7] The procedure involves gaining access to the hepatic arteries for targeted chemotherapy, theoretically allowing much lower doses of chemotherapeutic agents to be used with little to no systemic influence of the drug. Hepatic artery aneurysms developing after TACE have been reported, hypothesized to form secondary to trauma to vessels during the procedure or as a side effect of the chemotherapy itself, but no known literature has documented iliac aneurysmal growth rates after TACE.[@bib8], [@bib9] Although the growth rate of CIA aneurysms is debated, the maximum reported growth rate in the literature is defined as 0.32 cm/y.[@bib10], [@bib11] Our patient presented with significantly greater growth. The RCIA and LCIA grew 1.0 cm during a period of just 7 months, and 0.7 cm and 0.5 cm, respectively, in just 1 month after two rounds of TACE. Aneurysmal disease as a result of chemotherapy is debated, with a recent report demonstrating no association between aortic aneurysm growth and chemotherapy.[@bib12] Conclusions {#sec3} =========== Despite this study, there remains a broadly held concern regarding the effects of these cytotoxic agents often prompting aneurysm repair at an earlier time than would otherwise be considered. There is no prevailing explanation for aneurysmal growth with chemotherapy use; however, some have questioned altered generation of DNA, collagen and elastin, release of inflammatory markers, and disturbance in smooth muscle proliferation.[@bib12] In theory, TACE is a localized treatment and should not have systemic effects, as are believed to be seen with other cytotoxic treatments, but this first documented incidence indicates a need for further review and awareness. Author conflict of interest: M.S. is a speaker for Medtronic and Gore. The editors and reviewers of this article have no relevant financial relationships to disclose per the Journal policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest.

Introduction {#S1} ============ Self-renewal is the process by which cells make more of themselves and has been ascribed to both normal and malignant stem cell populations ([@R16]; [@R20]). In many cancers, tumor cells are hierarchically organized, with a distinct population of undifferentiated cells exclusively retaining self-renewal and long-term growth potential---these cells are commonly called tumor-propagating cells (TPCs). TPCs are often refractory to conventional chemotherapies, thereby leading to relapse and metastasis ([@R1]). While molecularly defined TPCs have now been identified in many cancers ([@R1]; [@R8]; [@R28]), the molecular pathways that drive cancer stemness, self-renewal, and differentiation arrest are not well understood. Rhabdomyosarcoma (RMS) is a common pediatric malignancy that shares morphologic and molecular features with embryonic skeletal muscle ([@R30]). RAS is a major oncogenic driver of the embryonal rhabdomyosarcoma (ERMS) subtype, with \>90% of patients having activation of this pathway ([@R4]; [@R13]; [@R24]). However, additional pathways are likely required for eliciting full transformation and imparting self-renewal to transformed cells. To date, only a single paper in the literature has defined acquired oncogenic pathways in regulating stem cell pathways in human ERMS, defining important roles for the Hedgehog signaling pathway in regulating TPC number in ERMS ([@R23]). However, despite this pathway being turned on in a fraction of human ERMS, activating mutations in the Hedgehog pathway are uncommon, and the extent to which this pathway contributes to ERMS to drive continued tumor growth by modulating TPC number is under active investigation. As with other tumors that sustain long-term growth through TPCs, it is likely that additional pathways regulate TPC number, growth, and maintenance in human rhabdomyosarcoma. Zebrafish have become an important animal model to uncover evolutionarily conserved pathways that regulate rhabdomyosarcoma growth, TPC cell number, and self-renewal ([@R5]; [@R8]; [@R13]; reviewed in [@R10]). Using transgenic approaches, we have generated zebrafish that develop embryonal rhabdomyosarcoma that are molecularly and histopathologically similar to human disease ([@R13]). The zebrafish model has also been used to assess tumor cell heterogeneity and to identify functions of molecularly defined cell fractions in driving tumor growth. For example, zebrafish can be created that express fluorescent proteins under control of muscle-specific promoters that are active only during specific states of muscle cell maturation ([@R8]). Using this approach, we have uncovered that *myf5:GFP+/mylz2-mCherry-negative* TPCs share similar characteristics with normal muscle satellite cells, can sustain tumor growth, and are regulated by similar molecular pathways ([@R5]; [@R8]; [@R13]; reviewed in [@R10]). Building on our knowledge that muscle development, regeneration, and stem cell self-renewal are regulated by the NOTCH1 pathway ([@R6]; [@R11]), we undertook experiments to assess a role for NOTCH1 in regulating human rhabdomyosarcoma growth through specifically affecting TPCs. Our work uncovered important roles for intracellular NOTCH1 (ICN1) signaling in regulating self-renewal, differentiation arrest, and growth in zebrafish, mouse xenografts, and human cell culture. Functional studies showed that SNAIL1 *(SNAI1)* is activated downstream of *ICN1* in human ERMS and stimulated self-renewal and growth, in part, by repressing expression of the muscle differentiation transcription factor *MEF2C*. Our data provide a mechanism by which oncogenic NOTCH1 regulates the overall number and properties of tumor-sustaining cell types in ERMS and provides therapeutic targets for this disease. Results {#S2} ======= The Notch1 Pathway Expands the Number of TPCs in Zebrafish ERMS {#S3} --------------------------------------------------------------- To assess a role for *NOTCH1* signaling in ERMS, we compared zebrafish ERMS cells that express *kRASG12D* with those that co-express both *kRASG12D* and *intracellular-activated NOTCH1* (*ICN1*)([Figures 1A and 1B](#F1){ref-type="fig"}). Gene expression analysis confirmed that transgenic *ICN1* was expressed at physiological levels found in normal development ([Figure S1A](#SD1){ref-type="supplementary-material"}). Primary ERMS onset, penetrance, and tumor size did not differ between tumors arising in *kRASG12D* or *kRASG12D+ICN1*-expressing fish ([Figures 1C and 1D](#F1){ref-type="fig"}). However, following the cell transplantation of equal numbers of ERMS cells into syngeneic CG1 recipients (1 × 10^4^ cells per fish), fluorescent-labeled *ICN1-*expressing tumors grew qualitatively faster and had increased penetrance of disease ([Figures 1E--1G](#F1){ref-type="fig"}; p = 0.012, log-rank/Mantel-Cox test). *ICN1*-expressing ERMS also expressed significantly higher levels of muscle stem cell genes, including *myf5*, *pax7a*, and *c-met* ([Figure 1H](#F1){ref-type="fig"}). This gene signature is highly and specifically expressed in the *myf5-GFP+* ERMS TPCs ([@R8]). *ICN1*-expressing ERMS also exhibited a 23-fold increase in TPCs when assessed by bulk limit dilution cell transplantation into syngeneic recipient animals ([Table S1](#SD1){ref-type="supplementary-material"}; n = 4 tumors per group; p \< 0.000001, extreme limiting dilution analysis \[ELDA\]). Our results have uncovered a dominant role for *NOTCH1* signaling in elevating the TPC number in zebrafish *kRASG12D*-induced ERMS. One explanation of our results is that *ICN1* may expand the number of previously defined *myf5-GFP+* TPCs ([@R8]). To directly test this hypothesis, we generated ERMS in syngeneic *myf5-GFP/mylz2-mCherry* transgenic fish. These fluorescent transgenic lines have been previously used to show that tumor-propagating activity is exclusively confined to the *myf5-GFP+/mylz2-negative* ERMS cells ([@R8]). Fluorescence-activated cell sorting (FACS) analysis revealed that primary *ICN1*-expressing ERMS had a 3-fold increase in the numbers of *myf5-GFP+/mylz2-negative* cells while also decreasing the more differentiated *mylz2-mcherry+* ERMS cells ([Figures 1I--1P](#F1){ref-type="fig"}; n = 5 tumors per group; p = 0.013, Student\'s t test). Similar results were observed in ERMS that developed in transplant recipient fish ([Figures 1Q--1V](#F1){ref-type="fig"}; n = 5 independent tumors per group; p \< 0.001, Student\'s t test). Importantly, the *myf5-GFP+/mylz2-negative* ERMS cells continued to retain tumor-propagating activity when assessed by limiting dilution cell transplantation ([Figure 2H](#F2){ref-type="fig"}; [Table S2](#SD1){ref-type="supplementary-material"}). Thus, ICN1 pathway activation expands the number of classically defined *myf5-GFP+/mylz2-negative* TPCs that have been previously shown to drive the growth of zebrafish *kRASG12D*-induced ERMS ([@R8]). *ICN1* Confers Tumor-Propagating Activity to Mid-differentiated ERMS Cells {#S4} -------------------------------------------------------------------------- *ICN1* increased molecularly defined *myf5-GFP+/mylz2-negative* TPCs 3-fold when compared with tumors that express only *kRASG12D*, yet *ICN1*-expressing ERMS cells had a 23-fold increase in TPCs when assessed by limiting dilution cell transplantation. These data suggested that *ICN1* could confer tumor-propagating ability to more differentiated ERMS cells. Mid-differentiated *myf5-GFP+/mylz2-mcherry+* ERMS from *ICN1*-expressing tumors expressed early muscle progenitor markers, including *myf5*, *c-met*, and *m-cadherin* yet retained more differentiated muscle gene expression, including *myoD*, *myogenin*, *mylz2*, *tnni2a*, *actc1b*,and *myh9a* ([Figures S1B--S1N](#SD1){ref-type="supplementary-material"}). We had previously shown that proliferation largely resided in the *myf5-GFP+/mylz2-mCherry*-*negative* ERMS population in *kRASG12D*-driven ERMS ([@R8]). By contrast, both the *myf5-GFP+/mylz2-mCherry*-*negative* and the mid-differentiated, double-positive cells from *ICN1*-expressing ERMS were highly proliferative when assessed for EDU incorporation following a 6-hr pulse ([Figures S1S--S1U](#SD1){ref-type="supplementary-material"}). These data suggest that mid-differentiated cells likely drive continued tumor growth in ICN1-expressing ERMS and that TPC function could be imparted to more differentiated ERMS cells. To directly assess whether more differentiated ICN1-expressing ERMS cells had gained tumor-propagating potential, highly purified ERMS sub-populations were isolated by FACS and implanted into syngeneic recipient fish at limiting dilution (purity \> 86.6%, and viability \> 86.9%; [Figure 2A](#F2){ref-type="fig"}). Consistent with previous findings ([@R8]), the *myf5-GFP+/mylz2-mCherry*-*negative* ERMS sub-population engrafted into recipient fish with no differences in engraftment frequencies between *kRASG12D* and *kRASG12D + ICN1*-expressing tumors ([Figures 2B--2D, 2H](#F2){ref-type="fig"}, and [S2A--S2E; Table S2](#SD1){ref-type="supplementary-material"}; n = 5 tumors analyzed). By contrast, the mid-differentiated *myf5-GFP+/mylz2-mCherry+ ICN1-*expressing ERMS cells could also now robustly engraft a tumor (p \< 0.01; [Figures 2E--2H](#F2){ref-type="fig"} and [S2F-- S2J; Table S2](#SD1){ref-type="supplementary-material"}). Serial transplantation experiments confirmed that both ICN1-expressing ERMS cell populations had long-term engraftment capacity ([Figures S2F--S2P; Table S2](#SD1){ref-type="supplementary-material"}). One potential cellular mechanism by which mid-differentiated ERMS cells can drive tumor growth is to undergo de-differentiation and become classically defined *myf5-GFP+/mylz2-mcherry-negative* self-renewing TPCs. To test this possibility, we isolated highly purified mid-differentiated *myf5-GFP+/mylz2-mCherry+* ERMS cells (97.5% sort purity, \>95% viable) and transplanted 10--20 cells into recipient fish. The calculated probability of en-grafting a tumor from a single TPC was calculated at \>99.7% ([Table S3](#SD1){ref-type="supplementary-material"}). Sort purity was independently confirmed by confocal microscopy (n = 100 of 100 tumor cells were G+R+ \[*myf5-GFP+/mylz2-mCherry+*\]), obviating the possibility of contamination by classically defined *myf5-GFP+/mylz2-mCherry-negative* TPCs. Highly purified double-positive ERMS cells engrafted robustly and made ERMS tumors that contained all fluorescent tumor cell subfractions, including the less differentiated *myf5-GFP+/mylz2-mCherry-negative* ERMS cells (n = 3 of 3; [Figures S2Q-- S2V; Table S3](#SD1){ref-type="supplementary-material"}). Taken together, we conclude that *ICN1* imparts tumor-propagating potential to mid-differentiated cells and enables these same cells to oscillate between cellular states, leading to the production of less differentiated ERMS cells that can self-renew and drive tumor growth. NOTCH1 Regulates Cell Growth, Self-Renewal, and Differentiation in Human ERMS {#S5} ----------------------------------------------------------------------------- To extend our findings to human ERMS, we first analyzed transcript expression of *NOTCH1* in primary patient tumors and uncovered that *NOTCH1* was highly expressed in 60% of both human alveolar rhabdomyosarcoma (ARMS) and ERMS when compared with normal muscle controls ([Figure 3A](#F3){ref-type="fig"}). Importantly, transcript expression of well-known downstream targets of NOTCH1 was also concordantly deregulated, including *NOTCH3*, *HEY1*, and *JAGGED1* ([Figures S3A--S3D](#SD1){ref-type="supplementary-material"}). Real-time qPCR of primary human rhabdomyosarcoma independently confirmed that *NOTCH1* was highly expressed in a majority of tumors (n = 8 of 12 samples expressed a \>4-fold increase in *NOTCH1* when compared with normal muscle; [Figure 3B](#F3){ref-type="fig"}; [Table S4](#SD1){ref-type="supplementary-material"}). Next, ICN1 protein expression was assessed in a panel of human rhabdomyosarcoma cell lines, and we uncovered that all rhabdomyosarcoma cell lines expressed activated ICN1 to varying degrees (n = 9; [Figure 4C](#F4){ref-type="fig"}). Finally, high *NOTCH1* expression was associated with reduced survival and poor outcome in human rhabdomyosarcoma patients (p = 0.013, log-rank statistic; [Figure 3C](#F3){ref-type="fig"}). Rhabdomyosarcoma subtypes also exhibited a trend toward worse outcome based on high NOTCH1 expression ([Figures S3E and S3F](#SD1){ref-type="supplementary-material"}), yet these analyses failed to reach statistical significance due to small sample sizes. We conclude that *NOTCH1* signaling is found in a large fraction of human ERMS, likely identifying a group of high-risk patients. To test the function of *ICN1* in human ERMS, we next performed knockdown of NOTCH1 using three different short hairpin RNAs (shRNAs) in RD and SMS-CTR cell lines, both of which are ERMS, have activating RAS mutations, and express high levels of ICN1 ([Figures 3D--3H](#F3){ref-type="fig"} and [S3G--S3J](#SD1){ref-type="supplementary-material"}). All three shRNA constructs reduced ICN1 expression ([Figures 3D](#F3){ref-type="fig"} and [S3G](#SD1){ref-type="supplementary-material"}). shRNA knockdown that reduced ICN1 expression by 90% led to loss of proliferation and cell death in RD lines ([Figure 3H](#F3){ref-type="fig"}). By contrast, knockdown of ICN1 using two additional shRNAs resulted in 41%--71% reduction in ICN1 protein expression, yet it had significant effects on reducing growth ([Figures 3F, 3G](#F3){ref-type="fig"}, and [S3H--S3J](#SD1){ref-type="supplementary-material"}). Following stable knockdown of ICN1 using these two shRNAs, both RD and SMS-CTR cells acquired morphological and gene expression changes associated with differentiation, while scrambled shRNA controls had no effect ([Figures 3I--3M](#F3){ref-type="fig"} and [S3K](#SD1){ref-type="supplementary-material"}). These results are consistent with those reported previously by the C.M.L. group who identified roles for NOTCH1 in regulating differentiation arrest in human rhabdomyosarcoma ([@R2]). To test the role of ICN1 on regulating TPC number, we next used sphere formation assays under minimal growth factor conditions, a surrogate for assessing tumor-propagating activity in vivo ([@R28]). Knockdown of NOTCH1 with two independent shRNAs resulted in significantly reduced sphere formation in both RD and SMS-CTR cells (p \< 0.01, Student\'s t test; [Figures 3N--3Q](#F3){ref-type="fig"} and [S3L--S3N](#SD1){ref-type="supplementary-material"}). Furthermore, pharmacologic inhibition of NOTCH1 using the gamma-secretase inhibitor dibenzaze-pine (DBZ) reduced sphere formation in a human PDX ERMS (p \< 0.05, Student\'s t test; [Figure 3R](#F3){ref-type="fig"}). These results were independently confirmed in RD cells, where DBZ treatment reduced ICN1 protein expression ([Figure 3S](#F3){ref-type="fig"}, bottom), enhanced differentiation as assessed by myosin heavy-chain (MF20) staining ([Figures S3P--S3R](#SD1){ref-type="supplementary-material"}), and reduced colony formation following 10 days of drug treatment (p \< 0.001, Student\'s t test; [Figure 3W](#F3){ref-type="fig"}). Conversely, gain-of-function experiments using constitutively active NOTCH1ΔE resulted in a 10-fold increase in sphere-forming ability in RD cells ([Figures 3S, 3U, and 3W](#F3){ref-type="fig"}; p \< 0.001, Student\'s t test). Because the membrane-bound NOTCH1ΔE requires cleavage by γ-secretase to become activated, we verified the specificity of our results by treating these cells with DBZ. DBZ treatment reduced sphere colony formation back to near-wild-type DMSO-treated control cells (p \< 0.0001, Student\'s t test; [Figures 3V and 3W](#F3){ref-type="fig"}). Our results establish a major role for the NOTCH1 signaling pathway in regulating TPC function and growth in human ERMS, consistent with our findings from the zebrafish model. *NOTCH1* Regulates *SNAI1* to Modulate TPC Function in Human ERMS {#S6} ----------------------------------------------------------------- Gene expression studies were completed to identify potential downstream targets of *ICN1*. Not surprisingly, a subset of known *NOTCH1* target genes were reduced following shRNA knockdown of *ICN1* in both RD and SMS-CTR cell lines (*NOTCH3*, p = 0.001; *HEY1*, p = 0.02; and *JAGGED1*, p = 0.001, Student\'s t test; [Figures 4A](#F4){ref-type="fig"} and [S4A](#SD1){ref-type="supplementary-material"}) ([@R2]; [@R22]). *SNAI1*, *SNAI2*, and *SOX9* were also highly expressed in ERMS tumors and cell lines, compared to normal muscle; however, only *SNAI1* mRNA and protein expression were reproducibly reduced following shRNA knockdown in both RD and SMS-CTR cells ([Figures 4A, 4B](#F4){ref-type="fig"}, [S4A, and S4B](#SD1){ref-type="supplementary-material"}). Transcriptional analysis of zebrafish ERMS had previously shown that *snai1a* and *snai2* were highly and specifically expressed in the TPC cell population in RAS-driven ERMS ([@R13]). These same factors were also upregulated in ICN1-expressing ERMS and were highly expressed in both the *myf5-GFP+/mylz2-mCherry*-*negative* and mid-differentiated *myf5-GFPmylz2-mCherry+* subpopulations in ICN1+ ERMS ([Figures S1Q and S1R](#SD1){ref-type="supplementary-material"}) ([@R13]). Since *SNAI1-*related family members have been implicated in regulating self-renewal in both normal and malignant stem cells in the breast epithelium ([@R15]; [@R31]), we reasoned that these factors might be downstream targets of ICN1 and regulate self-renewal in human ERMS. Remarkably, rhabdomyosarcoma cell lines expressing high ICN1 also co-expressed high SNAI1 protein, correlating well with graded gene expression between the two factors ([Figure 4C](#F4){ref-type="fig"}). Gene expression analysis also uncovered a high correlation of *SNAI1* and *NOTCH1* transcript co-expression in human primary rhabdomyosarcoma ([Figure 4D](#F4){ref-type="fig"}; p \< 0.0001; Pearson correlation, 0.604). This correlation was even higher than well-known *NOTCH1* target genes *NOTCH3* and *HEY1* ([Figure 4D](#F4){ref-type="fig"}). Additional gene expression studies confirmed that high-*NOTCH1*-expressing primary tumors also had high *SNAI1* ([Figure 4E](#F4){ref-type="fig"}; n = 8 of 8). Finally, chromatin immunoprecipitation (ChIP) experiments showed that *SNAI1* was a direct transcriptional target of NOTCH1, with NOTCH1 binding enriched \>15-fold within a 1-kb region upstream of the *SNAI1* promoter ([Figure 4F](#F4){ref-type="fig"}; p \< 0.01). We also assessed whether *NOTCH1* signaling was correlated with Hedgehog and canonical WNT/b-catenin signaling pathways previously shown to be associated with stemness and differentiation in rhabdomyosarcoma ([@R5]; [@R23]; [@R33]). We found that *NOTCH1* transcript expression highly correlated with *GLI3* and, to a lesser extent, with *GLI1*; however, *NOTCH1* expression did not correlate with either *PTCH1* or *PTCH2*, or with Hedgehog-associated embryonic stem cell gene *NANOG*, suggesting that Notch and Hedgehog programs may regulate distinct self-renewal programs in rhabdomyosarcoma ([Figures S4D and S4E](#SD1){ref-type="supplementary-material"}). Similarly, there was no correlation between *NOTCH1* and canonical WNT target genes *AXIN2* and *DKK1* or stem cell genes previously identified to drive TPC growth in human rhabdomyosarcoma, including *NANOG*, *POU5F1*,or *SOX2*. Together, these results show that *SNAI1* is a bona fide downstream target of *NOTCH1* in ERMS and suggest non-overlapping roles for the *NOTCH1/SNAI1* axis in regulating stem cell programs that drive tumor growth. We next assessed roles for *SNAI1* in regulating ERMS differentiation and self-renewal using shRNA knockdown in both RD and SMS-CTR cells. Similar to NOTCH1 knockdown, SNAI1 loss also resulted in reductions in cell number and acquisition of morphological characteristics associated with differentiation (three independent shRNAs; [Figures 5A--5E](#F5){ref-type="fig"} and [S5A--S5E](#SD1){ref-type="supplementary-material"}). Stable knockdown moderately decreased SNAI1 protein expression ([Figure 5F](#F5){ref-type="fig"}) yet had profound effects on increasing differentiation and reducing ERMS self-renewal in sphere colony formation assays performed in RD cells (p \< 0.01, Student\'s t test; [Figures 5F--5N](#F5){ref-type="fig"}). Consistent with *SNAI1* regulating self-renewal, expressing *SNAI1* using tamoxifen-inducible *SNAI1* (SNAI1ERSS) ([@R9]) resulted in enhanced sphere formation (p \< 0.001, Student\'s t test; [Figures 5O](#F5){ref-type="fig"} and [S5F--S5H](#SD1){ref-type="supplementary-material"}). Finally, epistasis experiments were completed to show that *SNAI1* is downstream of NOTCH1. For example, stable knockdown of *ICN1* led to increased differentiation in RD and SMS-CTR cells. Following the addition of 4-hydroxytamoxifen and activation of SNAI1ERSS activity in these cells, differentiation was blocked ([Figure S5I](#SD1){ref-type="supplementary-material"}). Similarly, reducing ICN1 levels with DBZ in RD cells resulted in reduced sphere colony formation, which was rescued by activation of the SNAI1ERSS construct ([Figures 5P-5T](#F5){ref-type="fig"} and [S5J](#SD1){ref-type="supplementary-material"}; p \< 0.001, Student\'s t test). These experiments show that *SNAI1* is activated downstream of *NOTCH1* and is required for regulating self-renewal and differentiation of human ERMS. *NOTCH1* and *SNAI1* Are Required for ERMS Xenograft Growth and Maintenance {#S7} --------------------------------------------------------------------------- Given that *NOTCH1* and *SNAI1* have potent effects on ERMS self-renewal in vitro, we expected that inactivation of either factor would result in reduced xenograft growth in mice. As expected, knockdown of either *NOTCH1* or *SNAI1* significantly impaired RD xenograft growth when compared with scrambled-control-infected cells ([Figures 6A--6C, 6I--6K](#F6){ref-type="fig"}, [S6A--S6E, and S6F--S6J](#SD1){ref-type="supplementary-material"}; p \< 0.001). By 30 days post-transplantation, 9 of 12 *NOTCH1* knockdown tumors had fully regressed, while all control tumors had grown substantially ([Figures 6A and 6B](#F6){ref-type="fig"}). Similarly, knockdown of *SNAI1* in RD cells using two different shRNAs resulted in significantly reduced tumor growth in vivo when assessed by luciferase imaging at 21 days and after tumors were palpable ([Figures 6I--6K](#F6){ref-type="fig"} and [S6F--S6J](#SD1){ref-type="supplementary-material"}; p \< 0.001, two-way ANOVA followed by Dunnett\'s multiple comparisons test). At necropsy, tumors isolated from *NOTCH1* and *SNAI1* knockdown xenografts weighed significantly less ([Figures 6C and 6K](#F6){ref-type="fig"}; p \< 0.001, Student\'s t test). Tumors were stained by H&E and revealed loss of cellularity in both *ICN1* and *SNAI1* knockdown tumors ([Figures 6D, 6E, 6L, and 6M](#F6){ref-type="fig"}). Further, KI67 staining revealed that both *NOTCH1* and *SNAI1* knockdown tumors were significantly less proliferative when compared to scrambled-control-shRNA-expressing RD cells ([Figures 6F--6H, 6N--6P](#F6){ref-type="fig"}; p \< 0.002, Student\'s t test). Together, these data support roles for *ICN1* and *SNAI1* in regulating continued tumor growth and maintenance in vivo. *MEF2C* Is Repressed by *NOTCH1/SNAI1* Signaling and, when Activated, Leads to ERMS Differentiation and a Loss of Self-Renewal {#S8} ------------------------------------------------------------------------------------------------------------------------------ Finally, we explored the downstream molecular pathways deregulated by *NOTCH1/SNAI1* to uncover how this signaling axis regulates differentiation and self-renewal. [@R14] have elegantly shown that promyogenic factors *JDP2*, *MEF2C*, and *RUNX1* are actively repressed in human ERMS RD cells and, when reactivated, can potently induce differentiation. Remarkably, expression of *RUNX1* and *JDP2* were unaffected by *NOTCH1 or SNAI1* knockdown, while only *MEF2C* was re-expressed ([Figures 7A and 7B](#F7){ref-type="fig"}). Further, gene expression analysis showed that *MEF2C* was expressed at lower levels in both human ERMS and ARMS when compared to normal muscle ([Figure 7C](#F7){ref-type="fig"}; p \< 0.001, Student\'s t test). An immunohistochemistry (IHC) analysis confirmed that MEF2C was expressed in only a small fraction of human ERMS cells ([Figure 7D](#F7){ref-type="fig"}), yet constitutively active NOTCH1ΔE could further reduce MEF2C+ cell numbers by 2.5-fold (p \< 0.01, Student\'s t test; [Figure 7D](#F7){ref-type="fig"}). Conversely, knockdown of either *NOTCH1 or SNAI1* resulted in increased numbers of MEF2C+ cells when assessed by IHC analysis (p \< 0.001; [Figures 7E and 7F](#F7){ref-type="fig"}). To confirm that MEF2C is a downstream target of the *NOTCH1/SNAI1* signaling axis, epistasis experiments were completed. Knockdown of MEF2C in the setting of NOTCH1 silencing lead to reduced differentiation of ERMS cells (p \< 0.01, [Figures 7G and 7H](#F7){ref-type="fig"}). To test whether MEF2C was able to modulate differentiation and self-renewal of ERMS, we next engineered cells to express a doxycycline-inducible form of MEF2C. Following the overexpression of MEF2C, RD cells underwent terminal differentiation and expressed high levels of myosin heavy chain ([Figure 7I and 7J](#F7){ref-type="fig"}). MEF2C also had important roles in modulating self-renewal in sphere colony formation assays where elevated MEF2C expression resulted in a 50% reduction in sphere colony formation ([Figure 7K](#F7){ref-type="fig"}; p \< 0.01, Student\'s t test). Finally, we performed additional epistasis experiments to show that SNAI1 is downstream of NOTCH1 and regulates MEF2C-induced differentiation ([Figure 7L](#F7){ref-type="fig"}). RD cells expressing a tamoxifen-inducible SNAI1 construct were treated with or without DBZ to suppress NOTCH1 activity. As expected, DBZ reduced ICN1 levels concomitant with elevated differentiation and expression of both MEF2C and myosin heavy chain. Following the reactivation of SNAI1 by treating cells with tamoxifen, differentiation was severely impaired, with reduction in both MEF2C and myosin heavy chain protein expression, indicating that NOTCH1 signaling through SNAI1 leads to the block of ERMS differentiation ([Figure 7L](#F7){ref-type="fig"}). Gene expression analysis of zebrafish ICN1-expressing ERMS also suggested the use of this molecular pathway in regulating differentiation and TPC self-renewal ([Figure S7](#SD1){ref-type="supplementary-material"}). In total, our experiments show that the *NOTCH1/SNAI1* axis suppresses *MEF2C* expression, locking cells in a less differentiated cell state while elevating the overall self-renewal potential of rhabdomyosarcoma. Discussion {#S9} ========== Only a single report in the literature has assessed a role for ICN1 in regulating human ERMS growth in vivo, which was largely attributed to regulation of the downstream transcriptional activation of *HEY1* ([@R2]). Despite these studies uncovering important roles for NOTCH1 in regulating rhabdomyosarcoma growth, roles for *NOTCH1* or *HEY1* in regulating stem cell programs that elevate overall numbers of TPCs was not reported. Rather, our work has uncovered prominent oncogenic roles for NOTCH1 in regulating the balance between ERMS self-renewal and differentiation, prominently impacting overall tumor growth. Remarkably, using the exceptional ability of our in vivo zebrafish ERMS model to differentially label ERMS tumor cells based on molecularly defined differentiation states, we also show that NOTCH1 pathway activation can break the rigid muscle stem cell hierarchies and modulate cell-state transitions between TPC and differentiated, non-proliferative cell populations. Importantly, these oscillating cell-state transitions are absent in ERMS expressing *kRAG12D* alone and differ markedly from the well-described roles for Notch1 in regulating symmetric stem cell divisions within the muscle satellite cell pool that increases the overall number of stem cells after injury ([@R6]; [@R11]). Our in vivo findings that the *NOTCH1* pathway breaks rigid stem cell hierarchies and can modulate cell-state transitions in ERMS was unexpected. Plasticity of the stem cell fate is an uncommon phenomenon, with only a few reports published in normal and malignant contexts. For example, in vivo cell-lineage tracing of intestinal epithelial cells was used to show that differentiated cells can repopulate the stem cell niche only after injury by de-differentiating into stem cells ([@R3]). Similar reports have now been seen in the lung and the liver where NOTCH1 can directly reprogram differentiated cells into less differentiated progenitors that drive regeneration ([@R12]; [@R32]). In melanoma, tumor cells can oscillate between stem-cell-like states by expressing JARIDB ([@R21]), reconciling the lack of cell-surface markers that identify pure populations of TPCs and suggesting that extreme cell-state transitions and plasticity can be found in a subset of cancers ([@R18]). To date, many of these studies lack plausible molecular mechanisms to account for these shifting cell fates. In ERMS, we show that the *NOTCH1/SNAI1/MEF2C* pathway regulates self-renewal and de-differentiation. Further, given the overall high conservation of molecular pathways used in normal and malignant muscle, we posit that ICN1 may have similar roles in de-differentiating myoblasts during injury into functional stem cells. We have found that *NOTCH1* transcriptionally regulates *SNAI1*, a neural crest cell and EMT (epithelial-to-mesenchymal transition)-expressed factor whose expression and function in muscle is largely unexplored. Rather surprisingly, we find that *SNAI1* is highly expressed in rhabdomyosarcoma tumors and that knocking it down reduces self-renewal and growth while elevating tumor differentiation. Our understanding of *SNAI1* function is largely defined in neural crest cells, cancers of epithelial origin, and endothelial cells, where it is found to function as a transcriptional repressor that is also a part of chromatin-modifying complexes that regulate cell motility and EMT ([@R17]; [@R26]). However, *SNAI1* and other EMT factors have also been recently implicated in regulating stemness in normal and malignant breast epithelial tissues. For example, [@R15] explored the relationship between EMT factors and stemness in immortalized human mammary epithelial cells and discovered that the EMT program can reinitiate stem cell features, including the ability to form mammo-spheres and to increase efficiency of engraftment in vivo. Building on these findings, [@R7] and [@R31] found that an EMT network directed by *SNAI2* can regulate mammary stem cell properties in vitro and that co-expression of *SNAI2* and *SOX9* in human breast cancer cells enhanced tumorigenic ability and metastatic spread. In addition to regulating stemness and EMT-associated processes in epithelial tumors, our findings point to *SNAI1* function in regulating stemness in ERMS. In ERMS, *SNAI1* modulates stem cell self-renewal programs by suppressing the expression of *MEF2C*, a well-known transcriptional activator that binds MYOD-binding sites and is required for robust terminal differentiation of myoblasts ([@R14]). Thus far, only a single report implicates a role for *SNAI1* in regulating *MYOD* transcriptional networks during muscle differentiation. In this report, [@R25] found that SNAI1 preferentially binds at GC-rich E-box elements enriched in differentiating myotubes, thereby competing with MYOD for access to enhancers and promoters that regulate myoblast differentiation. Our work has uncovered unexpected roles for *MEF2C* in blocking self-renewal in addition to enhancing differentiation in human ERMS, suggesting that *MEF2C* acts as a molecular switch that regulates both self-renewal and differentiation programs downstream of *NOTCH1* and *SNAI1*. Indeed, others have shown that *MEF2C* is poorly expressed in human ERMS and, when re-expressed, leads to potent differentiation of RD cells ([@R14]). Thus, it appears that differentiation is not just the default state of rhabdomyosarcoma cells that exit the cell cycle. Rather, once differentiation programs are initiated by MEF2C, self-renewal programs are actively turned off, suggesting important insights into how self-renewal and differentiation programs are reciprocally regulated in muscle and rhabdomyosarcoma growth. Our results are particularly important, as NOTCH1 inhibitory antibodies are now being assessed in preclinical models that have increased specificity and less gastrointestinal toxicity as pan-g-secretase inhibitors ([@R29]), raising hope that these strategies might be applied to the treatment of rhabdomyosarcoma in the future. Finally, given the importance of *NOTCH1* as an oncogene in other cancers ([@R19]), it is likely that similar effects on self-renewal, stemness, and cell-state transitions will be observed in other Notch-driven tumors. Experimental Procedures {#S10} ======================= Animals {#S11} ------- Animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care under protocols \#2011-N-000127 (zebrafish) and \#2013N000038 (mouse) and by the Partners Human Research Committee under institutional review board (IRB) protocol \#2009-P-002756 (human). Zebrafish used in this work include: CG1 strain, *myf5*-GFP transgenic zebrafish, *myf5-GFP/mylz2-mCherry* double-transgenic CG1-strain syngeneic zebrafish ([@R8]). NOD (non-obese diabetic)/SCID (severe combined immunodeficiency)/IL2g null mice used in this study were obtained from Jackson Laboratory. Micro-injection and ERMS Generation {#S12} ----------------------------------- The *rag2-kRASG12D*, *rag2-ICN1* (zebrafish intracellular notch1a), *rag2-GFP*, and *rag2-dsREDexpress* constructs were linearized with *Xho1*, phenol:chloroform extracted, ethanol precipitated, resuspended in 0.5 × Tris-EDTA + 0.1 M KCl, and injected into one-cell-stage embryos of the respective backgrounds, as previously described ([@R13]). Quantification of Zebrafish Rhabdomyosarcoma Size and Initiation {#S13} ---------------------------------------------------------------- Zebrafish were followed for tumor onset using an epifluorescent stereomicro-scope. Primary tumor size was quantified at 30 days of age using fluorescence intensity multiplied by the pixel area using the ImageJ software package as described previously ([@R5]). Kaplan-Meier tumor onset analysis was performed using GraphPad Prism software. FACS and ERMS Cell Transplantation {#S14} ---------------------------------- Zebrafish ERMS tumor cells were fluorescently labeled with GFP, dsRED, or mCherry and stained with DAPI to exclude dead cells and were sorted twice using a Laser BD FACSAria II Cell Sorter. Sort purity and viability were assessed after two rounds of sorting when possible, exceeding 85% and 95%, respectively. Sorted ERMS cells were transplanted into syngeneic CG1 fish and monitored for tumor engraftment under a fluorescent dissecting microscope from 10 to 120 days post-transplantation. TPC frequency was quantified using the Extreme Limiting Dilution Analysis software (<http://bioinf.wehi>. edu.au/software/elda/). Gene Expression Analysis {#S15} ------------------------ Real-time qPCR was completed using the Roche Lightcycler 480 machine. PCR primers and specific conditions are provided in [Table S5 and Supplemental Experimental Procedures](#SD1){ref-type="supplementary-material"}. RNA isolation and cDNA preparation were performed as previously described ([@R5]). Bioinformatic Analysis of Human Rhabdomyosarcoma Samples {#S16} -------------------------------------------------------- Previously published transcriptome data from 65 ERMS samples ([@R24]) were processed using a standard Tuxedo pipeline ([@R27]). The resulting gene expression were log~2~ transformed and standardized (*Z* scored) using a set of 63 normal tissue samples. Using the "Hmisc" package in R, the Pearson correlation was determined for specific genes. Human Rhabdomyosarcoma Cell Lines and PDX Tumor {#S17} ----------------------------------------------- The human RD cell line was obtained from the ATCC; SMS-CTR, 381T, Rh3, Rh5, and Rh30 cell lines were obtained from Dr. Corinne Linardic; the Rh18 (fusion-negative) cell line was obtained from Dr. Peter Houghton; and RMS176 and RMS559 ERMS cells were obtained from Dr. Jonathan Fletcher. The ERMS PDX tumor PCB00234 was obtained from Dr. Charles Keller under IRB protocol \#2009-P-002756 and Partners Institutional Biosafety Committee (PIBC) \#2012B000024. Western Blot Analysis {#S18} --------------------- Total cell lysates from human rhabdomyosarcoma cell lines and human myoblasts were obtained following lysis in 2% SDS lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology). Western blot analysis used primary antibodies: rabbit a-NOTCH1 (Abcam), a-Cleaved NOTCH1 (Cell Signaling Technology), a-SNAI1 goat pAB (R&D Systems), a-Myosin Heavy Chain mouse mAb (monoclonal antibody) myosin heavy chain (a-MF20, R&D), MEF2C rabbit mAb (CST); and secondary antibodies: HRP (horseradish peroxidase) anti-rabbit (CST, 7074) or HRP anti-mouse (GE Healthcare, NA93IV). Membranes were developed using an ECL reagent (Western Lightning Plus ECL, PerkinElmer; or sensitive SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Membranes were striped, rinsed, and re-probed with the respective internal control rabbit a-Lamin B1 (Abcam) or rabbit a-GAPDH (CST). ChIP Assay {#S19} ---------- Chromatin from 5 × 10^6^ RD cells was isolated and fixed with 1% formaldehyde, sonicated, and processed according to the manufacturer\'s protocols (ChIP Assay Kit, Millipore). Immunoprecipitation was performed using 5 mg rabbit anti-NOTCH1 antibody (Abcam) or rabbit immunoglobulin G (IgG) and Pierce Protein A/G Agarose (Thermo Scientific). The immunoprecipitated DNA was subjected to real-time PCR with primers that target the SNAI1 promoter with negative controls in a region 7.5 kb upstream and an ORF (open reading frame)-free region in chromosome 6. All signals were normalized against input by the percentage input calculation method and normalized to IgG signal. Significance was calculated by Student\'s t test. Immunofluorescence Staining {#S20} --------------------------- Cells were fixed at 72 hr post-transfection in 4% paraformaldehyde (PFA)/PBS, permeabilized in 0.5% Triton X-100/PBS, and incubated with rabbit a-MEF2C (CST) and a-myosin heavy chain (R&D) in 2% goat serum/PBS. Secondary antibody detection used Alexa 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit (Invitrogen). Cells were counterstained with DAPI (1:10,000) and imaged. Images were processed in ImageJ and Adobe Photoshop. Lentiviral, Retroviral, and siRNA Knockdown {#S21} ------------------------------------------- Scrambled control shRNA and gene-specific shRNAs were delivered on the pLKO.1-background vector and packaged using 293T cells. Retroviral particles were made in Plat-A packaging cells using FuGENE6 (Promega). rhabdomyosarcoma cells were infected with viral particles for 24 hr at 37°C with 4 μg/mL of polybrene (EMD Millipore). Gene-specific smart-pool or control small interfering RNAs (siRNAs) (Dharmacon, GE Life Sciences) (1 pmol) were reverse-transfected into cells using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) in flat, clear-bottom 96-well plates. Mouse Xenografts, Bioluminescent Imaging, and Caliper Measurements {#S22} ------------------------------------------------------------------ Luciferized RD cells were co-infected with shRNA lentivirus as outlined earlier. At 3 days post-infection, cells were collected, counted, and analyzed by flow cytometry to determine viability using DAPI. Equal numbers of viable cells were then embedded into Matrigel at a final concentration of 1 × 10^6^ cells per 100 μL and injected subcutaneously into anesthetized mice. Tumor growth was monitored weekly using bioluminescence imaging following injection using Luciferin at 75 mg/kg (15 mg/mL injected intraperitoneally). Comparison between groups was performed using a Student\'s t test. When palpable, tumors were measured using a caliper scale to measure the greatest diameter and length, which were then used to calculate tumor volume. Supplementary Material {#S23} ====================== This work was funded by NIH grants R01CA154923, R24OD016761, and U54CA168512 (to D.M.L.) and grants K99CA175184 (to M.S.I.); the Harvard Stem Cell Institute (to D.M.L.); Alex\'s Lemonade Stand Foundation (to D.M.L. and M.S.I.); a St. Baldrick\'s Research Grant (to D.M.L.); and a CPRIT grant RR160062 (to M.S.I.). We thank the Specialized Histopathology Services at Massachusetts General Hospital (MGH) and the Dana-Farber/Harvard Cancer Center (P30 CA06516), the MGH Cancer Center/Molecular Pathology Confocal Core, and the MGH CNY Flow Cytometry Core and Flow Image Analysis (1S10RR023440-01A1). **Supplemental Information:** Supplemental Information includes [Supplemental Experimental Procedures](#SD1){ref-type="supplementary-material"}, seven figures, and five tables and can be found with this article online at <http://dx.doi.org/10.1016/j.celrep.2017.05.061>. **Author Contributions:** M.S.I. and D.M.L. conceived, designed, and conducted the study; analyzed data; and prepared the manuscript. C.K., D.M.L., J.K., and D.M. recommended experiments and edited the manuscript. M.S.I., M.N.H., R.L., and K.M.M. performed most of the experiments. R.L., S.S., and J.K. performed bioinformatic and statistical analyses. E.Y.C. and P.G.N. performed histopathological analysis; P.S., A.J., Z.M., A.D.D., B.C.B., M.S.A., K.S., C.M.L., A.M., S.R., D.B., K.S., and D.M. helped with in vivo experiments, real-time qPCR, and data analysis. {ref-type="supplementary-material"}.](nihms880402f1){#F1} {ref-type="supplementary-material"}.](nihms880402f2){#F2} {ref-type="supplementary-material"}.](nihms880402f3){#F3} {ref-type="fig"}.\ (C) Western blot analysis showing ICN1 and SNAI1 co-expression across human rhabdomyosarcoma cell lines.\ (D) Pearson correlation between the expression of *NOTCH1*, *NOTCH3*, *SNAI1*, *SNAI2*, *HES1*, *HEY1*, and *MEF2C* in primary human ERMS assessed by RNA-sequencing analysis.\ (E) qPCR gene expression of *SNAI1* performed on skeletal muscle (SM), human rhabdomyosarcoma cell lines, and primary rhabdomyosarcoma. TBP, TATA box-binding protein.\ (F) ChIP assay in RD ERMS cells followed by qPCR gene expression for NOTCH1-binding regions in a region 1 kb upstream of the *SNAI1* transcription start site (TSS). Ctrl, control; Chr, chromosome; NG, non geneic.\ Error bars in (A), (E), and (F) represent ±1 SD. In (A), \*p \< 0.05. In (E), \*p \< 0.01. In (F), \*p \< 0.05; \*\*p \< 0.01, Student\'s t test; ns, not significant. See also [Figure S4](#SD1){ref-type="supplementary-material"}.](nihms880402f4){#F4} {ref-type="supplementary-material"}.](nihms880402f5){#F5} {ref-type="supplementary-material"}.](nihms880402f6){#F6} {ref-type="supplementary-material"}.](nihms880402f7){#F7} ###### Highlights - NOTCH1 expands the number of tumor-propagating cells (TPCs) in zebrafish and human ERMS - Notch1 drives the de-differentiation of zebrafish ERMS cells into self-renewing TPCs - A NOTCH1/SNAI1 pathway drives self-renewal and blocks MEF2C regulated differentiation - Self-renewal and differentiation pathways are linked and viable therapeutic targets [^1]: These authors contributed equally [^2]: Lead Contact

1. Introduction {#sec1} =============== To date, the number of menopausal women is continually growing. The World Health Organization (WHO) has estimated that the number of menopausal women worldwide will be 1200 million within 2030 \[[@B1]\]. It has been reported that cognitive decline is one of the important symptoms frequently observed especially in premature menopause \[[@B2]\]. Unfortunately, the current therapeutic strategy is still not in satisfaction level. The effect of hormone replacement therapy (HRT) on the cognitive function of menopausal women is controversial \[[@B3]--[@B7]\]. In addition, serious adverse effect such as breast cancer risk is reported in HRT \[[@B8], [@B9]\]. Therefore, the alternative strategy has gained much attention. Among various alternative strategies, plant-based therapy is very much popular \[[@B10]\]. In the recent years, the use of plant-based food supplement is increased in Thailand \[[@B11]\]. It has been demonstrated that dietary approaches are regarded as the safe and effective preventive intervention against neurodegeneration \[[@B12]\]. A pile of evidence has revealed that consumption of the polyphenol-rich supplements can enhance memory impairment \[[@B13]--[@B15]\]. Recent findings have demonstrated that the purple corn (*Zea mays* Linn., purple color) cob, an agricultural waste, can be served as an important natural resource of polyphenol \[[@B16]\]. It also exhibits potent antioxidant activity and can improve oxidative stress-related disorders \[[@B16], [@B17]\]. In addition to purple corn cob, pandan (*Pandanus amaryllifolius*), a commonly used culinary plant in Southeast Asia, also possesses high phenolic compound content and exhibits antioxidant activity \[[@B18]\]. An effervescent powder containing pandan also improves oxidative stress-related damage of the pancreas \[[@B19]\]. Based on these pieces of information and synergistic effect according to traditional folklore concept, the protective effect against oxidative stress-related brain damage and functional disorders of the combined extract of purple corn cob and pandan leaves (PCP) in menopausal women has been considered in order to produce an additive value of both plants. Currently, no data concerning this issue are available until now. Therefore, we aimed to determine the neuroprotective and memory-enhancing effects of the combined extract of purple corn cob and pandan leaves in experimental menopause in ovariectomized rats. 2. Materials and Methods {#sec2} ======================== 2.1. Chemicals and Reagents {#sec2.1} --------------------------- Thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), glacial acetic acid, N-butanol, pyridine, 1,3,3-tetraethoxypropane (TEP), cytochrome C, xanthine oxidase, xanthine, glutathione reductase, nicotinamide adenine dinucleotide phosphate (NADPH), hydrogen peroxide, superoxide dismutase, glutathione peroxidase, catalase, acetylthiocholine iodide (ATCI), acetylcholinesterase, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), cresyl violet, sodium acetate, sodium carbonate, 2,4,6-tripyridyl-striazine (TPTZ), Folin-Ciocalteu reagent, gallic acid, ascorbic acid, Trizma hydrochloride, potassium chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH), tris-hydrochloride, and sodium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemicals used in Western blot analysis were purchased from Bio-Rad Laboratories. Methanol and acetic acid (HPLC grade) were purchased from Fisher Scientific. 2.2. Plant Material Preparation and Extraction {#sec2.2} ---------------------------------------------- The cobs of purple waxy corn *(Zea mays*, open-pollinated cultivar) harvested during November--December 2012 were identified and kindly provided by Professor Kamol Lertrat and Assistant Professor Bhalang Suriharn, Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. Pandan (*Pandanus amaryllifolius*) leaves were harvested at the same period as *Z. mays* from the Khon Kaen province. The plant identification was performed by Mister Winai Somprasong, an expert agricultural scientist in the Botany and Plant Herbarium research group, Plant Varieties Protection Division, Ministry of Agriculture and Cooperatives. The cobs of *Z. mays* and the leaves of *P. amaryllifolius* were cleaned and cut into a small pieces; then, they were force dried by using an oven at 60°C overnight. The dried plants (2 kilograms of each plant) were twice extracted with 5 liters of distilled water. The percent yield of *Z. mays* and *P. amaryllifolius* were 2.4 and 8, respectively. 2.3. Preparation of a Polyphenol-Rich Functional Drink {#sec2.3} ------------------------------------------------------ Powder of various ingredients including 2% (*w*/*v*) combined extract of *Z. mays* and *P. amaryllifolius* (a ratio of both extracts was obtained from our unpublished in vitro data which provided optimum potential and under petit patent), 0.75% (*v*/*v*) sucralose, 1% (*v*/*v*) lemonade, 0.025% (*w*/*v*) salt, and 96.225% (*v*/*v*) water. All ingredients were mixed together and filtered through a cheesecloth, and the filtrate was used for further study. 2.4. Determination of Total Phenolic Compound Contents {#sec2.4} ------------------------------------------------------ The determination of total phenolic compounds content was carried out by using the Folin-Ciocalteu method \[[@B20]\]. In brief, an aliquot of combined extract beverage (20 *μ*l) was added to distilled water (1.58 ml) and 50% (*v*/*v*) Folin-Ciocalteu phenol reagent (0.1 ml) (Sigma-Aldrich). After 8 minutes of incubation, 20% sodium carbonate (0.3 ml) was added and mixed well. The mixture was kept in a dark room and incubated for 2 hours at room temperature. The absorbance was measured at 765 nm with a UV-spectrophotometer (Pharmacia LKB-Biochrom4060). Gallic acid at concentrations of 50--600 mg/l were used for preparing the standard calibration curve. The total phenolic compound was expressed as gallic acid equivalents per mg extract (mg/l GAE). 2.5. Assessment of DPPH Inhibition {#sec2.5} ---------------------------------- The scavenging activity against free radicals of the developed drink was assessed via DPPH assessment. Briefly, 0.15 mM DPPH in methanol (0.5 ml) and the functional drink (1 ml) were mixed and incubated at room temperature for 30 minutes. The absorbance was determined at 517 nm with a UV-spectrophotometer (Pharmacia LKB-Biochrom4060). The DPPH radical scavenging activity was calculated using the following equation: $$\begin{matrix} {\% Inhibition\, of\, DPPH = \left\lbrack \left( \frac{Abs\, control - Abs\, sample}{Abs\, control} \right) \right\rbrack \times 100.} \\ \end{matrix}$$ Abs control was the absorbance of methanol plus DPPH reagent while Abs sample was the absorbance of developed drink or standard. The linear portion of percentage inhibition of combined extract beverage was plotted against its concentration. The half maximal inhibitory concentration (IC~50~) was calculated by using the equation from its graph \[[@B21]\]. All determinations were performed in triplicate. 2.6. Determination of Ferric-Reducing Antioxidant Power (FRAP) Assay {#sec2.6} -------------------------------------------------------------------- The assessment of ferric-reducing antioxidant power (FRAP) was performed based on the ability of the tested substance to reduce ferric tripyridyl triazine (Fe III TPTZ) complex to ferrous form (intense blue color) at low pH by using a modified method of Benzie and Strin \[[@B22]\]. FRAP reagent was freshly prepared by mixing solution A (300 mM acetate buffer pH 3.6), solution B (10 mM 2,4,6-tripyridyl-striazine (TPTZ) in 40 mM HCl), and solution C (20 mM ferric chloride) together at a ratio of A : B : C; 10 : 1 : 1, respectively, and kept in water bath at 37°C. The tested substance (50 *μ*l) was added to FRAP reagent (1.45 ml), mixed thoroughly, and incubated at 37°C for 10 minutes. The absorbance was measured with spectrophotometer at 593 nm (Pharmacia LKB-Biochrom4060). FRAP reagent and L-ascorbic acid (100--1000 *μ*M) were used as blank and standard calibration, respectively. Data were expressed as *μ*M L-ascorbic acid equivalent. 2.7. Determination of Anthocyanin Content {#sec2.7} ----------------------------------------- Anthocyanin content was determined according to the official method of the Association of Official Analytical Chemists (AOAC) \[[@B23]\]. The tested sample (1 ml) was mixed with 0.025 M potassium chloride pH 1.0 (2 ml) or 0.4 M sodium acetate pH 4.5 (2 ml). After the incubation at room temperature for 10 minutes, the absorbance was determined at 520 and 720 nm using a UV-spectrophotometer (Pharmacia LKB-Biochrom 4060). All assessments were performed as triplicate. Anthocyanin content was calculated and expressed as mg/l cyaniding-3-glucoside equivalent/mg extract (mg/l CGE) as follows: $$\begin{matrix} {Anthocyanin\text{  }content\text{  }\left( \frac{cyanindin - 3 - glucoside\, equivalent,mg}{l} \right) = \frac{\left( {A \times MW \times DF \times 10^{3}} \right)}{\left( {ɛ \times 1} \right)},} \\ \end{matrix}$$where A = (A 520 nm--A 700 nm) pH 1.0 − (A 520 nm--A 700 nm) pH 4.5, MW (molecular weight) = 449.2 g/mol, DF = dilution factor obtained from the study, *ɛ* = 26,900 molar extinction coefficient, in l mol^−1^ cm^−1^, for cyanindin-3-glucoside, 10^3^ = factor for conversion from g to mg, and l = path length of the cuvette in cm (1 cm). 2.8. Assessment of Acetylcholinesterase Inhibitory (AChEI) Activity {#sec2.8} ------------------------------------------------------------------- Inhibition of acetylcholinesterase of the sample was determined according to the method previously described \[[@B24]\] using acetylthiocholine chloride iodide (ATCI) as a substrate. In brief, combined extract beverage (25 *μ*l) was incubated with 15 mM ATCI (25 *μ*l), 3 mM DTNB (5,5′-dithiobis\[2-nitrobenzoic acid\]) (75 *μ*l), and 50 mM Tris buffer (pH 8) (50 *μ*l) for 5 minutes at room temperature. The absorbance was measured with a microplate reader (iMark™ Microplate Absorbance Reader) at 415 nm before and after adding 0.25 units/ml acetylcholinesterase (AChE) (25 *μ*l) to the mixture. The elevation of yellow color from the reaction was obtained and the percentage inhibition was calculated by comparing the yellow color of extract to a noninhibition well (Tris buffer). All tests were conducted in triplicate. 2.9. Fingerprint Chromatogram Assessment {#sec2.9} ---------------------------------------- The fingerprint chromatogram of the developed drink was analyzed by using gradient high-performance liquid chromatography (HPLC) system. High-performance liquid chromatography (HPLC) system consisted of 515 HPLC pump and 2998 photodiode array detector (Water Company, USA). Chromatographic separation was performed using Purospher®STAR, C-18 endcapped (5 *μ*m), LiChroCART®250-4.6, and HPLC-Cartridge, sorbet lot number HX255346 (Merk, Germany) with guard column (Merk, Germany). Methanol (A) and 7.5% acetic acid in deionized (DI) water (B) were used as mobile phases. The gradient elution was carried out at a flow rate of 1.0 ml/min with the following gradient: 0--17 min, 70%A; 18--22 min, 100%A; 23--25, 50%A; and 26--30 min, 60%A. The sample was filtered (0.45 *μ*m, Millipore), and a direct injection of tested sample at the volume of 20 *μ*l on the column was performed. The chromatograms were recorded at 280 nm using a UV detector and data analysis was performed using EmpowerTM3. 2.10. Experimental Animals and Protocols {#sec2.10} ---------------------------------------- Female Wistar rats (Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand), weighing 200--250 g, were used as the experimental animals. They were randomly housed 6 per cage in a temperature-controlled room on a 12 h light/dark cycle with ad libitum access to food and water. All procedures in this experiment were strictly performed in accordance with the internationally accepted principles for laboratory use and care of the European Community (EEC directive of 1986; 86/609/EEC). The experiment protocols were approved by the Institutional Animal Care and Unit Committee, Khon Kaen University, Thailand (record number AEKKU 1/2556). The experimental rats were divided into 8 groups (*n* = 6/group) as follows: Group I: Naïve intact group; all rats received no treatment and were served as the control group. Group II: Sham operation + vehicle; all rats were subjected to sham operation surgery and received distilled water which was served as the vehicle in this study. Group III: OVX + vehicle; the experimental animals in this group were subjected to bilateral ovariectomy (OVX) and received the vehicle. Group IV: OVX + donepezil (3 mg/kg BW); the OVX rats were orally given donepezil, an acetylcholinesterase inhibitor, at a dose of 3 mg/kg and were served as the positive control-treated group. Group V: OVX + isoflavone; all OVX rats were orally given isoflavone, a well-known polyphenol substance with cognitive enhancing, at a dose of 20 mg/kg BW. Groups VI--VIII: OVX + PCP20, OVX + PCP40, and OVX + PCP80; the OVX rats in these groups received PCP at doses of 20, 40, and 80 mg/kg BW, respectively. The treatment programs of the assigned substances for rats in groups II--VIII were started since the first day after surgery and were maintained throughout a 28-day experimental period. All treatments were performed once daily in the morning with the total volume of 1.5 ml. The assessment of nonspatial memory was performed every 7 days throughout the study period while the determinations of oxidative stress markers including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), the activity of acetylcholinesterase (AChE), histology, and ERK1/2 expression in the prefrontal cortex were determined at the end of the study. 2.11. Ovariectomized Surgery Procedure {#sec2.11} -------------------------------------- The experimental animal was anesthetized with sodium thiopental at the dose of 60 mg/kg BW via intraperitoneal route prior to the ovariectomy. The ovariectomized (OVX) procedure was performed according to the method which had been previously described \[[@B25]\]. Briefly, the dorsolateral incisions were performed bilaterally, the ovarian blood vessels were tied off, and the ovaries were removed. Then, the skin was sutured and the rat was returned to their cage after postoperation care. Sham operation was carried out with the same procedures except that both the ovaries were kept intact. 2.12. Object Recognition Test {#sec2.12} ----------------------------- The object recognition test, the common test for evaluating nonspatial memory in rats, was used to assess the effect of PCP on nonspatial memory. This test was performed as previously described elsewhere with minor modification \[[@B26]\]. In brief, each rat was placed in an open field (80 cm long × 50 cm high × 60 cm wide) with two identical objects for 3 minutes (T1) and then was placed back to its home cage. Both objects should be placed in a symmetric position in the central line of the area. Then, the animal was orally given the assigned substance, and 30 minutes later, the second 3-minute trial was performed. In this session, one of the objects was replaced with the novel object which was totally different in shape and size at the same location. During the intertrial interval, the objects and open-field apparatus object were cleaned with 70% ethanol to avoid a confounding error induced by the influence of odor. The amount of time which the rat spent exploring each object was recorded and calculated as a novel object ratio (NOR) as the following equation: $$\begin{matrix} {NOR = \frac{\left( {T_{novel} - T_{familiar}} \right)}{\left( {T_{novel} + T_{familiar}} \right)},} \\ \end{matrix}$$where *T*~novel~ = time spent to explore the novel object and *T*~familiar~ = time spent to explore the familiar object. 2.13. Histological Study {#sec2.13} ------------------------ After perfusion, the brains were removed and fixed with 4% paraformaldehyde solution (pH 7.4) at 4°C. Then, they were cryoprotected in formalin-sucrose (30%) for 2-3 days. Serial sections of tissue containing prefrontal cortex were cut frozen on a cryostat (Thermo Scientific™ HM 525 Cryostat) at 20 *μ*m thick and mounted on slides coated with 0.3% aqueous solution of gelatin containing 0.05% aluminum potassium sulfate. To stain with cresyl violet, the slides were air dried; hydrated by successive immersion in 95, 70, and 50% ethanol; stained in 0.5% cresyl violet for 2 min at room temperature; dehydrated by successive immersion in 50, 70, 95, and 100% ethanol and xylene; and mounted with DPX. Three representative slides containing the prefrontal cortex were selected according to the stereotaxic coordinates anteroposterior 2.5--4.5 mm and mediolateral 0.2--1.0 mm from the rat brain atlas \[[@B27]\]. The analysis was performed by a blinded observer. The evaluation was performed via Olympus light microscope model BH-2 (Japan) at 40x magnification. The density of survival neurons in medial prefrontal cortex area (mPFC) was expressed as number of cells/255 *μ*m^2^. 2.14. Determination of Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Expression {#sec2.14} ------------------------------------------------------------------------------------ The prefrontal cortex was subjected to a 2-minute homogenization in 1/10 (*w*/*v*) M-PER mammalian protein extraction (Pierce Protein Biology Product, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma-Aldrich). Then, it was centrifuged at 14,000*g* for 10 minutes at 4°C. The supernatant was harvested and used for the determination of ERK1/2 expression. Protein concentration of the supernatant was quantified by using NanoDrop instrument (Thermo Fisher Scientific, Wilmington, Delaware USA). Total 30 *μ*g of brain samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V. All protein bands were transferred. The determination of ERK1/2 protein was performed according to the method previously described with a minor modification \[[@B28]\]. Total of 30 *μ*g of brain samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V. Proteins from the gel were transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and blocked with 5% nonfat dry milk in 0.1% tween 20 tris-buffered saline (TBS-T) for 30 minutes. After the blocking of a membrane, they were incubated with primary antibody which recognized ERK1/2 or monoclonal rabbit antiphosphorylated p44/p42 mitogen-activated protein kinase (MAPK) (Cell Signaling Technology Inc.; dilution, 1 : 1000) for 2 hours at room temperature. Then, they washed and incubated with secondary antibody conjugated with horseradish peroxidase or anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology Inc.; dilution, 1 : 2000) for 2 hours at room temperature. The band density was detected with an enhanced chemiluminescent (ECL) system (GE Healthcare, Piscataway, NJ). The analysis was performed using ImageQuant TL analysis software (GE Healthcare, Piscataway, NJ). The expression was normalized using antitotal ERK1/2. Data were presented as a relative density to the naïve control. 2.15. Biochemical Assays {#sec2.15} ------------------------ The prefrontal cortex, an important area in learning and memory, was isolated and prepared as brain homogenate by subjecting to homogenization with 50 volume of 0.1 M phosphate buffer saline. Then, the homogenate was used for the determination of acetylcholinesterase (AChE) activity and oxidative stress status including malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Protein concentration was assessed according to the Lowry method \[[@B29]\] and albumin bovine serum (2--20 mg/ml) was used as a standard. The determination of AChE was carried out to reflect the cholinergic function in OVX rats by the colorimetric method \[[@B30]\]. A reaction mixture of 200 *μ*l of 0.1 mM sodium phosphate buffer (pH 8.0), 10 *μ*l of 0.2 M DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)), and 20 *μ*l of the sample solution were incubated for 5 minutes, and the absorbance at 415 nm was recorded via microplate reader (iMark Microplate Absorbance Reader). Then, 10 *μ*l of acetylcholine thiochloride (ACTI) was added, incubated for 3 minutes, and recorded the absorbance at 415 nm. The activity of AChE was calculated according to the equation below and expressed as mmol/min/g protein. $$\begin{matrix} \left. AChE\, activity = \left( \mathrm{\Delta}A/1.36 \times 10^{4} \right) \times 1/\left( 20/230 \right)C, \right. \\ \end{matrix}$$where ∆*A* = the difference of absorbance/minute and *C* = protein concentration of brain homogenate. MDA level was assessed according to the method of Thiraphatthanavong et al. \[[@B31]\]. In brief, the mixture of 0.1 ml of brain homogenate, 0.1 ml of 8.1% (*w*/*v*) sodium dodecyl sulfate, 0.75 ml of 20% (*v*/*v*) acetic acid pH 3.5, 0.75 ml of 0.8% (*w*/*v*) thiobarbituric acid, and 0.3 ml of distilled water were mixed thoroughly and boiled at 95°C for 1 hour. After cooling, 0.5 ml of water and 2.5 ml of the mixture of n-butanol and pyridine at the ratio of 15 : 1 were added, mixed together, and centrifuged at 4000 rpm for 10 minutes. The pink layer was harvested and determined the optical density at 532 nm. 1,1,3,3-tetramethoxypropane (2--20 nmol) was served as a standard and MDA level was expressed as nmol/mg protein. SOD assessment was performed according to the method previously described elsewhere \[[@B32]\]. Briefly, 20 *μ*l of brain homogenate was mixed with the mixture which contained 216 mM potassium phosphate buffer (KH~2~PO~4~), 10.7 mM ethylenediaminetetraacetic acid, 1.1 mM cytochrome C, and 0.54 mM xanthine solution pH 7.4 at the ratio of 25 : 1 : 1 : 50. Then, 20 *μ*l of 0.05 units/ml of xanthine oxidase was added and incubated for 5 minutes at room temperature. The absorbance was measured at 490 nm using microplate reader. SOD enzyme activities at the concentrations of 0--10 units/ml were used as standards, and the results were expressed as units/mg protein. The activity of catalase (CAT) was evaluated indirectly by measuring the residual H~2~O~2~ which was titrated by potassium permanganate. In brief, 10 *μ*l of brain homogenate was mixed with 50 *μ*l of 30 mM H~2~O~2~, 25 *μ*l of 5 N H~2~SO~4~, and 150 *μ*l of 5 mM KMnO~4~. The mixture was shaken and the absorbance was measured at 490 nm. CAT enzyme at the concentration range of 0--10 units/ml was used as a standard and the result was expressed as units/mg protein \[[@B32]\]. Glutathione peroxidase activity was assessed using the colorimetric method. In brief, 10 *μ*l of brain homogenate was mixed with the mixture containing 50 *μ*l of 30 mM H~2~O~2~, 25 *μ*l of 5 N H~2~SO~4~, and 150 *μ*l of 5 mM KMnO~4~. The mixture was shaken and the absorbance was measured at 490 nm. The standard calibration curve was prepared by using CAT enzyme at the concentration range of 0--10 units/ml. CAT activity was expressed as units/mg protein \[[@B32]\]. 2.16. Statistical Analysis {#sec2.16} -------------------------- Data are presented as mean ± standard error of mean (SEM). The statistical analysis of the experiment was carried out using IBM SPSS Statistics (version 21). Data was analyzed using one-way analysis of variance (ANOVA), followed by Tukey\'s post hoc test. Probability levels less than 0.05 were accepted as significant. 3. Results {#sec3} ========== 3.1. Biological Activities of the Combined Extract {#sec3.1} -------------------------------------------------- Total phenolic compounds and anthocyanin content together with the biological activities including the antioxidant activity (DPPH and FRAP assay) and acetylcholinesterase inhibition activity of the combined extract were evaluated. The results showed that 1 ml of the combined extract contained the total phenolic compounds and anthocyanin contents of 184.00 ± 1.91 mg/l gallic acid equivalent and 25.66 ± 0.32 mg/l cyanidin-3-glucoside equivalent, respectively. IC~50~ of the antioxidant activity via 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was 56.37 ± 0.45 *μ*g/ml, whereas the antioxidant activity via ferric-reducing antioxidant power (FRAP) assay was 602.40 ± 2.33 *μ*M L-ascorbic acid equivalent. In addition, AChEI activity showed IC~50~ at a concentration of 1950 ± 16.02 *μ*g/ml as shown in [Table 1](#tab1){ref-type="table"}. 3.2. The Fingerprint of the Combined Extract {#sec3.2} -------------------------------------------- [Figure 1](#fig1){ref-type="fig"} shows the fingerprint chromatogram of PCP, the combined extract of purple corn cob and pandan leaves. More than 7 different peaks were observed in the chromatogram. Four of them were anthocyanins (peak 2--peak 5) including cyaniding-3-glucoside (peak 2), pelargonidin-3-glucoside (peak 3), cyanidin 3-O-(6″-malonyl-glucoside) (peak 4), and cyaniding-3-o-B-glucopyranoside (peak 5). It was found that the contents of anthocyanins mentioned earlier in PCP were 3.239 ± 0.014, 2.543 ± 0.011, 2.993 ± 0.024, and 2.335 ± 0.006 mg/ml, respectively. In addition to anthocyanins, gallic acid, rutin, and ferulic acid were observed at the concentrations of 0.180 ± 0.001, 0.337 ± 0.001, and 0.341 ± 0.027 mg/ml, respectively. 3.3. Effect of the Combined Extract on Nonspatial Memory in OVX Rats {#sec3.3} -------------------------------------------------------------------- Memory-enhancing effect of the combined extract on nonspatial memory was shown in [Figure 2](#fig2){ref-type="fig"}. It was found that sham operation rats showed no significant change on novel object ratio (NOR). OVX rats treated with vehicle significantly decreased NOR throughout the study period (*p* value \< 0.01, 0.01, 0.05, and 0.05, resp., compared with the sham operation group). OVX rats with isoflavone treatment attenuated the reduction of NOR induced by OVX throughout the study period (*p* value \< 0.01, 0.05, 0.01, and 0.01, resp., compared with OVX + vehicle). Donepezil also showed the mitigation effect on the reduction of NOR in OVX rats, but significant differences were observed only at 7- and 21-day treatment periods (*p* value \< 0.001 and 0.01, resp., compared with the OVX + vehicle-treated group). The combined extract at doses of 20 and 80 mg/kg BW significantly attenuated the decreased NOR in OVX rats at 7, 21, and 28 days of treatment (*p* value \< 0.001 and 0.01; 0.05 all; and 0.01 and 0.05, resp., compared with the OVX + vehicle-treated group). The significant mitigation effect of PCP at a dose of 40 mg/kg BW on NOR was also observed at 7 and 21 days of treatment (*p* value \< 0.001 and 0.01, resp., compared with the OVX + vehicle-treated group). The increased NOR in the PCP treatment groups were also observed at a 14-day study period, but no significant difference was revealed. 3.4. Histological Change in the Prefrontal Cortex {#sec3.4} ------------------------------------------------- Based on the previous finding that the prefrontal cortex played a crucial role on working memory in rodents \[[@B33]\], we also investigated the neuron density in this area and results were shown in [Figure 3](#fig3){ref-type="fig"}. The current results demonstrated that OVX treated with vehicle significantly reduced the neuron density in PFC (*p* value \< 0.05, compared with the sham operation group). Both donepezil and isoflavone could attenuate the reduction of neuron density in PFC of OVX rats (*p* value \< 0.01 all; compared with the OVX + vehicle-treated group). In addition, combined extract at the dosage range used in this study also significantly attenuated the reduction of neuron density in PFC of OVX rats (*p* value \< 0.01, 0.01, and 0.05; compared with the OVX + vehicle-treated group). 3.5. Biochemical Assays {#sec3.5} ----------------------- [Table 2](#tab2){ref-type="table"} shows the effect of PCP on oxidative stress markers including MDA level and the activities of SOD, CAT, and GSH-Px. Sham operation showed no significant changes of the mentioned parameters. OVX rats which received vehicle significantly increased MDA level (*p* value \< 0.05; compared to the sham operation group) in the prefrontal cortex (PFC). Donepezil treatment failed to produce the significant changes on all parameters just mentioned in OVX rats. However, isoflavone significantly decreased MDA level but enhanced SOD activity (*p* value \< 0.05 and 0.01, resp., compared to the OVX + vehicle-treated group). Interestingly, PCP at all doses used in this study significantly decreased MDA level in PFC (*p* value \< 0.001 all; compared to the OVX + vehicle-treated group). The elevation of SOD activity in PFC was observed in OVX rats which received PCP at the concentrations of 20 and 40 mg/kg BW (*p* value \< 0.001 and 0.01, resp., compared with the OVX + vehicle-treated group). Since the cholinergic system plays a crucial role on learning and memory \[[@B34]\], we also investigated the effect of PCP on the cholinergic system by using the suppression activity of AChE or AChEI activity as an indirect index, and results were shown in [Figure 4](#fig4){ref-type="fig"}. It was found that both sham operation and OVX rats which received vehicle failed to produce the significant changes of AChE. The decreased AChE activity was observed in OVX rats which received donepezil and combined extract at the dose of 20 mg/kg BW (*p* value \< 0.05 and 0.01, resp., compared with the OVX rats + vehicle-treated group). When compared with OVX rats which received vehicle, no significant changes of AChE were observed in the other groups. 3.6. ERK1/2 Signaling Pathway {#sec3.6} ----------------------------- Based on the crucial role of ERK1/2 on the survival neurons and memory enhancement \[[@B28]\], the effect of PCP on the phosphorylation ERK1/2 was also determined, and results were shown in [Figure 5](#fig5){ref-type="fig"}. The current data showed that OVX rats significantly decreased expression of phosphorylation ERK1/2 in PFC (*p* value \< 0.001; compared with the sham operation group). Isoflavone and medium dose of PCP produced the significant attenuation effect induced by OVX (*p* value \< 0.01 and 0.05, resp., compared with the OVX + vehicle-treated group). 4. Discussion {#sec4} ============= The current data showed that ovariectomy, a widely used model of menopause, increased oxidative stress status, impairment of the cholinergic system, and memory impairment which were in concordance with the previous study \[[@B35], [@B36]\]. In the present study, it was found that OVX increased MDA without changes of the main scavenger enzymes such as SOD, CAT, and GSH-Px. This was in agreement with the previous study \[[@B37]\]. These results suggested that the elevation of MDA level might occur either via the increased oxidative stress production or via the decreased function of the nonenzymatic antioxidant system. Interestingly, PCP, the combined extract of purple corn cob and pandan leaves attenuated the memory impairment evaluated by using the object recognition test. It has been reported that the prefrontal cortex plays a crucial role on nonspatial memory. Lesion of this area could induce nonspatial memory impairment \[[@B38]\]. In this study, we have found that this impairment was attenuated by both isoflavone and all doses of PCP. The reduction of MDA level which indicated the decreased oxidative stress status and the enhanced neuron density was also observed in OVX rats which received isoflavone and all doses of PCP. Therefore, we suggested that PCP and isoflavone might improve oxidative stress status leading to the enhanced neuron density in PFC resulting in the improved nonspatial memory. The decreased oxidative stress status in PFC observed in this study might occur partly via the enhanced function of a scavenger enzyme especially SOD. Since no closed relationship between the decreased MDA level and the enhanced SOD activity in PFC was observed, other factors such as the decreased nonenzymatic system and the decreased oxidative stress production mentioned earlier might also contribute to the role. In addition to oxidative stress, the cholinergic system in PFC also plays the crucial role on memory. Depletion of acetylcholine (ACh) together with the elevation of acetylcholinesterase (AChE) in the PFC induces memory impairment both in primates and in rodents \[[@B39], [@B40]\]. The cognitive-enhancing effect of donepezil, an AChEI, was observed without changes of oxidative stress markers. Our study also demonstrated that both donepezil and low dose of PCP also suppressed AChE activity in PFC. Therefore, the enhanced cholinergic function in PFC by suppressing AChE activity in the mentioned area also contributes to a role on the cognitive-enhancing effect of both substances. It has been reported that polyphenol \[[@B14]\] including anthocyanins \[[@B41]\] can improve memory impairment induced by scopolamine which exerts the effect at muscarinic receptors. Since PCP contains polyphenols and anthocyanins, it is also possible that PCP also exerts its influence on muscarinic receptor. However, this requires further investigation. Recently, it has been demonstrated that mitogen-activated protein kinase (MAPK) especially ERK1/2 contributes to the pivotal role on learning and memory \[[@B42]\]. The substances which improve ERK signaling also improve the impairment of object recognition memory \[[@B43]\]. Based on this information, we did suggest that the cognitive-enhancing effect of isoflavone and medium dose of PCP might also occur via the enhanced ERK1/2 signaling pathway. Anthocyanins, a member of flavonoids, have been shown to exert the neuroprotective and cognitive-enhancing effect \[[@B44]\]. Since our fingerprint of PCP showed that the main ingredient in PCP was anthocyanins (peak 2--peak 5), we suggested that the neuroprotective effect and cognitive-enhancing effects of PCP in this study might involve anthocyanins. However, the effect of other ingredients still cannot be omitted. Our data failed to show a dose-dependent manner of PCP. The possible explanation might be due to the nonsimple linear relationship between the concentration of PCP and the observed parameters. Since many factors exert the influences on the observed parameters in this study, no simple linear relationship was observed. In addition, PCP contained many ingredients, so the effect of an active ingredient could be masked by other ingredients. In this study, we have found that our data showed that although in vitro data showed that IC~50~ of AChEI of PCP was very high, low dose of PCP could exert the cognitive-enhancing effect via the suppression of AChE in the prefrontal cortex while the medium and high doses of PCP failed to exert this effect. The possible explanation might also occur as that mentioned earlier in the lack of a dose-dependent study. Taken all data together, our study highlights the neuroprotective and cognitive effects of PCP that might occur primarily via the decreased oxidative stress which in turn increased neuron density in the brain especially in PFC, an area playing an important role on learning and memory especially nonspatial memory, resulting in the improved nonspatial memory. However, the improved cholinergic function and signal transduction via ERK1/2 might also exert the roles especially at low and medium doses, respectively. 5. Conclusion {#sec5} ============= This study is the first study to demonstrate the neuroprotective and cognitive effects of PCP. We have shown that the combined extract of purple corn cob and pandan leaves can be served as functional ingredients for developing neuroprotectant and cognitive enhancer for menopausal women. Therefore, we highlight how to create the value for agricultural waste such as purple corn cob. However, chronic toxicity is required in order to assure the consumption safety before moving forward to a clinical trial study. This study was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food Research Cluster of Khon Kaen University, the Integrative Complementary Alternative Medicine Research and Development Center, and the Invitation Research Grant of the Faculty of Medicine (Grant no. IN58219), Khon Kaen University. Conflicts of Interest ===================== The authors declare that they have no conflicts of interest. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} ###### The biological activity of PCP including total phenolic compound, anthocyanin content, DPPH radical activity, FRAP activity, and AChEI activity. Test PCP Standard reference ------------------------------------------------- --------------- --------------------------- Total phenolic compound (mg/l GAE) 184 ± 1.91 --- Anthocyanin content (mg/l CGE) 25.66 ± 0.32 --- FRAP activity (*μ*M L-ascorbic acid equivalent) 602.40 ± 2.33 --- DPPH radical activity (IC~50~*μ*g/ml) 56.37 ± 0.45 Ascorbic acid 2.89 ± 0.01 AChEI activity (IC~50~*μ*g/ml) 1950 ± 16.02 Donepezil 0.51 ± 0.03 ###### The effect of PCP on oxidative stress markers in the prefrontal cortex (*n* = 6/group). Oxidative stress markers in the prefrontal cortex ------------------------------ --------------------------------------------------- --------------- -------------------- --------------- Naïve control 0.073 ± 0.0.001 0.661 ± 0.034 0.567 ± 0.164 2.707 ± 0.234 Sham operation 0.077 ± 0.007 0.576 ± 0.052 0.834 ± 0.256 2.516 ± 0.237 OVX + vehicle 0.095 ± 0.008^a^ 0.523 ± 0.034 0.309 ± 0.100 2.684 ± 0.216 OVX + donepezil 3 mg/kg BW 0.090 ± 0.007 0.714 ± 0.047 1.387 ± 0.344 2.650 ± 0.187 OVX + isoflavone 20 mg/kg BW 0.059 ± 0.005^∗^ 0.902 ± 0.111 2.220 ± 0.458^∗∗^ 2.130 ± 0.210 OVX + PCP 20 mg/kg BW 0.047 ± 0.012^∗∗∗^ 0.866 ± 0.186 3.080 ± 0.352^∗∗∗^ 2.447 ± 0.359 OVX + PCP 40 mg/kg BW 0.034 ± 0.002^∗∗∗^ 0.799 ± 0.070 2.047 ± 0.184^∗∗^ 2.151 ± 0.160 OVX + PCP 80 mg/kg BW 0.032 ± 0.004^∗∗∗^ 0.639 ± 0.029 1.068 ± 0.366 1.921 ± 0.092 ^a^ *p* value \< 0.05, compared with the sham operation group. ^∗^, ^∗∗^,  ^∗∗∗^*p* value \< 0.05, 0.01, and 0.001, respectively, compared with the OVX + vehicle-treated group. Data were expressed as mean ± SEM. [^1]: Academic Editor: Luciano Saso

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Introduction {#s1} ============ MS can be diagnosed using the McDonald criteria [@pone.0019702-McDonald1]. MRI is the imaging method of choice with inflammatory diseases of the CNS and the most sensible method in the diagnosis of MS [@pone.0019702-Polman1]. MRI serves to depict acute as well as chronic MS lesions and their volume and allows physicians to measure the volume of additional regions in the brain. Olfactory disorders increasingly occur with Parkinson and Alzheimer\'s disease, but with less frequency with other neurodegenerative diseases [@pone.0019702-Ponsen1], [@pone.0019702-Mesholam1]. They often occur as early symptoms of these diseases. There are some scientific studies already that report olfactory disorders in MS patients at a rate of 15% [@pone.0019702-Hawkes1], 22.5% [@pone.0019702-Zorzon1] and 38.5% [@pone.0019702-Doty1]. Our study wants to examine pathological tissue changes and volume changes of the olfactory brain and of the olfactory bulb (OB) of MS patients and correlate them with their olfactory and gustatory function. The aim is to investigate possible causes of the origin of olfactory disorders in MS patients in greater depth. For this purpose, volumetric measurements of the OB as well as the olfactory brain were performed as objective examination method for the first time. The volumes were determined with manual segmentation. The exact number, localisation and volume of the lesions were determined using a standardised MRI protocol specially developed for our study. Olfactory testing was performed using the tripartide Threshold-Discrimination-Identification-Test (TDI). Materials and Methods {#s2} ===================== The study was performed in the time from January 2009 until November 2009. Ethical approval and trial registration was obtained by the medical ethics comittee of Charité, University of Berlin. Written consent was required to participate in the study. 34 prospective patients (24 women, 10 men, 22--65 years, Ø 41 years, mean disease duration 6 years) were examined. 25 patients had relapsing-remitting MS, five patients primary progressive and four patients secondary progressive MS. The patients were included in resp. excluded from the study after an ENT and neurological examination as well as by completing two questionnaires. Patients with the diagnosis MS (McDonald criteria, revised version 2005) were included in the study. Exclusion criteria were: pregnancy, age below 18 or over 65, olfactory disorders with a different genesis (post-infectious, post-traumatic, sinunasal, infections of the upper respiratory tract, tumours treated with radiation or chemotherapy, allergies, patients suffering from depression, Parkinson\'s or Alzheimer\'s disease). Patients taking drugs that could cause olfactory dysfunction as for example methotrexat, amitryptilin, certain antibiotics and D-Penicillamine were excluded by the questionnaires. Furthermore patients receiving corticosteroid treatment up to six weeks before testing were excluded from the study because corticosteroids can have an effect on the OF [@pone.0019702-Heilmann1]. To exclude dementia, the prospective patients were submitted to the Mini Mental State Examination (MMSE) [@pone.0019702-Folstein1]. A total score of at least 24 points was defined as exclusion criterion. Patients with grave physical disabilities were excluded using the Expanded Disability Status Scale (EDSS) [@pone.0019702-Kurtzke1]. A value of below seven was the threshold value for participation in the study. To exclude depression, a Becks Depression Inventory test (BDI) was performed. The BDI is a self-evaluation method for recording the severity of symptoms of depression [@pone.0019702-Beck1]. MS patients show a higher depression level [@pone.0019702-Foley1]. Disease-related symptoms, e.g. increased fatigue, lead to false higher scores in the BDI test. Adapted to the patients in our study, we defined a score of below 15 points as exclusion criterion. Patients with test scores of above 15 were tested once again by a psychologist using the Hamilton Rating Scale for Depression and the Hamilton Anxiety Rating Scale in an external assessment. A complete ENT examination with endoscopy for exact anatomic evaluation of the nasal passage, the sinuses, damage to the nasal mucosa as well as the presence of polyps was performed to exclude olfactory disorders of another origin. Exemption criteria also applied for the participation in the MRI examination. The olfactory capacity was evaluated using the tripartide TDI test recommended by the "Working Group Olfactology and Gustology" of the German ENT Society (standardised, reliability r = 0.72) [@pone.0019702-Kobal1]. The Threshold test consists of 48 sniffing sticks with a 16-stage dilution series of n-butanol for determining the olfactory perception threshold of a patient. The discrimination test consists of 48 sniffing sticks to test the distinction of smells. Everyday smells have to be identified with the identification test. A TDI value of less than 16 means anosmia, up to 30 points hyposmia and above 30.5 points normosmia [@pone.0019702-Hummel1]. Paper taste test strips (TST) by Burghart, Wedel, Germany, were used to determine the sense of taste [@pone.0019702-Mueller1]. The 16-part test checks the four tastes sweet, sour, salty and bitter in four different concentrations each. The test strip is placed in the centre of the front third of the top of the tongue. Prior to each application, the mouth is rinsed with water. A test value of below 9 indicates a reduced sense of taste. The patients were examined on a 1.5 Tesla MRI system (Symphony Vision, Siemens, Erlangen) using a standardised protocol to visualise the OB and the olfactory brain. The protocol contained 3 mm T2 turbo spin echo sequences (TR 3070 msec, TE 107 msec, Matrix 256×192, FOV 250 mm). 3 mm proton density sequences (TR 3070 msec, TE 18 msec, Matrix 256×192, FOV 250 mm) were applied to determine the MS lesion load by using axial sections. 3 mm T1-weighted sequences (TR 600 msec, TE 14 msec, Matrix 256×192, FOV 250 mm) were used for volumetric measurements of the olfactory brain. Isotropic, 0.5 mm thick, high-resolution, strongly T2-weighed CISS sequences (constructive interference in steady state, TR 8.56 msec, TE 4.28 msec, Matrix 256×205, FOV 130 mm) were performed additionally for volumetry of the OB. Gadolinum was applied as contrast agent to allow a detailed assessment of the MS lesions. The olfactory brain was defined as the piriform and entorhinal cortex, front agranular regions of the insular lobe up to the anterior comissure, orbitofrontal cortex. White matter in between these regions was also considered as part of the olfactory brain in order to take into account the fact that connections could have been damaged by MS lesions [@pone.0019702-Hummel2]. The volume of the OB ([Figure 1](#pone-0019702-g001){ref-type="fig"}) and the olfactory brain was determined with MRI by circumnavigating the contours in axial sections with the computer software Amira 3.2. {#pone-0019702-g001} We determined the number and volume of the lesions in the olfactory brain as well as in the rest of the brain. The examiners were blinded to the patient\'s olfactory test and EDSS scores. MRI measurements, olfactory and neurological testing have been performed on the same day. Statistical analysis was performed with SPSS (17.0, Chicago, USA). Correlations were determined with a bivariate regression analysis. The results of the MRI evaluation were compared with smell and taste test values using the Pearson and Spearman correlation. The significance level was defined at ≤0.05. The Welch test and the Brown-Forsythe test were used as statistic test method to check the equality of the mean values of different test parameters in patients with different types of progression. The Mann-Whitney U-test for independent samples was used to compare different control samples. The patients were recruited continuously from the MS centre, Charité Campus Mitte. A healthy control group of 30 persons whose gender, age and smoking habits coincided with that of the patients were recruited among the patients\' family members as well as among the hospital staff. All participants of the control group had to complete the same questionnaires and received an ENT examination in order to exclude the same causes for olfactory dysfunction as for the MS patients. Results {#s3} ======= 34 MS patients and 30 healthy control persons were examined. The volume of the OB ([Table 1](#pone-0019702-t001){ref-type="table"}) of the 34 patients correlated with the number of MS lesions in the olfactory brain (r = −0.34, p\<0.05) as well with the volume of the MS lesions in the olfactory brain (r = −0.37, p\<0.05). 10.1371/journal.pone.0019702.t001 ###### Results of the MRI examination. {#pone-0019702-t001-1} Mean value Standard deviation ---------------------------------------------- ---------------- -------------------- Total volume OB 128.0 mm^3^ ±41.54 mm^3^ Volume OB right 62.35 mm^3^ ±21.08 mm^3^ Volume OB left 65.65 mm^3^ ±22.89 mm^3^ Volume Olfactory Brain 38896.79 mm^3^ ±7731.36 mm^3^ Number of lesions in the Olfactory Brain 2.38 ±3.62 Volume lesions in the Olfactory Brain 90.37 mm^3^ ±171.42 mm^3^ Number of lesions in the Non Olfactory Brain 43.35 ±25.40 Volume of lesions in the Non Olfactory Brain 11599.0 mm^3^ ±14333.61 mm^3^ The volume of the olfactory brain correlated with the volume of the lesions in the olfactory brain (r = −0.33, p = 0.05) and the EDSS value (r = −0.37, p\<0.05). A significant difference of p = 0.03 was observed between the average volumes of the patient\'s olfactory brain with different types of progression of MS. The volumes of the patients with primary and secondary progressive form were lesser than those with relapsing-remitting progressive form. Subjective olfactometry using sniffing sticks showed hyposmia in 41% of 34 MS patients ([Table 2](#pone-0019702-t002){ref-type="table"}). In the healthy control group, 8% of the control persons showed hyposmia. 71% of MS patients with a decreased OB volume and 83% with a decreased olfactory brain volume displayed hyposmia. Nine of 34 patients refused the taste strip test. 16% of 25 patients had a gustatory dysfunction. In the control group, 5% of the patients had a taste disorder. 10.1371/journal.pone.0019702.t002 ###### Patient data: Results of olfactometry, gustometry and neurological examinations. {#pone-0019702-t002-2} Mean value Standard deviation ----------------------------- ------------ -------------------- Age (years) 41.4 ±12.4 Duration of disease (years) 6.1 ±7.9 TDI 31.5 ±4.0 T (threshold) 6.3 ±1.8 D (Discrimination) 12.4 ±2.5 I (Identification) 12.8 ±1.7 TST 10.8 ±2.8 EDSS 3.3 ±2.1 BDI 7.3 ±6.4 MMSE 28.9 ±1.1 The TST value correlated with the number of lesions in the olfactory brain (r = −0.49, p\<0.05) as well as their volumes (r = −0.52, p\<0.05). The TDI value correlated with the EDSS score (r = −0.54, p\<0.01). Discussion {#s4} ========== The olfactory sense is of great importance in everyday life. The olfactory organ is a control function of the body and warns us about toxic substances and spoilt food. Some neurological disorders go hand in hand with olfactory disorders [@pone.0019702-Ponsen1], [@pone.0019702-Mesholam1]. Detecting olfactory disorders is becoming increasingly important in the research of neurodegenerative diseases [@pone.0019702-Hawkes1]. There is a substantial need for diagnosis and the significance of imaging the olfactory system is also increasing continuously [@pone.0019702-Abolmaali1]. Special attention is paid to the OB, which seems to be an image of the neuronal afference of smelling due to its high plasticity [@pone.0019702-Abolmaali2]. A significant correlation between the OB volume and the olfactory ability was determined in several studies [@pone.0019702-Rombaux1], [@pone.0019702-Goektas1], [@pone.0019702-Buschhter1]. The olfactory ability is reduced with increasing OB hypoplasia [@pone.0019702-Rombaux1], [@pone.0019702-Goektas1], [@pone.0019702-Buschhter1]. An OB volume of \<100 mm^3^ was determined as the cut-off value for a reduced OB volume [@pone.0019702-Bauknecht1]. In several studies, reduced OB volumes were determined in patients with olfactory disorders of a post-infectious, post-traumatic and sinunasal genesis [@pone.0019702-Mueller2], [@pone.0019702-Rombaux2], [@pone.0019702-Yousem1]. A reduced OB volume was also observed in patients suffering from Alzheimer\'s disease and schizophrenia [@pone.0019702-Turetski1], [@pone.0019702-Thomann1]. The OB volumes were not reduced in patients suffering from Parkinson\'s disease [@pone.0019702-Hummel3]. In our study, we investigated the volume of the OB and the olfactory brain in MS patients and compared this to the number and volume of pathological lesions. The OB volume of the 34 MS patients was diminished with a mean value of 128 mm^3^ compared to the standard values of the normal population with the same age (134 mm^3^) [@pone.0019702-Buschhter1]. The correlation of number and volume of the plaques in the olfactory brain with the OB volume could explain the variability of the OB volumes in MS patients. The OB volume decreases with an increasing number and volume of MS lesions in the olfactory brain. These results may help to explain the occurrence of olfactory disorders in MS patients. The olfactory brain volume correlated with the volume of MS lesions in the olfactory brain as well as the EDSS score. A reduced olfactory brain volume was detected in two studies with patients suffering from Parkinson\'s and schizophrenia [@pone.0019702-Wattendorf1], [@pone.0019702-Turetsky1]. The larger the volume of the MS lesions in the olfactory brain, the more pronounced was the hypoplasia of the olfactory brain. 83% of patients with an olfactory brain volume of below 30000 mm^3^ displayed hyposmia. This correlation may also serve to explain olfactory disorders in MS patients. The olfactory brain volumes in MS patients with primary and secondary progression were significantly smaller than those of MS patients with relapsing-remitting progression. Demyelination with axon destruction results with chronic progredient progression, which might explain this significant difference in volume. The olfactory brain volume as well as the TDI value correlated with the EDSS score, which provides information about the degree of disability of the MS patient. Several MS patients reported about a subjective improvement resp. deterioration of their olfactory sense in case of changed physical symptoms, e.g. during an acute episode. The TDI score seems to uncover changes in the olfactory capacity in MS patients. The TST values correlated with the number and volume of the lesions in the olfactory brain. On a cortical level, the gustatory and olfactory systems intersect, above all in the insular cortex, the amygdala and the orbitofrontal cortex. On downstream paths of the OB\'s mitral cells from the piriform to the orbitofrontal cortex, the olfactory stimuli are linked with gustatory information in the anterior insula (multimodal integration). In our study, we investigated the volume of the OB and olfactory brain in MS patients for the first time and compared this to the number and volume of MS lesions. The correlation of a higher number and volume of MS lesions in the olfactory brain with a decreased OB and olfactory brain volume might explain the occurrence of olfactory dysfunction in MS patients. The results should be verified in a longitudinal study with a higher number of patients. **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**The authors have no support or funding to report. [^1]: Conceived and designed the experiments: OG FF LH. Performed the experiments: OG FAS GB. Analyzed the data: FAS BD KE. Contributed reagents/materials/analysis tools: OG FF GB LH. Wrote the paper: OG FAS.

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1. Introduction {#sec1} =============== *Helicobacter pylori* (*H. pylori*) is a spiral shaped microaerophilic gram-negative bacterium that resides in the gastric epithelial mucosa and induces an inflammatory response leading to gastritis and peptic ulcer disease \[[@B1], [@B2]\]. It has been implicated as playing a role in gastrointestinal malignancies, especially gastric adenocarcinoma and MALToma until the latter could be treated with *H. pylori*eradication \[[@B3], [@B4]\]. *H. pylori* has a worldwide prevalence rate of about 50%, with a higher prevalence in developing countries \[[@B5], [@B6]\]. According to population-based studies, it has been shown that the *H. pylori* infection rate is very high in the Iranian population \[[@B7]\]. *H. pylori* detection can be made with diverse diagnostic tests, which are technically divided into invasive and noninvasive based on whether endoscopy is required or not. Invasive tests offer the possibility of obtaining tissue samples, which can be used for a rapid urease test (RUT), culture, polymerase chain reaction (PCR), and histopathologic evaluation. Noninvasive tests include serum *H. pylori* IgG antibody titer, the urea breath test (UBT), and *H. pylori* stool antigen assay. Compared to noninvasive diagnostic modes, however, invasive techniques are inconvenient for patients and also have higher cost \[[@B8]\]. A UBT diagnostic test is based on the fact that swallowed "labeled carbon-containing urea" is broken down to ammonia and carbon dioxide (CO~2~) by the urease-producing microorganism (*H. pylori*) in the gastric mucosa, and, finally, tagged carbon within the liberated CO~2~ is detected in exhaled breath samples \[[@B9], [@B10]\]. Between two carbon isotopes (^13^C and ^14^C), which are used for the UBT, the ^13^C isotope has the difficulty of requiring more complex equipment, such as a mass spectrophotometer and administration of a pretest meal such as citric acid. However, when the ^14^C isotope is utilized, the required equipment is only a portable compact beta-scintillation counter, which offers the convenience of performing the test in the office. Although the ^14^C isotope is radioactive, microdose (1 *μ*Ci) ^14^C has the minimal radiation of one day background exposure \[[@B11]\]. We encountered repeatedly, dyspeptic patients, in our clinical practice, whose UBT results were not consistent with histopathology about *H*. *pylori*infection; based on Helicobacter genetic polymorphisms and differences between *H. pylori* strains in different countries, and since clinical validation of Heliprobe ^14^C-UBT has not yet been investigated in an Iranian population, we conducted a prospective study to compare Heliprobe ^14^C-UBT performance against diagnostic gold standards \[[@B12]--[@B14]\]. 2. Materials and Methods {#sec2} ======================== We studied 125 consecutive patients with dyspepsia that had been referred for upper GI endoscopy. We defined dyspepsia, based on the Rome III criteria, as having one or more of the following conditions: postprandial fullness (termed postprandial distress syndrome), early satiation (inability to finish a normal-sized meal or postprandial fullness), and epigastric pain or burning (termed epigastric pain syndrome) \[[@B15]\]. We considered subjects aged 15 y--75 y. We excluded patients who had been using proton pump inhibitors, H2 blockers, or any antibiotics within four previous weeks of the endoscopic evaluation. Pregnant women, patients who had a history of *H. pylori* eradication, and those with any severe cardiopulmonary disorders or debilitating or life-threatening conditions were excluded as well. Study personnel were blinded as to patient test results. The study protocol was approved by the ethics committee of the Gastrointestinal and Liver Diseases Research Center of Guilan University of Medical Sciences, and written informed consents were obtained from each participant. After an overnight fast, patients underwent gastroscopy with a FUJINON endoscope, and four separate gastric biopsies were taken, three from the antrum and one from the corpus. One of the antral samples was used for a rapid urease test (RUT), and two antral samples plus the corpus biopsy sample were used for histopathologic examination. To perform the RUT, we utilized a home-made liquid rapid urease kit (Gastric Urease, Bahar Afshan Co., Iran). We put tissue samples in a yellow-colored reagent liquid, and results were read after 30 min, 60 min, and finally after 24 hours. Liquid color changes into deep red, purple, or violet indicated a positive result. Negative results were indicated by no color change. Tissue samples were prepared with standard hematoxylin and eosin (H&E) and Giemsa stainings for histopathologic investigation. The histopathology exam result was considered positive when *H*. *pylori* was detected in either of the stains and negative when the organism was not detected in any. One blood sample was obtained from each patient to be examined for anti *H. pylori*IgG titer. We utilized the ELIZA method (LDN, Nordhorn, Germany), and sera with titers that were \>11 international units (IU) were considered positive (test cut-off point: 10 IU, \<9 IU: negative, 9--11 IU: doubtful, \>11 IU: positive); we regarded doubtful results as negative. After gastroscopy, we performed Heliprobe ^14^C-UBT tests. In order to carry out the UBT, after an overnight fast, the patient swallowed a ^14^C-labeled urea-containing capsule (Helicap, Institute of Isotopes, Budapest, Hungary) with water. The overall activity of these capsules is as small as 1 *μ*Ci (37 KBq). After 15 minutes, the patient breathed out into a dry cartridge (Heliprobe breath card, Kibion AB, Uppsala, Sweden) through its mouthpiece until the color of the card indicator changed from orange to yellow, which took about 1 min to 2 min. Thereafter, the breath card was inserted into a small desktop Geiger Müller counter (Heliprobe Analyser, Kibion AB, Uppsala, Sweden), and the radioactivity of the breath samples was read after 250 seconds of an automated process. Finally, the test results were expressed on the LCD of the analyzer in a numeric fashion (0: patient not infected, 1: borderline result, 2: patient infected), which corresponded to radioactivity as count per minute (CPM): \<25 CPM: patient not infected, 25--50 CPM: borderline result, \>50 CPM: patient infected. We considered grades 0 and 1 as negative results in our study, and only samples with activities that were more than 50 CPM (expressed as no. 2 on the counter LCD) were regarded as positive. Descriptive analysis was done for demographic features. Gold standard for *H. pylori*positivity was defined as "positive results for any two of three diagnostic methods (histopathology, RUT, serology)." Sensitivity, specificity, negative and positive predictive values (NPV and PPV), and the accuracy of the UBT were computed against our GS; for categorical variables, 95% confidence interval (95% CI) was calculated. 3. Results {#sec3} ========== We enrolled 125 consecutive patients in our study according to the above mentioned inclusion and exclusion criteria; 65 (52%) were females, and 60 (48%) were males. Patient ages ranged from 18 y to 66 y with a mean of 35.81 ± 12.97 y. As a result of histopathologic evaluation of tissue specimens, 69 (55.2%) patients were found to be infected with *H. pylori,*and 56 (44.8%) were not infected. Serologic examination of patient sera samples for IgG antibody against *H. pylori*showed 87 (69.6%) positive results, while the remaining 38 (30.4%) were seronegative. The RUT results were positive in 63 (50.4%) patients and negative in 62 (49.2%) patients. *H. pylori*infection was found in 67 (53.6%) subjects by the ^14^C urea breath test (^14^C-UBT), and 58 (46.4%) subjects were negative. All four tests were positive or negative in 57 (45.6%) and 34 (27.2%) patients, respectively. Only 2 patients had no histopathologic evidence of *H. pylori* infection, whilst their RUT and serology results were positive. (Incidentally these two patients also had positive UBTs.); We think that this might be the result of errors during sampling or histopathologic examination. 20 patients showed solitary positive serology tests which indicated recent past infection. [Table 1](#tab1){ref-type="table"} shows other discordances between our test results. Compared to our GS, the UBT could correctly detect 67 of 71 *H. pylori* infected subjects with 94% sensitivity (95% CI: 85--98%) and 100% positive predictive value (PPV; 95% CI: 93--100%). The UBT also excluded correctly all 54 uninfected patients with 100% specificity (95% CI: 92--100%) and negative predictive value (NPV) of 93% (95% CI: 82--98%). The UBT showed 97% accuracy ([Table 2](#tab2){ref-type="table"}). 4. Discussion {#sec4} ============= *H. pylori* has a high prevalence rate in developing countries, such as Iran. According to data reported by Derakhshan et al., *H. pylori*must be considered a risk factor for noncardiac gastric adenocarcinoma in Iranian patient \[[@B16]\]. Eradication of *H. pylori*could lower the incidence rate of gastric cancer \[[@B17]\]. A more precise diagnosis of *H. pylori,* allowing an earlier eradicating treatment, may play a crucial role in cancer prevention strategies. Currently, there is an increasing need for an easy-to-perform, accurate, and readily available diagnostic technique in *H. pylori*prevalent populations. Considering economic concerns and the availability of tests in developing countries, as well as testing possibilities in medical clinics, we recommend use of the Heliprobe ^14^C-UBT over other noninvasive and invasive tests. Based on the data that suggest that the combination of tests increases their overall diagnostic power, we combined *H. pylori* serologic assessment and the RUT with histopathology in order to account for the possibility of very few false negative results from microscopic examination (which were seen in two cases in our study) \[[@B18]\]. We discovered that the ^14^C-UBT has 94% sensitivity, 100% specificity, 100% PPV, 93% NPV, and 97% accuracy, compared with GS. These findings are compatible with Jonaitis et al.\'s study results, with their defined *H. pylori* positive gold standard as "at least one positive test of RUT or histopathology". They found that Heliprobe had 92% sensitivity, 100% specificity, 100% PPV, 84% NPV, and 94% accuracy \[[@B19]\]. Our results are also in accordance with Ozdemir et al.\'s results for Heliprobe performance (96.6% sensitivity, 100% specificity, 93.7% PPV, 100% NPV, and 97.7% accuracy), although their *H. pylori*positive gold standard was defined as "positivity of any two of the three following tests (RUT, PCR, and histopathology)" \[[@B20]\]. In Rasool et al.\'s study regarding histopathology alone as GS, Heliprobe sensitivity, specificity, NPV, PPV, and accuracy were 92%, 93%, 84%, 97%, and 93%, respectively, which were consistent with our results although the studies\' gold standards are slightly different \[[@B21]\]. In another study, conducted by Öztürk et al. that is again regarding only histopathology as GS, Heliprobe had higher sensitivity (100%), but its specificity (76%) was lower than that of our study \[[@B22]\]. People who undergo medical diagnostic tests using radioisotopes are often worried about radiation exposure. The half life of the ^14^C isotope is about 5000 years, but, with regards to the short biologic half life of urea, more than two-thirds of the tagged urea will be excreted in the urine within the following three days of the test; moreover, the total dose of the ^14^C used in the test is very low, and activity of this quantity of isotope was evaluated as 1 *μ*Ci. Accordingly, based on the published data, about 800 breath testing episodes must be carried out for one person to receive an effective dose equivalent to the amount that an average person absorbs from natural sources in one year \[[@B23]\]. Considering the few times a person needs to be tested with the ^14^C-UBT, the lifelong cumulative radiation of the test is negligible. Even in conditions of repeated UBT testing, radiation exposure risk is very low. Previously, ^14^C-UBT was not used in children because of the concerns about the radiation hazards; however, diverse studies have established its safety in pediatric patients \[[@B11], [@B24]\]. Although no experimental study has yet been done to assess ^14^C-UBT safety in pregnancy, Bentur et al. have claimed that, in view of the insignificant ^14^C radioactivity, fetal radiation exposure is extremely lower than teratogenic thresholds \[[@B25]\]. One of the advantages of the Heliprobe system is that it can be used in a clinical setting, allowing the preparation of test results on-site in less than one hour. The portable beta-scintillation counter that is used in this test could simply be placed on a desktop; however, the ^13^C-UBT needs a sophisticated mass spectrophotometer to read the results. Of course, ^13^C-UBT has some advantages over ^14^C-UBT such that the former utilizes a nonradioactive isotope that makes it suitable to use in pregnant women and children. Although some studies, as mentioned earlier, have already emphasized on ^14^C-UBT safety in children, ^13^C-UBT is still the preferred method in them. Considering ^13^C safety, it is also a better isotope than ^14^C for epidemiologic studies, as some studies used it to investigate *H. pylori*routes of transmission in preschool age \[[@B26], [@B27]\]. Stool antigen is another sensitive and specific noninvasive diagnostic test for *H. pylori* \[[@B28]\]. Although it is competitive with the ^14^C-UBT in terms of accuracy, but it is not appropriate for office use because it is a time consuming exam regarding sampling limitations and off-site test interpretation. Conclusively, compared to invasive gold standards, the Heliprobe ^14^C-UBT is an accurate, sensitive, and specific test for *H*. *pylori*diagnosis. The main advantages of the Heliprobe ^14^C-UBT are its rapidity and patient convenience. Furthermore, in view of the very low radioactivity of the Heliprobe ^14^C-UBT and its portability, this test seems to be a more suitable option for office use than a nonradioactive, complex and off-site ^13^C-UBT as well as other invasive diagnostic modalities. There are no potential conflict of interests that any of the authors have identified relevant to this paper. The authors thank the Gastrointestinal and Liver Diseases Research Center (GLDRC) of Guilan University of Medical Sciences and Beta Medical Engineering Co., which supported this work in part (<http://www.betamedgroup.com/>), especially Mr. Ali Rafat-Zand for financial support for this work. ###### Distribution of *H. pylori*diagnostic test discordant results. Patients (*n*) Histology RUT Serology UBT ---------------- ----------- ----- ---------- ----- 57 \+ \+ \+ \+ 4 \+ \+ − \+ 4 \+ − \+ \+ 4 \+ − \+ − 2 − \+ \+ \+ 20 − − \+ − 34 − − − − RUT: rapid urease test; UBT: urea breath test; +: positive; −: negative; *n*: number. ###### Diagnostic performance of Heliprobe ^14^C-UBT against gold standard. Heliprope ^14^C-UBT compared to: Sensitivity (95% CI) Specificity (95% CI) PPV (95% CI) NPV (95% CI) Accuracy (95% CI) ---------------------------------- ---------------------- ---------------------- ----------------- --------------- ------------------- Gold standard 94% (85--98%) 100% (92--100%) 100% (93--100%) 93% (82--98%) 97% ^14^C-UBT: urea breath test with labeled carbon-14; RUT: rapid urease test; PPV: positive predictive value; NPV: negative predictive value; CI: confidence interval. For gold standard definitions refer to the text. [^1]: Academic Editor: Y. Yamaoka

1. Introduction {#sec1} =============== Periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. Prostaglandin E~2~ (PGE~2~), interleukin- (IL-)6, and IL-8 are known to play important roles in inflammatory responses and tissue degradation. PGE~2~ has several functions in vasodilation, the enhancement of vascular permeability and pain, and the induction of osteoclastogenesis and is believed to play important roles in inflammatory responses and alveolar bone resorption in periodontal disease \[[@B1]\]. IL-6 has the ability to induce osteoclastogenesis \[[@B2], [@B3]\]. IL-8 acts as a chemoattractant for neutrophils \[[@B4]\] that produce proteases such as cathepsin, elastase, and matrix metalloproteinase- (MMP-)8, leading to tissue degradation. Recently, we reported that several kampo medicines, shosaikoto \[[@B5]\], hangeshashinto \[[@B6]\], and orento \[[@B7]\], suppress lipopolysaccharide- (LPS-) induced PGE~2~ production by HGFs and suggested that these kampo medicines have anti-inflammatory effects in periodontal disease. Another kampo medicine, kakkonto (TJ-1), has been clinically used for various diseases such as the common cold, coryza, the initial stage of febrile diseases, and inflammatory diseases. There are several reports that kakkonto shows antiallergic effects \[[@B8], [@B9]\] and antiviral effects \[[@B10]--[@B13]\] in animal and *in vitro* experimental models. For anti-inflammatory effects, kakkonto has been reported to decrease PGE~2~ production in cultured rabbit astrocytes \[[@B14]\]. Therefore, we considered the possibility that kakkonto decreases PGE~2~ production by human gingival fibroblasts (HGFs) and has anti-inflammatory effects with respect to periodontal disease. However, the anti-inflammatory effects of kakkonto are not adequately understood. HGFs are the most prominent cells in periodontal tissue. Moreover, LPS-treated HGFs produce inflammatory chemical mediators such as PGE~2~ and inflammatory cytokines such as IL-6 and IL-8 \[[@B2], [@B15], [@B16]\]. Moreover, because HGFs have sustained production of PGE~2~ \[[@B17]\], IL-6, and IL-8 \[[@B18]\] in the presence of LPS, these mediators and cytokines in periodontal tissues are thought to be derived from HGFs. Therefore, we believe that examining the effects of drugs on HGFs, as well as on monocytes and macrophages, is important in the study of periodontal disease. In the present study, we examined the effect of kakkonto on LPS-induced PGE~2~, IL-6, and IL-8 production using this *in vitro* model. 2. Materials and Methods {#sec2} ======================== 2.1. Reagents {#sec2.1} ------------- Kakkonto was purchased from Tsumura & Co. (Tokyo, Japan; lot number: D23122), and its components are listed in [Table 1](#tab1){ref-type="table"}. Kakkonto was suspended in Dulbecco\'s modified Eagle\'s medium (D-MEM, Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal calf serum, 100 units/mL penicillin, and 100 mg/mL streptomycin (culture medium) and was rotated at 4°C overnight. Then, the suspension was centrifuged and the supernatant was filtrated through a 0.45 *μ*m-pore membrane. Phorbol 12-myristate 13-acetate (PMA) purchased from Sigma. Other reagents were purchased from Nacalai tesque (Kyoto, Japan). LPS from *Porphyromonas gingivalis* 381 was provided by Professor Nobuhiro Hanada (School of Dental Medicine, Tsurumi University, Japan). 2.2. Cells {#sec2.2} ---------- HGFs were prepared as described previously \[[@B6]\]. In brief, HGFs were prepared from free gingiva during the extraction of an impacted tooth with the informed consent of the subjects who consulted Matsumoto Dental University Hospital. The free gingival tissues were cut into pieces and seeded onto 24-well plates (AGC Techno Glass Co., Chiba, Japan). HGFs were maintained in culture medium at 37°C in a humidified atmosphere of 5% CO. For passage, HGFs were trypsinized, suspended, and plated into new cultures in a 1 : 3 dilution ratio. HGFs were used between the 10th and 15th passages in the assays. This study was approved by the Ethical Committee of Matsumoto Dental University (number 0063). 2.3. Cell Viability {#sec2.3} ------------------- The numbers of cells were measured using WST-8 (Cell Counting Kit-8; Dojindo, Kumamoto, Japan) according to the manufacturer\'s instructions. In brief, HGFs (10,000 cells/well) were seeded in 96-well plates (AGC Techno Glass Co., Chiba, Japan) and incubated in serum-containing medium at 37°C overnight. Then, the cells were treated with various concentrations of kakkonto (0, 0.5, 1, 2, 5, and 10 mg/mL) in the absence or presence of LPS (10 ng/mL) for 24 h (200 *μ*L each well) in quadruplicate for each sample. Then, the media were removed by aspiration and the cells were treated with 100 *μ*L of mixture of WST-8 with culture medium for 2 h at 37°C in CO incubator. Optical density was measured (measured wavelength at 450 nm and reference wavelength at 655 nm) using an iMark microplate reader (Bio-Rad, Hercules, CA, USA), and the mean background value was subtracted from each value. Data is represented as means ± SD (*n* = 4). 2.4. Enzyme-Linked Immunosorbent Assay (ELISA) {#sec2.4} ---------------------------------------------- HGFs (10,000 cells/well) were seeded in 96-well plates and incubated in serum-containing medium at 37°C overnight. Then, the cells were treated with various concentrations of kakkonto (0, 0.01, 0.03, 0.1, 0.3, and 1 mg/mL) in the absence or presence of LPS (10 ng/mL) for 24 h (200 *μ*L each well) in triplicate for each sample. After the culture supernatants were collected, viable cell numbers were measured using WST-8 as described above. The concentrations of PGE~2~, IL-6, and IL-8 in the culture supernatants were measured by ELISA according to the manufacturer\'s instructions (PGE~2~, Cayman Chemical, Ann Arbor, MI, USA; IL-6 and IL-8, Biosource International Inc., Camarillo, CA, USA) and were adjusted by the number of viable cells. Data are represented as ng or pg per 10,000 cells (mean ± SD, *n* = 3). 2.5. Cyclooxygenase Activity {#sec2.5} ---------------------------- The effects of kakkonto on the activities of cyclooxygenase (COX)-1 and COX-2 were analyzed using a COX inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer\'s instructions. COX activities were evaluated by the measurement of prostaglandin produced from arachidonic acid by COX-1 or COX-2. These values were normalized to a relative value of 100% for cells without LPS or kakkonto treatments, and are represented as means ± SD (*n* = 3). 2.6. Western Blotting {#sec2.6} --------------------- HGFs were cultured in 60 mm dishes and treated with combinations of LPS and kakkonto for the indicated times. Then, cells were washed twice with Tris-buffered saline, transferred into microcentrifuge tubes, and centrifuged at 6,000 ×g for 5 min at 4°C. Supernatants were aspirated and cells were lysed on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethyleneglycol bis(2-aminoethylether) tetraacetic acid (EGTA), 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 *μ*g/mL aprotinin, 5 *μ*g/mL leupeptin, and 1 *μ*g/mL pepstatin) for 30 min at 4°C. Then, samples were centrifuged at 12,000 ×gfor 15 min at 4°C, and supernatants were collected. The protein concentration was measured using a BCA Protein Assay Reagent Kit (Pierce Chemical Co., Rockford, IL, USA). The samples (10 *μ*g of protein) were fractionated in a polyacrylamide gel under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane (Hybond-P; GE Healthcare, Uppsala, Sweden). The membranes were blocked with 5% ovalbumin for 1 h at room temperature and incubated with primary antibody for an additional 1 h. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized with an ECL Kit (GE Healthcare). Antibodies against COX-2 (sc-1745, 1 : 500 dilution), cytoplasmic phospholipase A~2~ (cPLA~2~, sc-438, 1 : 200 dilution), annexin1 (sc-11387, 1 : 1,000 dilution), and actin (sc-1616, 1 : 1,000 dilution), which detects a broad range of actin isoforms, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK; p44/42 MAP kinase antibody, 1 : 1,000 dilution) and phosphorylated ERK (Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) monoclonal antibody, 1 : 2,000 dilution) were from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-goat IgG (sc-2020, 1 : 20,000 dilution) was from Santa Cruz, and anti-rabbit IgG (1 : 20,000 dilution) and anti-mouse IgG (1 : 20,000 dilution) were from DakoCytomation (Glostrup, Denmark). 2.7. Statistical Analysis {#sec2.7} ------------------------- Differences between groups were evaluated by the two-tailed pairwise comparison test with a pooled variance, followed by correction with the Holm method (total 16 null hypotheses; 5 null hypotheses without kakkonto versus with kakkonto in the absence of LPS, 5 null hypotheses without kakkonto versus with kakkonto in the presence of LPS, and 6 null hypotheses without LPS versus with LPS) (Figures [1](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}). Differences between the control group and experimental groups were evaluated by a two-tailed Dunnett\'s test ([Figure 3](#fig3){ref-type="fig"}). All computations were performed with the statistical program R (<http://www.r-project.org/>). Dunnett\'s test was performed using the "glht" function in the "multcomp" package. Values with *P* \< 0.05 were considered significantly different. 3. Results {#sec3} ========== 3.1. Effects of Kakkonto on HGFs Viability {#sec3.1} ------------------------------------------ First, we examined the effect of kakkonto on HGFs viability. The viability of HGFs was approximately 90% at up to 1 mg/mL of kakkonto for a 24 h treatment in the absence or presence of LPS ([Figure 1](#fig1){ref-type="fig"}). The viabilities were approximately 70% and 20% at 5 mg/mL and 10 mg/mL of kakkonto, respectively ([Figure 1](#fig1){ref-type="fig"}). Therefore, we used kakkonto at the concentrations of up to 1 mg/mL in further experiments. 3.2. Effects of Kakkonto on PGE~2~, IL-6, and IL-8 Production {#sec3.2} ------------------------------------------------------------- We examined whether kakkonto affects the production of PGE~2~ and inflammatory cytokines (IL-6 and IL-8) by HGFs. Because kakkonto affects cell viability, the concentrations of PGE~2~, IL-6, and IL-8 needed to be adjusted according to viable cell number. When HGFs were treated with 10 ng/mL of LPS, HGFs produced large amounts of PGE~2~, IL-6, and IL-8. Indomethacin decreased LPS-induced PGE~2~ production in a concentration-dependent manner but slightly decreased LPS-induced IL-6 and IL-8 production (data not shown). Kakkonto significantly decreased PGE~2~ production in a concentration-dependent manner ([Figure 2(a)](#fig2){ref-type="fig"}). In the absence of LPS, kakkonto had no effect on PGE~2~ production ([Figure 2(a)](#fig2){ref-type="fig"}). In contrast, kakkonto increased LPS-induced IL-6 and IL-8 production (Figures [2(b)](#fig2){ref-type="fig"} and [2(c)](#fig2){ref-type="fig"}). In the absence of LPS, up to 0.1 mg/mL of kakkonto did not affect IL-6 and IL-8 production, but above 0.3 mg/mL of kakkonto, their concentrations were increased (Figures [2(b)](#fig2){ref-type="fig"} and [2(c)](#fig2){ref-type="fig"}). Similar results were obtained using human skin fibroblast TIG-103 cells (data not shown). 3.3. Effects of Kakkonto on COX Activities {#sec3.3} ------------------------------------------ Because PGE~2~ production is regulated by COX enzymes and suppressed by acid NSAIDs such as aspirin and diclofenac sodium, which inhibit COX activities, we examined whether kakkonto inhibits COX-1 and COX-2 activities. Kakkonto decreased COX-1 activity to approximately 70% at 1 mg/mL but did not affect COX-2 activity ([Figure 3](#fig3){ref-type="fig"}). 3.4. Effects of Kakkonto on Molecular Expression in the Arachidonic Acid Cascade {#sec3.4} -------------------------------------------------------------------------------- We examined whether kakkonto affects the expression of molecules in the arachidonic acid cascade. cPLA~2~ is the most upstream enzyme in the arachidonic acid cascade and releases arachidonic acid from plasma membranes. Kakkonto did not alter cPLA~2~ expression in the absence or presence of LPS ([Figure 4](#fig4){ref-type="fig"}). COX-2 was not detected in the absence of LPS. Treatment with kakkonto alone increased COX-2 expression. However, kakkonto did not alter LPS-induced COX-2 expression ([Figure 4](#fig4){ref-type="fig"}). Annexin1, also named lipocortin1, is an anti-inflammatory mediator produced by glucocorticoids that inhibit cPLA~2~ activity \[[@B19], [@B20]\]. However, neither LPS nor kakkonto showed an effect on annexin1 expression ([Figure 4](#fig4){ref-type="fig"}). 3.5. Effects of Kakkonto on ERK Phosphorylation {#sec3.5} ----------------------------------------------- cPLA~2~ is reported to be directly phosphorylated at Ser505 by ERK, resulting in cPLA~2~ activation \[[@B21], [@B22]\]. Therefore, we examined whether kakkonto suppresses LPS-induced ERK phosphorylation. ERK phosphorylation was enhanced at 0.5 h after LPS treatment and thereafter was attenuated. One mg/mL of kakkonto suppressed LPS-induced ERK phosphorylation at 0.5 h to 2 h ([Figure 5](#fig5){ref-type="fig"}). 4. Discussion {#sec4} ============= In the present study, we examined the effect of kakkonto on LPS-induced PGE~2~, IL-6, and IL-8 production by HGFs. Kakkonto concentration dependently decreased LPS-induced PGE~2~ production but did not affect PGE~2~ production without LPS treatment, similar to shosaikoto, hangeshashinto, and orento \[[@B5]--[@B7]\]. Moreover, kakkonto suppressed LPS-induced ERK phosphorylation. In contrast, kakkonto increased LPS-induced IL-6 and IL-8 production. It is widely known that PGE~2~ leads to inflammatory responses such as vasodilation, enhanced vascular permeability, and pain generation \[[@B1]\]. Acid non-steroidal anti-inflammatory drugs NSAIDs show anti-inflammatory effects by suppression of PGE~2~ production, even though they do not affect IL-6 and IL-8 production. Our findings showing that kakkonto decreases LPS-induced PGE~2~ production suggest that kakkonto also has anti-inflammatory effects in periodontal disease and that its effects are mainly mediated by suppression of PGE~2~ production even though kakkonto increased LPS-induced IL-6 and IL-8 production. Our results showed that kakkonto suppressed LPS-induced ERK phosphorylation in HGFs. Previously, we demonstrated that orento inhibits LPS-induced ERK phosphorylation and cPLA~2~ activation, leading to the suppression of PGE~2~ production in HGFs \[[@B7]\]. Therefore, we consider that kakkonto decreased LPS-induced PGE~2~ production through the suppression of ERK phosphorylation in HGFs. Although kakkonto increased COX-2 expression in the absence of LPS, kakkonto did not alter PGE~2~ production. We consider a likely reason to be the suppression of cPLA~2~ activation through the inhibition of ERK phosphorylation and/or the suppression of COX-1 activity. However, the components that induce COX-2 expression remain unknown. Our results showed that kakkonto increased LPS-induced IL-6 and IL-8 production by HGFs. Previously, we reported that the activation of the protein kinase A (PKA) pathway by adrenaline or aminophylline increases LPS-induced IL-6 and IL-8 production in HGFs \[[@B23]\] and that H-89, a PKA inhibitor, decreases LPS-induced IL-6 and IL-8 production \[[@B23], [@B24]\]. Therefore, kakkonto may activate the PKA pathway. In general, steroidal anti-inflammatory drugs (SAIDs) suppress the expression of cPLA~2~, COX-2, and inflammatory cytokines (such as IL-6 and IL-8) and induce the expression of annexin1. However, kakkonto did not affect cPLA~2~, annexin1, or LPS-induced COX-2 expression, and it increased IL-6 and IL-8 production. This therefore suggests that the mechanism by which kakkonto decreases PGE~2~ production is different from that of SAIDs. Many studies have demonstrated that NSAID administration prevents gingival inflammation \[[@B25]\] and several clinical studies have indicated that the concentration of PGE~2~ in gingival crevicular fluid (GCF) is increased in periodontal disease \[[@B26]\] and is decreased by oral administration or mouthwash with NSAIDs \[[@B27], [@B28]\]. Considering that both NSAIDs and kakkonto suppress PGE~2~ production, it is possible that administration of kakkonto also decreases the PGE~2~ concentration in GCF and results in the improvement of gingival inflammation. Therefore, kakkonto may be useful for the improvement of gingival inflammation in periodontal disease. Importantly, kakkonto did not affect the basal level of PGE~2~, although kakkonto decreased COX-1 activity to approximately 70%. Because PGE~2~ produced by COX-1 protects gastric mucosa, these results suggest that kakkonto may cause minimal gastrointestinal dysfunction. 5. Conclusion {#sec5} ============= We demonstrated that kakkonto suppresses LPS-induced ERK phosphorylation, resulting in the suppression cPLA~2~ activation and further PGE~2~ production by HGFs. These results suggest that kakkonto is clinically useful for the improvement of inflammatory responses in periodontal disease. The authors thank Professor Nobuo Yoshinari (Department of Periodontology) for HGFs preparation. The study was aided by funding from the Nagano Society for the Promotion of Science and a Scientific Research Special Grant from Matsumoto Dental University. Ethical Approval ================ This study was approved by the Ethical Committee of Matsumoto Dental University (no. 0063). Conflict of Interests ===================== The authors have no conflict of interests to disclose. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} ###### Ingredients of the kakkonto formula. Japanese name Latin name Amount (g) Amount\* (g/g of product) --------------- ---------------------- ------------ --------------------------- Kakkon *Puerariae Radix* 4.0 0.111 Taiso *Zizyphi Fructus* 3.0 0.083 Mao *Ephedrae Herba* 3.0 0.083 Kanzo *Glycyrrhizae Radix* 2.0 0.056 Keihi *Cinnamomi Cortex* 2.0 0.056 Shakuyaku *Paeoniae Radix* 2.0 0.056 Shokyo *Zingiberis Rhizoma* 2.0 0.056 Total   18.0 0.500 \*7.5 g of kakkonto product contains 3.75 g of a dried extract of the mixed crude drugs. [^1]: Academic Editors: A. Pittaluga and E. M. Urbanska

1. Introduction {#sec1-molecules-17-05339} =============== 1,4-Dihydropyridines as analogues of nicotinamide adenine dinucleotide (NADH) coenzymes exhibit a wide range of biological activities, such as calcium channel blocking, and today they are widely used in pharmacology \[[@B1-molecules-17-05339]\]. Acridines which possess the 1,4-dihydropyridine parent nucleus have interesting pharmaceutical properties such as a positive iontropic effects promoting the entry of calcium to the intracellular space \[[@B2-molecules-17-05339]\], and 1,8-(2*H*,5*H*)-acridinediones are known as laser dyes \[[@B3-molecules-17-05339]\]. 1,8-(2*H*,5*H*)-Acridinediones were synthesized by the adoption of the Hantzsch procedure, *i.e.*, by the thermal reaction of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) with an aldehyde and ammonia. Most of the methods reported previously usually require long reaction times, afford 1,4-dihydropyridines in relatively low yield, and suffer from utilizing harmful organic solvents in most cases \[[@B4-molecules-17-05339],[@B5-molecules-17-05339],[@B6-molecules-17-05339],[@B7-molecules-17-05339],[@B8-molecules-17-05339],[@B9-molecules-17-05339],[@B10-molecules-17-05339],[@B11-molecules-17-05339]\]. On the other hand, with the increasing environmental concerns, green chemistry has attracted intensive attention in recent years. Multicomponent one-pot reactions as a kind of economical and efficient procedure has been widely used for the synthesis of heteroatom-containing compounds \[[@B12-molecules-17-05339],[@B13-molecules-17-05339],[@B14-molecules-17-05339]\]. Moreover, organic reactions in water as the reaction medium, which represents a clean, economical, and environmental-safe protocol, has attracted considerable attention. We have already reported the one-pot synthesis of 10-unsubstituted 1,8-(2*H*,5*H*)-acridinediones by the thermal reaction of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) with an aldehyde and ammonium acetate in pure water without any additives \[[@B15-molecules-17-05339]\]. In this paper, ammonium acetate has been replaced by aniline and 4-methylaniline for the synthesis of *N*-substituted 1,8-(2*H*,5*H*)-acridinediones by the Hantzsch reaction in refluxing water. 2. Results and Discussion {#sec2-molecules-17-05339} ========================= 1,8-(2*H*,5*H*)-Acridinediones were synthesized by the one-pot three-component Hantzsch condensation for a designated time of paraformaldehyde, dimedone and NH~4~HCO~3~, or NH~4~OAc, or aniline, or 4-methyl-aniline in refluxing water without any additives. The reaction times and yields are listed in [Table 1](#molecules-17-05339-t001){ref-type="table"}. From [Table 1](#molecules-17-05339-t001){ref-type="table"}, one can see that the reaction yields of ammonium compounds (entry 1 and 2) are higher than for aromatic amines (entry 3 and 4). This is probably due to the lower solubility of the aromatic amines in water and lower reactivity than that of the ammonium compounds. It should be mentioned that the amount of the amine in the reactions was different. When an aromatic amine was used (entry 3 and 4), the molar ratio of **1**, **2**, and **3** was 1:2:1; when an ammonium compound was used (entry 1 and 2), the molar ratio of **1**, **2**, and **3** was 1:2:4. The increasing of the dosage of ammonium compound is because of the easy resolvability. In order to obtain a higher yield, we tried to add a phase-transfer catalyst (PTC) to the reaction mixtures. Cetyltrimethylammonium bromide (CTAB) was previously applied by us to the aqueous Michael reaction of dimedone with chalcones \[[@B16-molecules-17-05339]\], and now in this Hantzsch condensation of aldehyde, dimedone and aniline, addition of 10% mol of CTAB produced *N*-substituted 1,8-(2*H*,5*H*)-acridinediones in much better yield. The reaction yields, as well as the melting points for the Hantzsch reactions with the molar ratio of aromatic aldehydes **1**, dimedone (**2**), and aniline or 4-methylaniline **3**, and CTAB as 1:2:1:0.1 in refluxing water are listed in [Table 2](#molecules-17-05339-t002){ref-type="table"}. Reaction times are all 90 min. In our protocol, no organic solvents were used during the reaction process. Furthermore, since the product is solid and precipitates out from the reaction mixtures, the work-up procedure involves simple filtration. The desired products of high purity were obtained by column chromatography or recrystallization. From [Table 2](#molecules-17-05339-t002){ref-type="table"}, one can see that all these reactions gave good to excellent yields, reflecting that dimedone has high solubility in water and high reactivity. The yields of *N*-substituted acridinediones for the aromatic aldehydes with an electron-withdrawing group are higher than those for aromatic aldehydes with electron-donating groups. We also tried 3,4-dimethoxybenzaldehyde, but the reaction yield is lower than 50%. We have attempted to extend the above methods to different 1,3-dicarbonyl compounds for the synthesis of unsymmetrically substituted 1,4-dihydropyridines such as polyhydroquinoline derivatives. Ethyl acetoacetate and 1,3-cyclohexanedione smoothly undergo a four-component reaction with aromatic aldehyde and ammonium acetate to produce polyhydroquinoline derivatives in refluxing water catalyzed by CTAB. Because of the lower activity for the synthesis of unsymmetrically substituted 1,4-dihydropyridines than the symmetrically substituted 1,4-dihydropyridines, ammonium acetate was used to replace the aromatic amines. The reaction yields, as well as the melting points for the Hantzsch reaction with the molar ratio of aromatic aldehydes **1**, dimedone (**2**), ethyl acetoacetate (**6**), ammonium acetate (**3**), and CTAB as 1:1:1:4:0.1 in refluxing water are listed in [Table 3](#molecules-17-05339-t003){ref-type="table"}. Reaction times are all 90 min. Among the acridinediones and polyhydroquinoline products **5c** and **5f** were unknown compounds and were characterized by their melting point, IR, ^1^H-NMR, ^13^C-NMR spectra and elemental analysis. Structures of known compounds were confirmed by comparison of their melting points and ^1^H-NMR spectra with the reported data. It should be mentioned that the melting point of **5d** was obviously higher than the reported in the literature \[[@B17-molecules-17-05339]\]. However, our measured IR, ^1^H-NMR, ^13^C-NMR spectral data and elemental analysis are consistent with the series of acridinediones products, exhibiting the same identities. The different mp might be ascribed to the presence of an impurity or solvent in product **5d** from the reported literature, because even a trace amount could have a great effect on the m.p. of a compound with high m.p. 3. Experimental {#sec3-molecules-17-05339} =============== 3.1. General {#sec3dot1-molecules-17-05339} ------------ ^1^H-NMR spectra were recorded on a Bruker Avance-400 (400 MHz) spectrometer (Bruker, Switzerland), and chemical shifts (δ) are reported in parts per million relative to tetramethylsilane and coupling constants (*J*) in Hz. Splitting patterns are designated as s, singlet; d, doublet; br, broad. ^13^C-NMR spectra were recorded on the same spectrometer (at 100 MHz) with complete proton decoupling, and chemical shifts are reported in parts per million relative to the solvent resonance used as the internal standard (CDCl~3~, δ 77.16 ppm; DMSO-d6, δ 39.52 ppm). IR spectra were taken on a Bruker Vector-22 spectrometer (Bruker, Switzerland) in KBr pellets and are reported in cm^−1^. Melting points were determined on an XT-4 apparatus (Beijing Tech Instrument Co., Beijing, China). Analytical TLC and column chromatography were performed on silica gel GF254 and silica gel H60, respectively. 3.2. Typical Procedure for the Synthesis of Acridinediones: 3,3,6,6-Tetramethyl-9,10-diphenyl-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridinedione *(**5a**)* {#sec3dot2-molecules-17-05339} --------------------------------------------------------------------------------------------------------------------------------------------------------- A mixture of **1a** (106.1 mg, 1 mmol), **2** (280.4 mg, 2 mmol), **3c** (93.1 mg, 1 mmol) and CTAB (36.4 mg, 0.1 mmol) in water (4 mL) was vigorously stirred under reflux. The reaction was completed after 90 min, as monitored by TLC. Due to the high yields of **5a**, the work-up procedure involved simple filtration and washing twice with water (10 mL). The obtained solid products were nearly pure. The desired product of high purity were further achieved by column chromatography with petroleum ether/ethyl acetate or recrystallization from 75% aqueous ethanol. *3*,*3*,*6*,*6-Tetramethyl-9-(4-cyanophenyl)-10-phenyl-3*,*4*,*6*,*7*,*9*,*10-hexahydro-1*,*8-(2H*,*5H)-acridinedione* (**5c**). IR (KBr): υ 2956 (s), 2872 (m), 2223 (m), 1643 (s), 1576 (s), 1492 (m), 1362 (s), 1297 (w), 1262 (m), 1174 (w), 1144 (m), 1122 (w), 1000 (m), 851 (s), 704 (s), 569 (s) cm^−1^; ^1^H-NMR (CDCl~3~): δ 0.79 (s, 6H, CH~3~), 0.95 (s, 6H, CH~3~), 1.85 (d, *J* = 17.5 Hz, 2H, CH~2~), 2.11 (d, *J* = 17.5 Hz, 2H, CH~2~), 2.12 (d, *J* = 16.2 Hz, 2H, CH~2~), 2.21 (d, *J* = 16.2 Hz, 2H, CH~2~), 5.31 (s, 1H, CH), 7.25 (d, *J* = 8.0 Hz, 2H, ArH), 7.54 (d, *J* = 8.6 Hz, 2H, ArH), 7.58 (d, *J* = 8.0 Hz, 2H, ArH), 7.59 (m, 3H, ArH); ^13^C-NMR (CDCl~3~): δ 195.8, 151.6, 150.6, 138.6, 131.9, 129.7, 128.8, 119.3, 113.4, 109.5, 50.1, 41.8, 33.7, 32.4, 29.6, 26.7; Anal. Calcd. for C~30~H~30~N~2~O~2~: C, 79.97; H, 6.71; N, 6.22; Found: C, 79.97; H, 6.80; N, 6.17. *3*,*3*,*6*,*6-Tetramethyl-9-(4-nitrophenyl)-10-phenyl-3*,*4*,*6*,*7*,*9*,*10-hexahydro-1*,*8-(2H*,*5H)-acridinedione* (**5d**). IR (KBr): υ 2956 (m), 1635 (s), 1594 (w), 1514 (m), 1349 (s), 1224 (m), 1176 (w), 1144 (w), 1113 (w), 1003 (m), 864 (w), 830 (w), 703 (m), 572 (w), 513 (w) cm^−1^; ^1^H-NMR (CDCl~3~): δ 0.72 (s, 6H, CH~3~), 0.88 (s, 6H, CH~3~), 1.77 (d, *J* = 17.5 Hz, 2H, CH~2~), 2.03 (d, *J* = 17.5 Hz, 2H, CH~2~), 2.04 (d, *J* = 16.3 Hz, 2H, CH~2~), 2.14 (d, *J* = 16.3 Hz, 2H, CH~2~), 5.28 (s, 1H, CH), 7.18 (d, *J* = 8.2 Hz, 2H, ArH), 7.53 (d, *J* = 8.2 Hz, 2H, ArH), 7.54 (s, 1H, ArH), 7.55 (d, *J* = 8.8 Hz, 2H, ArH), 8.05 (d, *J* = 8.8 Hz, 2H, ArH); ^13^C-NMR (CDCl~3~): δ 195.6, 153.6, 150.4, 146.2, 138.7, 129.7, 128.8, 123.5, 113.6, 50.1, 41.9, 33.6, 32.4, 29.6, 26.7; Anal. Calcd. for C~29~H~30~N~2~O~4~: C, 74.02; H, 6.43; N, 5.95; Found: C, 73.63; H, 6.43; N, 5.91. *3*,*3*,*6*,*6-Tetramethyl-9-(3*,*4-dichlorophenyl)-10-phenyl-3*,*4*,*6*,*7*,*9*,*10-hexahydro-1*,*8-(2H,5H)-acridine-dione* (**5f**). IR (KBr): υ 2962 (m), 2948 (m), 1650 (s), 1638 (s), 1573 (m), 1471 (m), 1360 (s), 1224 (s), 1143 (m), 1027 (w), 1002 (w), 878 (m), 704 (s), 574 (m) cm^−1^; ^1^H-NMR (CDCl~3~): δ 0.80 (s, 6H, CH~3~), 0.93 (s, 6H, CH~3~), 1.83 (d, *J* = 17.6 Hz, 2H, CH~2~), 2.08 (d, *J* = 17.6 Hz, 2H, CH~2~), 2.17 (m, 4H, CH~2~), 5.21 (s, 1H, CH), 7.22 (m, 2H, ArH), 7.28 (m, 2H, ArH), 7.50 (s 1H, ArH), 7.56 (m, 3H, ArH); ^13^C-NMR (CDCl~3~): δ 195.7, 150.3, 146.6, 138.7, 131.9, 130.0, 129.9, 129.6, 127.6, 113.8, 50.1, 41.8, 32.4, 29.7, 26.8; Anal. Calcd. for C~29~H~29~Cl~2~NO~2~: C, 70.44; H, 5.91; N, 2.83; Found: C, 70.40; H, 6.01; N, 2.78. 4. Conclusions {#sec4-molecules-17-05339} ============== In summary, acridinediones and polyhydroquinoline derivatives were synthesized by the one-pot Hantzsch condensation of an aromatic aldehyde, 5,5-dimethyl-1,3-cyclohexanedione, ethyl acetoacetate and amine. It represents a straightforward protocol for the eco-friendly and efficient synthesis of a series of 1,4-dihydropyridines with potential biological activities. We are grateful for financial support from the Natural Science Foundation of Anhui Province Education Department (KJ2010B049) and Anhui Provincial Natural Science Foundation (1208085QB24). *Sample Availability*: Samples of the compounds **5a**--**n**, **7a**--**d** are available from the authors. Supplementary materials can be accessed at: <http://www.mdpi.com/1420-3049/17/5/5339/s1>. ###### Click here for additional data file. molecules-17-05339-t001_Table 1 ###### Reaction time and yield for the reaction of different amines/ammonium salts.  Entry Amine Reaction time/min Product R Yield / % ^a^ ------- ------------------------ ------------------- --------- ------------------ --------------- 1 NH~4~OAc 40 min **4a** H 86 2 NH~4~HCO~3~ 40 min **4b** H 83 3 C~6~H~5~-NH~2~ 90 min **4c** C~6~H~5~ 71 4 4-CH~3~-C~6~H~5~-NH~2~ 90 min **4d** 4-CH~3~-C~6~H~4~ 69 ^a^ Isolated yield of the pure product recrystallized from 75% aqueous ethanol. molecules-17-05339-t002_Table 2 ###### Yields and melting points for the one-pot synthesis of *N*-substituted acridinediones.  Entry R R' Product Yield / % ^a^ m.p. (lit.) / °C ------- --------------------- ------------------ --------- --------------- --------------------------------------------------- 1 C~6~H~5~ C~6~H~5~ **5a** 80 220--222 (200--205) \[[@B17-molecules-17-05339]\] 2 4-Cl-C~6~H~4~ C~6~H~5~ **5b** 86 243--245 (233--235) \[[@B18-molecules-17-05339]\] 3 4-CN-C~6~H~4~ C~6~H~5~ **5c** 88 265--267 4 4-NO~2~-C~6~H~4~ C~6~H~5~ **5d** 85 281--282 (216--218) \[[@B17-molecules-17-05339]\] 5 3- NO~2~-C~6~H~4~ C~6~H~5~ **5e** 84 272--274 (276--278) \[[@B19-molecules-17-05339]\] 6 3,4- Cl~2~-C~6~H~3~ C~6~H~5~ **5f** 90 274--275 7 4-CH~3~O-C~6~H~4~ C~6~H~5~ **5g** 70 291--293 (290--291) \[[@B20-molecules-17-05339]\] 8 C~6~H~5~ 4-CH~3~-C~6~H~4~ **5h** 81 261--263 (264--266) \[[@B21-molecules-17-05339]\] 9 4-Cl-C~6~H~4~ 4-CH~3~-C~6~H~4~ **5i** 85 270--272 (271--272) \[[@B21-molecules-17-05339]\] 10 4-CN-C~6~H~4~ 4-CH~3~-C~6~H~4~ **5j** 88 268--270 (273--275) \[[@B21-molecules-17-05339]\] 11 4-NO~2~-C~6~H~4~ 4-CH~3~-C~6~H~4~ **5k** 83 \>300 (\>300) \[[@B22-molecules-17-05339]\] 12 3-NO~2~-C~6~H~4~ 4-CH~3~-C~6~H~4~ **5l** 85 281--283 (283--284) \[[@B23-molecules-17-05339]\] 13 3,4-Cl~2~-C~6~H~3~ 4-CH~3~-C~6~H~4~ **5m** 88 253--255 (250--252) \[[@B23-molecules-17-05339]\] 14 4-CH~3~O-C~6~H~4~ 4-CH~3~-C~6~H~4~ **5n** 72 280--282 (281--283) \[[@B23-molecules-17-05339]\] ^a^ Isolated yield of the pure product recrystallized from 75% aqueous ethanol. molecules-17-05339-t003_Table 3 ###### Yields and melting points for the one-pot synthesis of polyhydroquinoline derivatives.  Entry R Product Yield / % ^a^ m.p. (lit.) / °C ------- ------------------- --------- --------------- --------------------------------------------------- 1 C~6~H~5~ **7a** 85 224--226 (228--229) \[[@B24-molecules-17-05339]\] 2 4-CH~3~O-C~6~H~4~ **7b** 81 257--259 (260--262) \[[@B24-molecules-17-05339]\] 3 4-Cl-C~6~H~4~ **7c** 90 244--266 (245--246) \[[@B24-molecules-17-05339]\] 4 4-NO~2~-C~6~H~4~ **7d** 88 242--244 (241--242) \[[@B24-molecules-17-05339]\] ^a^ Isolated yield of the pure product recrystallized from 75% aqueous ethanol.

In the second edition of the special issue titled "Immunometabolism: Molecular Mechanisms, Diseases, and Therapies," a total of 37 manuscripts were received, and 16 of these were accepted. This issue demonstrated that nonresolving, chronic low-intensity inflammation not only is involved in the development and maintenance of several metabolic diseases such as visceral obesity, type 2 diabetes, dyslipidemias, atherosclerosis, hypertension, and cancer but also acts as an important linking factor between those conditions. Metabolic disease-associated inflammation is characterized by the recruitment of cells from both the innate and adaptive immune system to metabolic tissues followed by their polarization to a proinflammatory profile, resulting in the exacerbated local production of inflammatory mediators. These processes are mainly activated by the canonical Toll-like receptor- (TLR-) NF*κ*B signaling pathway, which can be triggered by several molecules such as the lipopolysaccharide from gut microbiota and saturated fatty acids, among others. This special issue successfully attracted several interesting original and review articles addressing different aspects of the intricate relationships between metabolism and inflammation in health and disease. E. Nishida et al., for example, exploring the association between cardiovascular diseases and periodontitis, a chronic inflammatory disease that affects the periodontium, demonstrated that the acute-phase protein serum amyloid A, which is elevated in the liver and blood of apolipoprotein E-deficient, atherosclerosis-prone mice, promotes the expression of adhesion molecules in human aortic endothelial cells via TLR2, being therefore a candidate linking factor between periodontal disease and atherosclerosis. Subsequently, in an interesting, mechanistic study, Z.-Z. Guo et al. established a role for the c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family which is activated by inflammatory signals and other stress stimuli, as an important mediator involved in the formation of abdominal aortic aneurysm induced by angiotensin II plus nicotine, a major chemical component of cigarettes. These findings indicate that JNK inhibition may hold promise as a pharmacological target to attenuate smoking-induced abdominal aortic aneurysm formation. In an elegant study exploring the mechanisms underlying the immunomodulatory actions of bilirubin, K. F. Corral-Jara et al. found strong evidence indicating that, during hepatitis A virus infection, conjugated bilirubin differentially regulates CD4+ T lymphocytes and Tregs function by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing hepatitis A virus cellular receptor (HAVCR1/TIM-1). These findings may help in the elucidation of the mechanisms involved in hepatitis virus infection. This special issue also gathered several studies that investigated the underlying mechanisms by which some nutrients and modulation of tissue oxygen levels may attenuate the chronic low-grade inflammation associated with metabolic diseases. Indeed, S. I. Pærregaard et al. investigated the involvement of membrane-free fatty acid receptor-4 (FFAR4), also known as GPR120, as mediator of the beneficial actions of *ω*3 fatty acids on metabolic health. By feeding *Ffar4* knockouts and heterozygous mice with either a control or an *ω*3-rich diet for 36 weeks, S. I. Pærregaard et al. show that FFAR4 signaling is not required for the anti-inflammatory and insulin-sensitizing effects mediated by *ω*3 fatty acids. G. Guan et al., on the other hand, presented interesting findings indicating that intake of a diet containing chitosan, polysaccharides found in insects, fungi, squid, oysters, krill, clams, and shellfish changes the composition of mice intestinal microflora by suppressing NF-*κ*B, TNF-*α*, and IL-6 and inducing a better control of inflammation and resolution of infection with *C. rodentium*. Moreover, V. Oliveira et al. showed that oligofructose supplementation in pregnancy and lactation induces the proinflammatory markers in dams and 90-day-old offspring and reduction in adiponectin pathway. In addition, F. Elbers et al. reported an impairment in the immunomodulatory and antimicrobial actions of the hepatic enzyme tryptophan 2,3-dioxygenase, which converts tryptophan, the essential amino acid for hosts and pathogens, to N-formylkynurenine, the precursor of the immune-relevant kynurenines, upon hypoxic conditions mimicking those occurring in vivo during infection and cancer, for example. These findings indicate that hypoxia might be detrimental for the appropriate host immune response towards relevant pathogens. In contrast to the deleterious effects of hypoxia, in an interesting study, S. Novak et al. reported that hyperbaric oxygenation treatment, which increases tissue oxygen content, oxidative metabolism, and production of reactive oxygen species (ROS), significantly reduces the severity of dextran sodium sulphate-induced colitis improving the inflammatory microenvironment in the gut mucosa, such an effect that seems to be mediated in part by the transcriptional factor hypoxic inducible factor 1 *α* (HIF-1*α*). Finally, regarding alternative treatments to inflammatory disease, W. Ren et al. reported that supplementation of drinking water of rodents with interferon tau, a type I interferon produced by trophectoderm cells of conceptuses of ruminant species, increased microbial diversity in the jejunum and ileum and decreased the expression of IL-17 in the intestines of normal and pathogen-infected mice, being therefore a candidate therapy strategy to treat the inflammatory intestinal diseases. Two studies in this special issue were performed in humans aiming to further characterize obesity-associated inflammation. F.-I. Corona-Meraz et al., by evaluating the inflammatory and metabolic phenotype of subjects with obesity and insulin resistance, found that chemerin, an adipokine related to adiposity levels and fat distribution, is associated with obesity, dyslipidemia, and insulin resistance, whereas its receptor chemerin chemokine-like receptor 1 (CMKLR1) is associated only with obesity. C. Wang et al., on the other hand, by evaluating the gene expression profile of the visceral adipose tissue from lean and obese subjects from the Uyghur population, found a correlation between reduced expression of A2b adenosine receptor and the transcriptional factors Kruppel-like factors 4 and 5 with obesity-associated dyslipidemia and inflammation. This special issue also brings some interesting review articles addressing several aspects of metabolic disease-associated inflammation. D. Ortuño-Sahagún et al., for example, reviewed recent evidence supporting the link between obesity and the pathogenesis of multiple sclerosis, a chronic autoimmune and inflammatory disease. P.-F. Bryan et al., on the other hand, reviewed the mechanisms involved in the regulation of cytokine profile by sphingolipids, the role of gut microbiota in providing signaling molecules that favor the communication between resident bacteria and intestinal cells, and the potential of sphingolipids and gut microbiota as targets or therapeutic agents for inflammatory bowel disease. H. J. Coelho Junior et al. reviewed the possible molecular mechanisms associated with muscle atrophy in stroke patients, as well as the modulatory effect of inflammation in this condition. G. van Niekerk et al. reviewed evidence suggesting that sickness-associated anorexia may be a mechanism by which autophagic flux is upregulated systemically and claim that some patients might benefit from permissive underfeeding. Finally, U. Nydegger et al. reviewed some approaches to sort out from big data the relevant results for patient care in precision medicine. Therefore, in our opinion, this special issue brings new insights into the intricate mechanisms driving the inflammatory processes associated with metabolic diseases. We hope that these information will help to pave the way for the development of efficient strategies to prevent and treat these diseases. *Jose C. Rosa Neto* *Jose C. Rosa Neto* *Fabio S. Lira* *Fabio S. Lira* *Soumen Roy* *Soumen Roy* *William Festuccia* *William Festuccia*

Introduction ============ With introduction of intracytoplasmic sperm injection (ICSI), new era was introduced for treatment of male factor infertility ([@B1]). In this procedure, an immobilized sperm with normal morphology is injected into a mature oocyte. Since inception of ICSI, more attentions have been given to the recognition and selection of high quality spermatozoa to improve the outcome of assisted reproduction technology (ART) treatment. It is well known that sperm quality affects the quality of generated embryo ([@B2]). Sperm morphology has been shown as the best prognostic factor, among sperm parameters, to predict spontaneous pregnancy, intra-uterine insemination (IUI) and conventional in vitro fertilization (IVF) success rates ([@B3]-[@B5]). On the other hand, injection of spermatozoa with abnormal morphology is associated with lower fertilization and implantation rates ([@B6]). In addition, the sperm morphology is a powerful tool that can be used to determine the right ART method for treating infertility ([@B7]), is the best indicator of male fertility ([@B8]) and considered a sensitive prognostic tool in IUI and IVF ([@B9]). The selection of sperm for ICSI is according to rough morphological characteristics. Meanwhile, the assessment of sperm morphology before ICSI is difficult because of sperm motility and not precise due to observation under low magnification. There may be some sperm morphological abnormalities that are not determined, when examined at routine magnification for ICSI (×200 or ×400) or even after fixation and staining (×1000). Recently several techniques have been introduced for evaluation of sperm morphology, including light microscopy, atomic force microscopy and electron microscopy. While these are not considered as real time techniques, Bartoov et al. (2001) developed a new tool for evaluation of non-fixed sperm morphology. In this technique, using a high power inverted light microscope with magnification of \>×6000 sperm morphology is evaluated in real time more precisely and at the sub-cellular level named motile sperm organelle morphology examination (MSOME) ([@B10]). It has been shown that there is a relationship between normal morphology of spermatozoa determined by MSOME and fertilization rate, embryo quality, as well as implantation, pregnancy rates and chance of having normal child ([@B2], [@B11]-[@B22]). To the best of our knowledge, there have not been any studies regarding the comparison of MSOME criteria between IVF and ICSI cycles. We aimed to compare the sperm morphology at high magnification between cycles with successful and failed pregnancy in IVF as well as ICSI cases. Materials and methods ===================== **Patients** Sixty-six infertile couples were included from October 2013 to August 2014 in this cross sectional prospective study that were treated by IVF (n=31) or ICSI (n=35) at Research and Clinical Center for Infertility, Yazd, Iran. Third party assisted reproduction cycles, patients with less than three oocytes, spermatozoa with only round head or double tail, testicular or epididymal sperm, and maternal age of \>40 years were excluded. The study was approved by the ethics committee of Yazd Reproductive Sciences institute. Signed informed consents were obtained from the patients. **Semen collection and sperm preparation** Semen samples were collected in sterile wide-mouth containers by masturbation following 2-5 days of abstinence. Semen analysis and preparation was performed according to standard world health organization (WHO) guidelines ([@B23]). Immediately after liquefaction, the semen samples were prepared by two layer (80/40) density gradient centrifugation method. The final pellet was resuspended in 0.5 ml pre-warmed equilibrated Ham's F10 supplemented with 5 mg/ml human serum albumin and incubated at 37^°^C with 5% CO2 until use. Makler counting chamber was used for evaluation of sperm count and motility and sperm morphology was assessed by Diff-Quick staining method. **Motile sperm organelle morphology examination (MSOME)** Several 5µ microdroplets of polivinylpyrrolidone (8% PVP, Vitrolife, Goteborg, Sweden) were placed on a sterile glass bottom dish (WillCo-Dish, Amsterdam, Netherlands) and overlaid withmineraloil (Ovoil 100, Vitrolife, Goteborg, Sweden). Then, 1µ of prepared sperm suspension was added to a forementioned microdroplets. The inverted microscope (TE300; Nikon, Tokyo, Japan) equipped with high power differential interference contrast optics was used for MSOME (6600×). The image's capture and video recording for further analysis was performed with software (OCTAX PolarAIDE; Octax). MSOME classification was defined according to Cassuto-Barak classification (2). Accordingly, the sperm morphology was evaluated based on the head shape, presence of vacuole in the nucleus and the head base. The score of each spermatozoon was determined as: (2×Head) + (3×Vacuole) + (Base). The scoring for each sperm cell ranged between 0 up to 6. Class I was high quality spermatozoa scored 4 to 6, Class II was medium quality spermatozoa scored 1 to 3, Class III was low quality sperm scored 0. We also defined MSOME parameters as follows: vacuoles (none, small: \<4%, medium: 4≥ \<15%, large: ≥15% of sperm head area, mix: having more than two vacuoles) ([Figures I](#F1){ref-type="fig"} and [II](#F2){ref-type="fig"}), head size (anterior-posterior diameter): normal: 5±0.2µm, small: ≤4.7µm, large: ≥5.3µm, head shape: normal: oval, abnormal: non-oval, cytoplasmic droplet: absence or presence ([Figure III](#F3){ref-type="fig"}). At least 200 motile spermatozoa per patient were assessed, and each parameter was reported as percentage. The same researcher in a blind manner performed all sperm analysis, so inter observer variability was not assessed. MSOME parameters were compared between successful and failed pregnancy. **ICSI and IVF** The type of ART was selected for each case based of etiology of infertility, sperm parameters, female age, and patient's history. IVF and ICSI techniques were performed as described elsewhere ([@B24]-[@B25]). Standard long protocol was used for controlled ovarian hyperstimulation which previously described ([@B26]). The injected oocytes after microinjection and conventionally inseminated COCs were evaluated 16-18 hrs later for presence of two pronuclei and two polar bodies to determine normal fertilization. Evaluations of embryo quality and embryo transfer were performed on day two. Embryo grading was previously described ([@B27]). Grade A and B embryos were considered as high quality embryos. Clinical pregnancy was confirmed after detection of fetal heart beat seven weeks after embryo transfer. **Statistical analysis** The data are presented as mean±SD and median (min-max) for quantitative and percentage for qualitative parameters. Shapiro-Wilk was used for evaluating normal distribution for quantitative data. Mann-Whitney U-test was applied in order to compare the MSOME parameters between IVF and ICSI groups. Chi-square test was used to compare proportions between two groups. Pearson test was performed for evaluation of correlation between different morphological parameters. The significant level was defined at p \< 0.05. Results ======= There were no significant differences for patient's age, sperm concentration, progressive motility, normal morphology, metaphase II oocytes, fertilized oocyte, formed embryos between successful and failed pregnancy cycles in IVF and ICSI groups ([Table I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}). Regarding MSOME parameters in IVF group, the data showed no significant difference for Cassuto-Barak classification, cytoplasmic droplet, and medium vacuole between successful and failed pregnancy cycles ([Table III](#T3){ref-type="table"}). However, the rate of LNV was significantly lower in successful pregnancies compared to the negative cycles in IVF group (7.38±4.4 vs 13.81±9.7, respectively, p=0.045), while this difference was not significant in ICSI group. Sperm shape and size were also the same between two groups. In ICSI group, only the rate of sperm shape normalcy was significantly higher in failed pregnancy compared to successful pregnancy ([Table IV](#T4){ref-type="table"}). In IVF group, data showed a negative correlation between LNV and sperm shape normalcy (r=-0.48, p=0.006). There was also a positive correlation between the rate of combination of non-vacuole and small vacuole spermatozoa and sperm shape normalcy (r= 0.55, p=0.001). A significant positive correlation was also found between the rate of sperm normal head and normal shape(r=0.36, p=0.04) with progressive motility (r=0.38, p=0.03). In ICSI group, a negative correlation was established between cytoplasmic droplet and sperm shape normalcy (r=-0.34, p=0.04). A positive correlation between sperm shape normalcy and non-vacuolated spermatozoa (r=0.45, p=0.006), positive correlation between LNV and non-progressive motility (r=0.38, p=0.02), negative correlation between large head and normal shape (r=-0.43, p=0.009) were also found. ###### Demographic characteristics and outcomes in IVF cycles with successful and failed pregnancies ----------------------------------------------------------------------------------------------------- **Variables** **Groups** **p-value** ---------------------------------------------------------------- -------------- --------------- ----- Male age (year) [a](#TFN1){ref-type="table-fn"} 34.2±4.9 32.8±5.9 0.6 Female age (year) [a](#TFN1){ref-type="table-fn"} 29.1±3.6 30.4±5 0.1 Sperm concentration (106 ml-1) [b](#TFN2){ref-type="table-fn"} 98.4±22.7\ 97.7±42.7\ 0.2 105 (60-120) 89.5 (50-210) Progressive motility (%)[b](#TFN2){ref-type="table-fn"} 54.1±10.4\ 50.7±10\ 0.6 52 (38-68) 52.5 (27-70) Normal morphology (%)[b](#TFN2){ref-type="table-fn"} 16.2±5.1\ 15.7±6.4\ 0.8 20 (10-20) 15 (8-27) Metaphase II oocyte[b](#TFN2){ref-type="table-fn"} 13.6±5\ 9.7±4.8\ 0.2 12 (9-25) 11.5 (1-20) Fertilized oocytes[b](#TFN2){ref-type="table-fn"} 8.6±4.7\ 8±4.4\ 0.9 7.5 (5-19) 7.5 (1-20) Formed embryos[b](#TFN2){ref-type="table-fn"} 7.9±4.1\ 7.6±4.5\ 0.7 6.5 (5-17) 7 (1-20) Transferred embryos[b](#TFN2){ref-type="table-fn"} 2.4±0.5\ 2.1±0.5\ 0.4 2 (2-3) 2 (1-3) Good quality embryos (%)[\*](#TFN3){ref-type="table-fn"} 62.5 46.7 0.6 ----------------------------------------------------------------------------------------------------- : Data are shown as mean±SD (Independent sample t-test). : Data are shown as mean±SD, median (min-max) (Man-Whitney U test). Chi-square test ###### Demographic characteristics and outcome in ICSI cycles with successful and failed pregnancies --------------------------------------------------------------------------------------------------- **Variables** **Groups** **p-value** ---------------------------------------------------------------- ------------- ------------- ------ Male age (year) [a](#TFN4){ref-type="table-fn"} 34.7±4.6 36.1±6 0.4 Female age (year) [a](#TFN4){ref-type="table-fn"} 31±2.8 30.3±4.9 0.7 Sperm concentration (106 ml-1) [b](#TFN5){ref-type="table-fn"} 87.1±59.7\ 67.9±32.9\ 0.4 80 (20-200) 60 (10-150) Progressive motility (%) [b](#TFN5){ref-type="table-fn"} 23.3±19.7\ 38.8±19.5\ 0.06 21 (0-53) 42 (1-71) Normal morphology (%) [b](#TFN5){ref-type="table-fn"} 8.1±9.3\ 12.6±8.4\ 0.1 5 (1-28) 12 (0-30) Metaphase II oocyte[b](#TFN5){ref-type="table-fn"} 10.6±5.1\ 8.52±4.8\ 0.3 9 (4-18) 7 (3-19) Fertilized oocytes[b](#TFN5){ref-type="table-fn"} 7.4±3.6\ 5.9±3.6\ 0.2 6 (3-12) 5 (2-16) Formed embryos[b](#TFN5){ref-type="table-fn"} 7.4±3.6\ 5±2.7\ 0.1 6 (3-12) 6 (3-12) Transferred embryos[b](#TFN5){ref-type="table-fn"} 2.4±0.5\ 2.3±0.4\ 0.8 2 (2-3) 2 (2-3) Good quality embryos (%)[\*](#TFN6){ref-type="table-fn"} 85.7 43.5 0.08 --------------------------------------------------------------------------------------------------- : Data are shown as mean±SD (Independent sample t-test). : Data are shown as mean±SD, median (min-max) (Man-Whitney U test). Chi-square test ###### MSOME criteria and Cassuto-Barak classification in IVF group ------------------------------------------------------------------------------------ **Parameters** **Groups** **p-value** ----------------------------------------------- -------------- -------------- ------ Class I [\*](#TFN7){ref-type="table-fn"} 7.4±3.5\ 8.9±7.3\ 1 7.5 (2-12) 7.5 (0-33) Class II[\*\*](#TFN8){ref-type="table-fn"} 52.4±14.9\ 45.9±8.3\ 0.1 52 (23-76) 47 (25-56) Class III[\*\*\*](#TFN9){ref-type="table-fn"} 40.4±16.7\ 45.2±11.7\ 0.1 38 (17-75) 45.5 (15-69) Class I + Class II 59.7±16.8\ 54.7±11.7\ 0.1 62 (25-83) 54.5 (31-85) Cytoplasmic droplet 20.5±6.6\ 23.9±10.4\ 0.4 20 (10-28) 21.5 (11-47) Large vacuole 7.4±4.4\ 13.8±9.7\ 0.04 6 (1-15) 12 (3-42) Medium vacuole 22.5±9.8\ 26.7±8.8\ 0.3 23.5 (8-39) 25.5 (15-48) Small vacuole 28.6±5.6\ 20±8.6\ 0.01 28.5 (20-36) 17.5 (4-40) Non-vacuole 18.6±13.4\ 16.7±10.4\ 0.9 14.5 (6-43) 12.5 (7-45) Mixed vacuole 22.9±8.2\ 23.7±9.6\ 0.7 20.5 (13-38) 24.5 (9-37) Normal head 55.9±13.4\ 54.2±8.6\ 0.6 55 (30-78) 56 (39-67) Large head 3.5±4\ 3.8±3\ 0.4 3 (0-13) 4 (0-10) Small head 39.4±17.6\ 42±8.6\ 0.7 43 (9-69) 42 (25-57) Normal shape 28.7±9.8\ 31.1±13.9\ 0.6 26.5 (19-46) 32 (11-56) ------------------------------------------------------------------------------------ Data are shown as mean±SD, median (min-max) (Man-Whitney U test). Spermatozoa with high quality Spermatozoa with medium quality Spermatozoa with low quality ###### MSOME criteria and Cassuto-Barak classification in ICSI group ----------------------------------------------------------------------------------- **Parameters** **Groups** **p-value** ------------------------------------------------ ------------- ------------- ------ Class I[\*](#TFN10){ref-type="table-fn"} 2.4±2.8\ 4.5±3.2\ 0.08 2 (0-8) 4 (0-14) Class II[\*\*](#TFN11){ref-type="table-fn"} 28.9±21.5\ 30.5±12.4\ 0.9 38 (3-55) 32 (8-65) Class III[\*\*\*](#TFN12){ref-type="table-fn"} 68.7±23.8\ 65±14.7\ 0.9 60 (42-97) 62 (21-91) Class I +Class II 31.3±23.8\ 35±14.7\ 0.9 40 (3-58) 38 (9-79) Cytoplasmic droplet 32.9±19.2\ 25±10.5\ 0.3 25 (10-60) 22 (12-53) Large vacuole 11.7±3.3\ 9.7±7\ 0.1 12 (6-16) 8 (2-35) Medium vacuole 27.3±9.5\ 21.9±7.2\ 0.1 27 (15-42) 21 (9-37) Small vacuole 19.9±9.4\ 23.3±6\ 0.3 19 (6-30) 24 (11-33) Non-vacuole 7.7±8.1\ 11.8±5.5\ 0.07 5 (1-24) 11 (0-24) Mixed vacuole 33.4±8.7\ 32.8±8.9\ 0.9 31 (25-50) 32 (14-50) Normal head 26±14.9\ 34.6±18.7\ 0.4 28 (0-46) 31 (8-82) Large head 6.4±9.9\ 8.7±5.3\ 0.1 2 (0-27) 8 (0-18) Small head 67.6±15.8\ 56.1±18.3\ 0.2 62 (53-100) 60 (16-90) Normal shape 7.4±6.6\ 15.3±7.5\ 0.01 7 (0-20) 15 (1-30) ----------------------------------------------------------------------------------- Data are shown as mean±SD, median (min-max) (Man-Whitney U test). Spermatozoa with high quality Spermatozoa with medium quality Spermatozoa with low quality {#F1} {#F2} {#F3} Discussion ========== Application of MSOME has not become a routine practice in ART clinics yet. In general, MSOME can evaluate the acrosome, post-acrosomal lamina, neck, tail, mitochondria and nucleus, in which the sperm nucleus is the most important organelle ([@B28]). Chromatin content can also be assessed by MSOME, as it was shown that the homogenous chromatin contains no more than one vacuole that occupies less than four percent of nuclear area ([@B11]). In addition to determination of overall view of sperm structure, MSOME can detect sperm nuclear vacuoles in more details. Previous studies have shown that nuclear vacuoles have detrimental effect on fertilization, embryo development and pregnancy outcome ([@B29]-[@B30]), while we did not find any correlation between MSOME parameters and fertilization and embryo development, the rate of LNVs were significantly higher in patients with successful pregnancies as compared to the patients with failed pregnancies. One of the proposed indications of MSOME is its predictive value for pregnancy in IUI ([@B31]). Evaluation of sperm morphology at high magnification for IVF cycles is recommended, because sperm morphology is an important factor for IVF success. To our knowledge, this is the first report regarding the MSOME criteria compared between IVF and ICSI cycles. Recently, De Vos et al. (2013) analyzed 330 semen samples from ICSI cycles. They stated that 18.1% of spermatozoa had normal shape with no vacuole as compared to our results of 11.8% in failed pregnancy cycles ([@B32]). Berkovitz et al. (2006) indicated that prevalence of LNV in spermatozoa from patients undergoing ICSI is 30-40%, which is higher compared to our results (11.7%) ([@B29]). One probable cause for this discrepancy would be due to different definitions of LNV that are used in different studies. We defined the LNV as the vacuole occupying\>15% of nuclear area; while, others have defined a vacuole area \>4%, \>13%, \> 25% or\> 50% of head area ([@B29], [@B33]-[@B35]). This needs to be standardized if this technique wants to be integrated in routine practice in infertility diagnosis and treatment. We have also defined medium size vacuole (4≥ and \<15% of head area) in order to standardize definition of vacuole. Our results also showed that the percent of abnormal sperm head size and shape, spermatozoa with cytoplasmic droplet and class III (according to Cassuto-Barak classification) were significantly higher in men who had abnormal semen parameters (ICSI group) compared to normal semen parameters (IVF group) (data not shown). Perdrix et al. (2012) evaluated MSOME criteria and conventional semen analysis in a population of 440 males. They found that the sperm head width and area as well as nuclear vacuoles number and area are significantly higher in men with abnormal semen (n=331) as compared to those with normal semen parameters (n=109) ([@B36]). Paternal age is considered as one of the main factors that affects sperm morphology. A recent study showed that prevalence of large and small vacuoles will increase with increasing paternal age ([@B37]). In our study, paternal age between successful and failed cycles both in IVF and ICSI were the same. Regarding the association between morphology evaluated with MSOME and conventional semen analysis, it was shown that there was no significant correlation between normal morphology by WHO and the ones defined by MSOME (26.1±7.2 vs 2.9±0.5, respectively) ([@B11]). While other investigators have found that MSOME might be more strict than Tygerberg criteria ([@B8]). Our data showed that the presence of LNV affects the normalcy of sperm shape whereas there was positive correlation between the rate of small and non-vacuole and sperm shape normalcy. It might be concluded that LNV should be considered as a definite parameter for evaluation of sperm shape. Interestingly, our results showed that there is significant correlation between sperm normal head and progressive motility. Theoretically, based on hydrodynamic rules, shape of sperm head is the most determinant factor for motility and in case of sperm head abnormality, sperm motility could be impaired. The impact of LNV on progressive motility is our another important finding that shows the other detrimental effect of LNV is inhibition of sperm motility. Conclusion ========== Although, MSOME is a time consuming method with no cost benefit, and demanding experienced operator, it can provide more details regarding fine sperm morphology in a real time manner. MSOME can be used as a predictive factor, and in case of high LNV rate, the cycle may not be considered for IVF, but substituted with ICSI. Therefore, MSOME should be considered as a non-invasive adjunct technology assisting ART performance. This study was extracted from PhD thesis of Shahin Ghazali. The authors would like to thank Yazd Reproductive Sciences Institute for providing the grant for performing this study. Note ==== ***This article extracted from Ph.D. thesis. (Shahin Ghazali)*** Conflict of interest ==================== The authors declare that there is no conflict of interest.

Students of the early modern period and its literature will be familiar with the 'barber-surgeon', usually glossed as a medical practitioner who performed a range of services from grooming to bloodletting. Eleanor Decamp seeks to deepen our understanding of this figure, but also to place pressure on the hyphen so that we can see that barbery and surgery were discrete if related occupations. In the process of explaining what made the barber and the surgeon recognisable to their contemporaries, she teaches us how to notice these figures on the landscape of representation. The barber wore an apron -- a garment he shared with other tradesmen -- but also carried recognisable equipment, linking him to 'the visible, legible, material world of the tradesman' (p. 46). While the surgeon might be recognised by his robe and coif, he was a 'mobile, transient figure who emerges, both literally and figuratively, at moments of crisis' (p. 47). Barbers were associated with a range of readily identified equipment: razor, scissors, basin, fleam (or lance), soapballs (with attendant puns), cupping glasses, ear-picker, comb. Surgeons had tools, of course, but these were less readily inventoried because they were constantly changing and were idiosyncratic, varying from practitioner to practitioner. In short, while props made the barber, they did not make the surgeon. The barber was often depicted in his shop, announced by a basin, a pole, even strings of teeth. In contrast, surgeons went to patients, working at the site of injuries on ships and battlefields, in households and workplaces, and sometimes in hospitals or their own homes. As a consequence, on the stage, Decamp shows, the surgeon has no place and no iconic props. Because surgeons opened bodies, the stakes of their practice were higher: 'To play a surgeon is a diabolical act; to play with barbery is harmless foolery' (p. 90). Barbers were often associated with deception; barbery is a show, even a put on. In contrast, perhaps simply because playmakers knew less about surgeons than they did about barbers, surgery is a secret and occurs off-stage. If we see it, that's because it is irregular or questionable in some way. In contrast to the chatty barber, the discreet 'surgeon's lack of spoken language *is* his narrative' (p. 190). Insisting that we always contrast the barber and the surgeon in this way, Decamp models a double vision that defines each occupation in terms of the other. Rich texture and evidentiary range distinguish this book, rather than bold argument. Decamp draws on an extraordinary range of primary sources, creatively gathered and interpreted. For instance, she several times makes good use of artefacts excavated from the wreck of the *Mary Rose* (a Tudor ship). She also consults wills, inventories, language manuals and dictionaries, medical treatises and ballads. Above all, she mines a wide range of plays, as the word 'performing' in her title might signal. First and last, she considers Lyly's *Midas*. But she also discusses, among others, Jonson's *Epicoene* and *Staple of News*, Webster's *Devil's Law Case*, Marston's *Dutch Courtes*an, Beaumont's *Knight of the Burning Pestle*, Middleton's *Fair Quarrel*, Markham and Sampson's *Herod and Antipater*, Fletcher and Massinger's *Sea Voyage*, Fletcher's *Monsieur Thomas*, and Chettle's *Hoffman*. She devotes the central chapter to case studies of Shakespeare's *Titus Andronicus* and *King Lear*. The non-dramatic texts to which she devotes sustained attention are Thomas Nashe's pamphlet *Have at You Saffron Walden* and an anonymously authored attack on Nashe, *The Trimming of Thomas Nashe*. While her discussion ranges from her earliest play, Richard Edwards's *Damon and Pithias* (first performed 1564--5) to the end of Restoration, the weight of her evidence comes from the period we associate with the flourishing of the drama: the later sixteenth century to the mid-seventeenth century. Her focus is London and the writers and cultural institutions centred there. Why is drama the central body of evidence here? Decamp links her choice to 'the meaning-making of drama, which is interested in questions of legitimacy, the correspondences between constitutional and corporeal bodies, the consequences of attempting to 'reach beyond' your lot, and the hazards of being misread and falsely labelled (directly or indirectly)' (p. 4). Barbers and surgeons were 'in the business of altering, amending, reinventing, and reshaping bodies and therefore identities' (p. 18), which was also a preoccupation of the stage. She also argues that 'forms of disguise which accompany the representation of a specific character (a barber or surgeon), as opposed to a specific actor, showcase production itself, and makes those characters discernable theatrical constructs, on or off the stage' (p. 63). The moments at which Decamp discusses the close connections between her topic and her archive are among the most interesting and useful in the book. In four of her five chapters, Decamp contrasts depictions of barbers and surgeons with respect to props, disguise, sound and language. In the chapter at the centre of the book, she proceeds a bit differently, offering case studies of how the associations around 'hazardous barbery' intensify moments of high crisis in Shakespeare's *King Lear* and *Titus Andronicus*. Although Decamp here addresses the best-known of the plays she has chosen, the chapter's argument is the book's most argument-driven and provocative. Decamp focuses on the scene in *Titus* in which Lavinia assists her father in murdering her rapists, Chiron and Demetrius, and making them into a pie to feed their mother Tamora. Decamp focuses our attention on the basin in which Lavinia collects the blood, an object associated with barbers, who used it for shaving and for bloodletting. In *King Lear*, Decamp again focuses on a scene and a prop, here the blinding of Gloucester and the chair to which he is first bound. Both objects, she argues persuasively, would have evoked the medical side of barbery and customers' terror at their vulnerability to the person who collected their blood in a basin or tied them to a chair to extract teeth. Neither play or scene mentions 'barber-surgery', that hybrid space in which barbers performed services we would now consider medicine. Yet, Decamp argues, the plays depend on a 'lurking medical and civic satire' (99) of the barbarism of such commonplace practices. While other playwrights portray such practices in city comedies, Shakespeare draws on them in these two tragedies, indirectly evoking them to heighten trauma. In the course of the book, Decamp suggests that barbery was a literary resource in a way that surgery was not. The barber figure moves from medieval drama, through popular dramatic scenes culled from earlier plays for performance during the interregnum, to the Restoration. This is not true for the surgeon. 'Put crudely, barbery material was evidently popular so playmakers made ample use of it to stuff their works: filling subplots, creating interludes' (p. 177), just as barbers purportedly used the hair they cut off to stuff tennis balls. The barber is also closely associated with the playmaker and the barber's shop with the theatre. Like the playmaker, the barber 'was endowed with the ability both to expose and to conceal or reconstruct a client's social standing and/or very nature' (p. 64). Just as playgoers are still called audiences or hearers, so the barber's shop was a 'sound-market' where 'ears were treated, entertained, and abused' (p. 136) and news and gossip exchanged. Decamp's thorough and erudite book will not only train readers to notice both barbers and surgeons in early modern drama and early modern English culture more generally. It also tunes our ears, as barbers might have, to the complex, historically specific resonances of objects (such as basins), words (including 'trim' and 'barbarous'), and names including Lavinia (associated with washing) and Chiron (who shares with the chirurgeon a root in the Greek word for hand).

The authors wish to make the following changes to their paper \[[@B1-genes-09-00315]\]. Due to an undetected mistake in the references management, certain errors appeared in the reference list and a reference was duplicated in [Table 1](#genes-09-00315-t001){ref-type="table"}. Consequently, three references have been changed as follows: Reference \[51\] "51. Kumar, N.; Mariappan, V.; Baddam, R.; Lankapalli, A.K.; Shaik, S.; Goh, K.L.; Loke, M.F.; Perkins, T.; Benghezal, M.; Hasnain, S.E.; et al. Comparative Genomic Analysis of *Helicobacter pylori* from Malaysia Identifies Three Distinct Lineages Suggestive of Differential Evolution. *Nucleic Acids Res.* **2015**, *43*, 324--335." has been replaced by: "51. Qumar, S.; Majid, M.; Kumar, N.; Tiwari, S.K.; Semmler, T.; Devi, S.; Baddam, R.; Hussain, A.; Shaik, S.; Ahmed, N. Genome Dynamics and Molecular Infection Epidemiology of Multidrug-Resistant *Helicobacter pullorum* Isolates Obtained from Broiler and Free-Range Chickens in India. *Appl. Environ. Microbiol*. **2017**, *83*, e02305-16." Reference \[44\] "44. Rehman, M.U.; Zhang, H.; Iqbal, M.K.; Mehmood, K.; Huang, S.; Nabi, F.; Luo, H.; Lan, Y.; Li, J. Antibiotic Resistance, Serogroups, Virulence Genes, and Phylogenetic Groups of *Escherichia coli* Isolated from Yaks with Diarrhea in Qinghai Plateau, China. Gut Pathog. **2017**, *9*, 1--11." has been replaced by "44. Rehman, M.A.; Yin, X.; Lepp, D.; Laing, C.; Ziebell, K.; Talbot, G.; Topp, E.; Diarra, M.S. Genomic Analysis of Third Generation Cephalosporin Resistant *Escherichia coli* from Dairy Cow Manure. *Vet. Sci.* **2017**, *4*, 4, doi:10.3390/vetsci4040057." Reference \[60\] "60. Wang, W.; Baloch, Z.; Peng, Z.; Hu, Y.; Xu, J.; Fanning, S.; Li, F. Genomic Characterization of a Large Plasmid Containing a *bla~NDM-1~* Gene Carried on *Salmonella enterica* Serovar Indiana C629 Isolate from China. *BMC Infect. Dis.* **2017**, *17*, 1--8." has been replaced by "60. Wang, J.; Li, X.; Li, J.; Hurley, D.; Bai, X.; Yu, Z.; Cao, Y.; Wall, E.; Fanning, S.; Bai, L. Complete Genetic Analysis of a *Salmonella enterica* serovar Indiana Isolate Accompanying Four Plasmids Carrying *mcr-1*, ESBL and Other Resistance Genes in China. *Vet. Microbiol*. **2017**, *210*, 142--146, doi:10.1016/j.vetmic.2017.08.024." Two references have been added, as they were omitted in error: Reference \[66\] in [Table 1](#genes-09-00315-t001){ref-type="table"} was wrongly cited, therefore it has been substituted by the new reference \[66\] "66. Li, B.; Yang, X.; Tan, H.; Ke, B.; He, D.; Wang, H.; Chen, Q.; Ke, C.; Zhang, Y. Whole Genome Sequencing Analysis of *Salmonella enterica* Serovar Weltevreden Isolated from Human Stool and Contaminated Food Samples Collected from the Southern Coastal Area of China. *Int. J. Food Microbiol.* **2018**, *266*, 317--323, doi:10.1016/j.ijfoodmicro.2017.10.032." Reference \[8\] in [Table 1](#genes-09-00315-t001){ref-type="table"} was wrongly cited, therefore it has been substituted by the new reference \[71\] "71. Flórez, A.B.; Mayo, B. Antibiotic Resistance-Susceptibility Profiles of *Streptococcus thermophilus* Isolated from Raw Milk and Genome Analysis of the Genetic Basis of Acquired Resistances. *Front. Microbiol.* **2017**, *8*, 2608, doi:10.3389/fmicb.2017.02608." Due to this correction, reference numbers were adjusted to follow a numerical order. In \[[@B1-genes-09-00315]\], the previous references \[66\] and \[71\] are now \[139\] and \[72\], respectively. The 31st row from [Table 1](#genes-09-00315-t001){ref-type="table"}, about *S. enterica* with origin in Dairy cattle and humans, was eliminated because it was a duplicate of row 28; the corrected table is: The authors would like to apologize for any inconvenience caused to the readers by these changes. genes-09-00315-t001_Table 1 ###### Main research studies published in recent years applying whole genome sequencing (WGS) to characterize antimicrobial resistance (AMR) in foodborne bacteria. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Reference Microbial Species Number of Isolates Sequenced Origin Main Findings in Relation to AMR ----------- ---------------------------------------------------------------------------------------------------------------- ------------------------------ ------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \[36\] *Aeromonas salmonicida* 101 Fish All sequenced isolates harbored three AMR genes against beta-lactam antibiotics encoded on the chromosome.\ Some isolates also harbored several other plasmid encoded resistance genes against trimethoprim, sulphonamide, and aminoglycoside antibiotics. \[37\] *Campylobacter* spp*.* 589 Retail poultry meat The following AMR genes were identified: *tetO*, *bla*~OXA-61~, *aph(2″)-Ic*, *aph(2″)-If*, *aph(2″)-Ig*, *aph(3′)-III*, *ant(6)-1a*, *aadE*, *aph(3″)-VIIa*, and *Inu(C)*.\ Mutations in housekeeping genes (*gyrA* at position 86, 23S rRNA at position 2074 and 2075) associated with AMR phenotypes were also identified. \[38\] *Campylobacter* spp. 114 Humans, retail meats, and cecal samples from food production animals Eighteen resistance genes, including *tet(O)*, *blaOXA-61*, *catA*, *lnu(C)*, *aph(2″)-Ib*, *aph(2″)-Ic*, *aph(2′)-If*, *aph(2″)-Ig*, *aph(2″)-Ih*, *aac(6′)-Ie-aph(2″)-Ia*, *aac(6′)-Ie-aph(2″)-If*, *aac(6′)-Im*, *aadE*, *sat4*, *ant(6′)*, *aad9*, *aph(3′)-Ic*, and *aph(3′)-IIIa*, and mutations in two housekeeping genes (*gyrA* and 23S rRNA), were identified. \[26\] *Campylobacter coli* 2 Retail meats A self-transmissible plasmid carrying multiple antibiotic resistance genes was identified, carrying genes encoding resistance to gentamicin, kanamycin, streptomycin, streptothricin, and tetracycline.\ Gentamicin resistance was due to a phosphotransferase gene, *aph(2″)-Ig*, not described previously. \[39\] *Clostridium difficile* 40 Human and porcine origin AMR genotypes were characterized by resistance to tetracycline \[*tetM*, *tetA(P)*, *tetB(P)*, and *tetW*\], clindamycin/erythromycin (*ermB*), and aminoglycosides (*aph3-III-Sat4A-ant6-Ia*).\ Resistance was mediated by clinically important mobile genetic elements, most notably Tn*6194* (harboring *ermB*) and a novel variant of Tn*5397* (harboring *tetM*). \[40\] *C. difficile* 2 Ground pork Identification of vancomycin (*vanW*, *vanA*, *vanR*, *vanS*, *vex2*, *vex3*, *vncR*, *vncS*); fluoroquinolones (*gyrA* and *gyrB*); tetracyclines (*tetM*, translation elongation factor G); beta-lactams (*blaZ*); and macrolides (macrolide efflux protein, macrolide glycosyltransferase) resistance genes, and multiple multidrug resistance efflux pump genes. \[31\] *Enterococcus* spp. 197 Various animal and food sources Resistance genotypes correlated with resistance phenotypes in 96.5% of cases for the 11 drugs investigated. \[21\] *Enterococcus faecalis*, *Enterococcus faecium*, *Escherichia coli*, *Salmonella enterica* serovar Typhimurium 200 Pigs High concordance (99.74%) between phenotypic and predicted antimicrobial susceptibility was observed.\ Correlation between MLST type and resistance profiles was only observed in *S. enterica* serovar Typhimurium, where isolates belonging to sequence type (ST) 34 were more resistant than ST19 isolates. \[41\] ESBL-producing *Enterobacteriaceae* 24 Fish and environmental samples Nine of eleven sequenced fish isolates had the *bla*~CTX-M-15~ gene, whereas 12/13 from environment carried *bla*~CTX-M-15~*.*\ AMR genes encoding resistance to sulfonamides (*sul1*/*sul2*), tetracyclines \[*tet(A)/tet(B)*\], fluoroquinolones \[e.g., *aac(6′)-Ib-cr*, *qnrS1*\], aminoglycosides \[e.g., *aac(3)-lld*, *strB*, *strA*\], and trimethoprim (e.g., *dfrA14*) were detected. \[42\] *E. coli* 17 Retail chicken meat All strains carried an IncK plasmid with a *bla*~CMY-2~ gene. \[43\] *E. coli* 168 Broilers and free-range retail poultry (meat/ceca) The prevalence rates of ESBL producing *E. coli* among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%.\ *E. coli* from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively). \[44\] *E. coli* 18 Dairy cow manure All sequenced isolates carried at least one β-lactamase *bla* gene: *TEM-1*, *TEM-81*, *CTX-M115*, *CTX-M15*, *OXA-1*, or *CMY-2*.\ Several other AMR genes were detected in the sequenced isolates and all of them harbored AMR plasmids belonging to classic Inc groups. \[45\] *E. coli* 16 Swine farm *bla*~NDM-5~ and *mcr-1* were located on two different plasmids, which showed 100% nucleotide identity in all 16 strains. \[46\] *E. coli* 26 Humans, cows, pigs, horse, rabbit, goat, environments and food A total of 39 plasmids were identified.\ Eight plasmids carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines, and resistance to heavy metals.\ Two plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. \[47\] *E. coli* 42 Feedlot cattle 70% of the cattle strains carried at least one AMR gene \[48\] *E. coli* 3 Dairy cows The *mcr-1* gene (linked to colistin resistance) coexisted with multiple resistance genes in a plasmid (pXGE1mcr) \[49\] *E. coli*, *Salmonella* spp. 463 Retail meats and farm local samples To improve the concordance between genotypic and phenotypic data, it was proposed to reduce the phenotypic cut-off values for streptomycin to ≥32 µg mL^−1^ for both *Salmonella* and *E. coli.* \[50\] *Helicobacter pullorum* 4 Chicken meat AMR-associated SNPs were detected (linked to resistance to fluoroquinolones, macrolides, and tetracyclines). \[51\] *H. pullorum* 11 Broiler and free-range chicken WGS revealed the presence of five or six well characterized AMR genes, including those encoding a resistance-nodulation-division efflux pump \[30\] *Klebsiella pneumoniae* 7 Pig and human samples at abbatoirs AMR genes associated with resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolides, lincosamide, streptogramins, rifampicin, sulfonamides, trimethoprim, phenicols, and tetracycline were identified. \[29\] *K. pneumoniae* 44 Chicken, turkey and pork meat Meat-source isolates were significantly more likely to be multidrug resistant and resistant to tetracycline and gentamicin than clinical isolates.\ Four sequence types occurred among both meat-source and clinical isolates. \[52\] *Listeria monocytogenes* 2 Ready-to-eat food Seven antibiotic and efflux pump related genes which may confer resistance against lincomycin, erythromycin, fosfomycin, quinolones, tetracycline, penicillin, and macrolides were identified in the genomes of both strains. \[53\] *L. monocytogenes* 5 Environments from pork processing plants Strains of a particular sequence type were shown to contain the BAC resistance transposon Tn*6188*, conveying resistance to quaternary ammonium compounds. \[54\] *Proteus mirabilis* 8 Food-producing animals Seven integrative and conjugative elements were identical to ICEPmiJpn1, carrying the cephalosporinase gene *bla*~CMY-2~. \[55\] Non-typhoidal *Salmonella* 536 Retail meat A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified.\ First finding of extended-spectrum β-lactamases (ESBLs) (*bla*~CTX-M1~ and *bla*~SHV2a~) in retail meat isolates of *Salmonella* in the United States. \[56\] Non-typhoidal *Salmonella* 1738 Animal, food and human sources The Minimum Inhibitory Concentration (MIC) predictions were correlated with the ResFinder database.\ The genotypic cut-off values were established for 13 antimicrobials against *Salmonella*. \[20\] Non-typhoidal *Salmonella* 3491 Received by Public Health England's Gastrointestinal Bacteria Reference Unit from different origins for surveillance purposes Discrepancies between phenotypic and genotypic profiles for one or more antimicrobials were detected for 76 isolates (2.18%).\ Only 88/52,365 (0.17%) isolate/antimicrobial combinations were discordant.\ Pan-susceptibility to antimicrobials was observed in 2190 isolates (62.73%). \[33\] *S. enterica* 90 Dairy cattle and humans Genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.2 and specificity of 85.2. \[57\] *S.* *enterica* serovar *Typhimurium* 984 Swine Multiple genotypic resistance determinants were predominant, including resistance against ampicillin, streptomycin, sulfonamides, and tetracyclines.\ Phenotypic resistance to enrofloxacin and ceftiofur was found in conjunction with the presence of plasmid-mediated AMR genes. \[58\] *S.* *enterica* serovar Typhimurium 1 Swine carcass The following AMR genes were identified: *tetA*, *aac3IIa*, *aadA1*, *strA*, *strB*, *bla*~TEM-1B~, *qnrE*, *sul1*, *drfA1*, and *floR*. \[59\] *S.* *enterica* serovar Heidelberg 113 Humans, abbatoir poultry and retail poultry CMY-2 plasmids, all belonging to incompatibility group I1, were identified in cefoxitin-resistant isolates.\ Analysis of IncI1 plasmid sequences revealed high identity (95% to 99%) to a previously described plasmid (pCVM29188_101) found in *S. enterica* serovar Kentucky*.* \[60\] *S.* *enterica* serovar Indiana 1 Poultry slaughterhouse 24 multi-drug resistance (MDR) genes, located on 4 plasmids, were identified, including the *mcr-1* gene (linked to colistin resistance). \[61\] *S.* *enterica* serovar Infantis 12 Humans, food-producing animals and meat Some isolates harbored a conjugative megaplasmid (\~280--320 Kb) which carried the ESBL gene *blaCTX-M-1*, and additional genes \[*tet(A)*, *sul1*, *dfrA1* and *dfrA14*\] mediating cefotaxime, tetracycline, sulfonamide, and trimethoprim resistance. \[62\] *S.* *enterica* serovar Muenster 2 Dairy farm environments The plasmid-mediated *qnrB19* gene and IncR plasmid type were identified in both isolates. \[63\] *S.* *enterica* serovar Typhimurium 225 Humans, animals, feed, and food The non-clinical use of narrow-spectrum penicillins (e.g., benzylpenicillin) might have favored the diffusion of plasmids carrying the *bla~TEM-1~* gene in *S. enterica* serotype Typhimurium in the late 1950s. \[64\] *S.* *enterica* serovar Typhimurium 4 Poultry and humans The following AMR genes were identified: *strA*, *strB*, and *aadA1* (aminoglycosides); *bla*~TEM-1B~ (β-lactams); *catA1* (phenicols); *sul1* and *sul2* (sulphonamides); *tet B* (tetracyclines); and *dfrA1* (trimethoprim)*.* \[65\] *S.* *enterica* serovar Typhimurium and *S.* *enterica* serovar Kentucky 2 Chicken carcasses A total of five plasmids conveying AMR genes were found. \[66\] *S.* *enterica* serovar Weltevreden 44 Human stool and contaminated food samples AMR genes were only identified in eight isolates, linked to resistance to tetracycline, ciprofloxacin or ampicillin. \[67\] *Staphylococcus aureus* 66 Retail meats Eleven *spa* types were represented.\ The majority of MRSA (84.8%) possessed SCC*mec* IV. \[68\] *S. aureus* 9 Pork, chicken and turkey meat Multiple resistance genes/mutations were detected.\ All livestock-associated methicillin-resistant *S. aureus* (LA-MRSA) harbored *tet(M)* (±*tet(K)* and *tet(L)*), and only seven of these additionally harbored multi-drug resistance to beta-lactams, quinolones, and macrolides. \[69\] *S. aureus* 12 Livestock animals Most isolates harbored resistance genes to ≥3 antimicrobial classes in addition to β-lactams. Heavy metal resistance genes were detected in most European *ccrC* positive isolates, with \>80% harboring *czrC*, encoding zinc and cadmium resistance. \[70\] *S. aureus* 15 Bulk milk A divergent *mec*A homologue (*mec*A~LGA251~), later named as *mec*C, was identified. \[71\] *Streptococcus thermophilus* 5 Raw milk *tet(S)* and *ermB* identified as determinants of AMR. \[72\] Carbapenem-resistant bacteria 28 Dairy cattle Isolates included: 3 *E. coli* harboring *bla*~CMY-2~ and truncated *ompF* genes; 8 *Aeromonas* harboring *bla*~cphA~-like genes; 1 *Acinetobacter baumannii* harboring a novel *bla*~OXA~ gene (*bla*~OXA-497~); and 6 *Pseudomonas* with conserved domains of various carbapenemase-producing genes. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [^1]: These authors contributed equally to this work.

Introduction {#sec1-1} ============ *Vata* is considered as a chief factor for the physiological maintenance of the body. Factors provoking *Vata* result in the instantaneous manifestation of diseases, which can even prove to be fatal. Therefore, the *Vataja nanatmaja vyadhis* are of utmost importance, rather than the vyadhis produced by the other two *doshas*. Contradictory approaches to pacify this vitiated state have to be restored to, to maintain the equilibrium. In the modern point of view, the diseases involving the neurological, musculoskeletal, psychosomatic, and gastrointestinal system disorders have more similarity with the *Vata vyadhi*. It indicates the wide ranging involvement of *Vata* in various systems of the body. The economy of the country relies on its work force. *Apabahuka* is one such disease that hampers the day-to-day activity of an individual. The fact that *Vata vyadhi* is one among the *Ashta maha gada*,\[[@ref1]\] is itself explanatory, with regard to the consequences caused by *Apabahuka*. Even though a definite factor responsible for the manifestation of this disease is not mentioned, a set of etiological factors can be interpreted. On analyzing the etiopathology, it may be interpreted that the disease *Apabahuka* manifests due to the depletion of tissue elements (*dhatu kshaya*) as well as *Samsrushta dosha*. *Apabahuka* is considered to be a disease that usually affects the shoulder joint (*amsa sandhi*) and is produced by the *Vata dosha*. Even though the term *Apabahuka* is not mentioned in the *nanatmaja Vata vyadhi*, Acharya Sushruta and others have considered *Apabahuka* as a *Vata vyadhi*.\[[@ref2]\] *Amsa shosha* can be considered as the preliminary stage of the disease where loss or dryness of the *Shleshaka Kapha* from the shoulder joint occurs. The next stage, that is, Apabahuka, occurs due to the loss of Shleshaka Kapha and symptoms like shoola during movement, restricted movement, and so on, are manifested. Even as this is commented on in the *Madhukosha teeka*, it is mentioned that Amsa shosha is produced by *dhatukshaya*, that is, *shuddha Vata janya*, and *Apabahuka* is *Vata Kapha janya*.\[[@ref3]\] Etiopathogenesis {#sec2-1} ---------------- ### Etiology (Nidana) {#sec3-1} The causes (*hetu*) of *Apabahuka* may be classified into two groups. (i) *Bahya hetu* --- Causing injury to the vital parts of the body (*marma*) or the region surrounding the *amsa sandhi*, which is also known as *bahya abhigataja* that manifests the *vyadhi* or disease first; (ii) *Abhyantara hetu* --- Indulging in the etiological factors that aggravate Vata leading to the vitiation of *vata* in that region and is also known as *dosha prokopajanya (Samshraya)*, which in turn leads to *karmahani* of *bahu*. The descriptions of *Nidana* are given below: ### Aharaja {#sec3-2} *Katu, Tikta, kashaya rasas, laghu sukshma*, and *sheeta guna* cause vitiation of the *Vata*. ### Viharaja {#sec3-3} The exercises directly or indirectly influencing the shoulder or *amsa desha* should be considered here: ### Plavana {#sec3-4} Results in *Vata kopa* due to overexertion in the joint. ### Bharavahana {#sec3-5} Carrying heavy loads over the shoulders will cause *Vata prakopa* and deformity in the joint capsule. This leads to disease formation. ### Balawat Vigraha {#sec3-6} Wrestling with a person who is more powerful will cause *agahata* to the *amsapradesha* and *Vataparkopa* takes place. This manifests the disease. ### Dukkha Shayya {#sec3-7} Improper posture that creates a great amount of more pressure on the *amsasandhi* will disturb the muscular integrity, and provokes *vata*. This manifests the disease. Other *viharaja nidanas* as reported in the *Vatavyadhi* context may influence the condition by provocating *Vatadosha*. Even though a specific *bahya hetu* (external cause) has not been mentioned for *Apabahuka*, the general factors reported for *Vata prakopa* have to be analyzed and elicited. To summarize, the above-mentioned *nidanas*, under *vihara*, especially involving the *amsa sandhi* and *marmabhighata* to *amsa*, lead to the development of *Apabahuka*. ### Samprapti {#sec3-8} In case of *Apabahuka*, two ways of vitiation of the *vata* can be considered. The etiological factors like *ruksha, laghu* and so on, and *atibharavahana* and the like cause vitiation of the *vata* directly. In another way, *Kapha prakopaka nidanas* like taking of *atisnigdha, atiguru dravya*, and so on cause an increase in the *vikruta Kapha*, which produces the *Kaphavrita-vata* condition. In both ways, the *vikruta vata dosha* gets accumulated in the bio channels (*srotas*) and manifests symptoms like the *stabdha poorna kosthata*. In the *prakopa avasta*, the *Vata* produces symptoms like *koshtha toda* and *sancharana* and *prasara avastha* symptoms like *atopa* may also be produced. However, *Ashukaritva* being one among the symptoms of *Vata*, the symptoms produced by it are very quick in onset, and hence, the *dosha kriyakalas* of the *vyadhi* are ill-defined and cannot be observed properly. *Sthana samsraya avastha* of the *vyadhi* occurs with the localization of the aggravated *Vata* in the specific *dhatu*, that is, *dosha dushya sammurachana*, which occurs in the specific organ of the body where *kha vaigunya* has previously taken place by the specific part of the *nidanas*, simultaneously with the *dosha vikruti. Shiro marma* is considered as the *uttamanga* and is mentioned as the seat or *moola* of all *indriyas. Shiro marma* performs all types of *cheshta* in the presence of the normal *vata* because, among the three *doshas*, only the *Vata* is responssible for all types of *cheshta*. The *Charaka samhita* has mentioned that when *shiro marma* gets affected, it produces symptoms like *chestahani*. In this case, the *sthana samshraya* of the *dosha* can be taken, as in *siro pradesha*. Usually in the *sthana samshraya avasta*, the *poorvaroopa* of the *vyadhi* are manifested. As *Apabahuka* is considered as a *vata vyadhi*, and *Vata* has the *ashukari guna*, the *poorvaroopas* like *bahupraspandita hara* and *shoola* may manifest mildly or are totally absent. However, the above-mentioned symptoms are clearly manifested in the *vyaktha avastha* or in the *roopa avastha* of the *vyadhis* in the vyakta sthana, that is, in the *amsa pradesha*. In this stage the *amsa pradesha* gets affected by the aggravated *Vata*, on account of which *Amsashosha* occurs in the initial stage by the decrease of the *Shleshaka Kapha*, which further leads to the manifestation of *Apabahuka*, with symptoms like *bahupraspandita hara* and *shoola*. Therefore, in the *Madhukosha* commentary of *Madhava nidana* it is mentioned that *amsa shosha* and *Apabahuka* are the two stages of the *vyadhi*. ### Marma abhighata {#sec3-9} Morbid *vyana Vata* in another way may cause *abhyantara marmabhighata* or any external trauma to *amsapradesha* may also cause *bahya marmbhighata* to *the amsa marma* present in *amsadesha*. As *amsa marma* is a *snayu* and *vaikalyakara marma*, afflicting *snayu* will manifest *bahupraspanditahara*. Even in modern medical science, the partial loss of blood supply in the area of insertion of tendons or some idiopathic cause can produce localized degeneration of the collagen. This induces an autoimmune response and cause a tear or distortion of the tendinous sheaths and ligaments. This obliterates the integral stability of the joint and results in restricted movement with painful and stiffened joints. ### Chikitsa {#sec3-10} The general line of treatment mentioned for *Vatavyadhi* in Ayurvedic classics include *Snehana* (both internal and external), *Swedana, Mrudusamshodhana, Basti, sirobasti Nasya*, and so on. Charaka further states that, depending on the location and *dushya* (tissue element vitiated by *Vata*) each patient should be given specific therapies. Vagbhata has mentioned *Nasyakarma* in the *jatroordhva Vatavikaras*.\[[@ref4]\] Three major approaches are followed in the management of *Vatavyadhi*. Treatment of *Kevala Vata*Treatment of *Samsrusta Vata*Treatment of *Avruta Vata* Ayurvedic classics explain the *chikitsa* of *Apabahuka* as follows. *Nasya* and *uttarabhaktika Snehapana* are useful in the management of *Apabahuka*.*Astanga Sangraha* mentions *Navana Nasya* and *sneha pana* for *Apabahuka*.Sushrutacharya advises *Vatavyadhi chikitsa* for *Apabahuka*, except *siravyadha*.*Chikitsa sara sangraha* advises *Nasya, Uttara bhaktika Snehapana*, and *Sweda* for the treatment of *Apabahuka*.*Brumhana nasya* is indicated in *Apabahuka* by Vagbhata. By considering the above references, the following can be stated as the line of treatment of *Apabahuka*. Nidana parivarjanaAbhyangaSwedanaUttarabhaktika snehapanaNasyakarmaShamanoushadhi Modern description {#sec2-2} ------------------ There are some clinical conditions of modern science, which may be compared with that of *Apabahuka*. These may be categorized as follows: Periarthritis, frozen shoulder or adhesive capsulitisSubacromial or subdeltoid bursitisSubcoracoid bursitisPainful shoulderBicipital tendinitisOsteoarthritis of shoulder jointBrachial plexes neuropathies However, in this study, the clinical condition, namely, periarthitis or frozen shoulder or adhesive capsulitis has been taken to correlate with *Apabahuka*. Periarthitis or frozen shoulder or adhesive capsulitis\[[@ref5][@ref6]\] {#sec2-3} ------------------------------------------------------------------------ This is a descriptive term used to indicate a clinical syndrome wherein the patient has a restricted range of active and passive glenohumeral motion. The Simmonds have reported on the tight inelastic tissues around the shoulder joint. They believed that the pathological changes in frozen shoulder were due to degeneration and focal necrosis of the supraspinous tendon. With revascularization, the tendon pathology could resolve. With inadequate vascular response, the tendons would continue to degenerate, developing tears of varying size, or a secondary biceps tendinitis could develop. In this condition, pain and stiffness of the shoulder joint are the cardinal symptoms leading to inability or loss of function of the affected upper limb. This may be achieved in three phases. Painful phaseStiffening phaseThawn / Resolving phase The patient gives a history of having noticed a slight painful catch in the region of the shoulder and upper arm for several months, gradually becoming aware of the inability to perform certain tasks, because of stiffness of the arm. Night pain, often awakening him after he has fallen asleep, is a common complaint. Frequently it radiates down the arm to the hand without being localized to any nerve distribution. Stiffness of the shoulder increases until all movements are lost. In Ayurveda, therapies like *abhyanga, swedana, snehapana nasya karma, vasti karma*, and *shamana Aushadhi* are mentioned to combat the *Vata vyadhi*. In the present study *nasyakarma* with *Laghumasha taila*, in the form of *Marsha nasya* have been advised for patients suffering from *Apabahuka. Laghumasha taila* contains drugs like *Kapikacchu, Bala, Shatavari, Sita, Punarnava, Saindhava, Jingini, Sarshapa taila*, and *Masha*.\[[@ref7]\] Aims and Objectives {#sec1-2} =================== To study the effect of *Laghumasha taila Marsha nasya* in *Apabahuka*.To study the literature of the disease in view of Ayurveda and Modern medical science. Materials and Methods {#sec1-3} ===================== Source of data {#sec2-4} -------------- Patients of either sex diagnosed with *Apabahuka* from the OPD and IPD of the ALN Rao Memorial Ayurvedic Medical College and Hospital, Koppa, were selected for the study. Out of the 18 patients, three patients were dropped in the initial stages of the study and 15 patients completed the course of treatment. Criteria for selection of the patients {#sec2-5} -------------------------------------- The patients presenting with the signs and symptoms of *Apabahuka* according to Ayurvedic texts were selected for the study. Patients of both sexes in the age group of 20 -- 60 years were selected. The main criteria for diagnosis was the presence of clinical symptoms of *Apabahuka*, that is, *Bahupraspandita hara* and *shoola*. The symptoms of *Srotodushti* were also assessed along with the main symptoms for the selection of the patients. Inclusion criteria {#sec2-6} ------------------ *Apabahuka* diagnosed according to the classical signs and symptoms described in Ayurveda.Patients of both sexes within the age group of 20 -- 60 years. Exclusion criteria {#sec2-7} ------------------ Systemic diseases presented with *Apabahuka* as a complicationPatients with a history of fracture of the affected handPregnancy and lactating women Laboratory investigations {#sec2-8} ------------------------- The modern laboratory investigations included for the clinical study are Blood for Hemoglobin %,Total count,Differential count, Erythrocyte sedimentation rate, Random blood sugar as routine investigations; Urine for sugar, albumin, and microscopic test; and X-Ray Shoulder joint --- Anterio-posterior, lateral view --- to rule out the history of fracture. Study design {#sec2-9} ------------ A randomized standard single blind clinical study has been adopted. Treatment schedule {#sec2-10} ------------------ After diagnosis, the randomly selected patients were treated with *Marsha nasya* with *Laghumasha taila* in doses of 6, 8, or 10 drops, as required by the patients, for seven days. Out of the 18 selected patients, three patients were dropped from the study and the remaining 15 patients completed the course of the study. ### Pratimarsha / Marsha nasya {#sec3-11} When the *nasya dravya* is used in the minimum quantity (two *bindus*), it is called *Pratimarsha*. Usually the *sneha dravyas* are used. This is different from *marsha nasya* where in the quantity used will be 6, 8, or 12 *bindus*. Method of Nasya Karma {#sec2-11} --------------------- *Nasya Karma* can be explained in the following three headings as reported in the classics. ### Poorva Karma {#sec3-12} This encompasses the following points like *Oushadhi sangraha, Nasya yantra, Atura vaya, Kala, Atura siddhata*, and so on. The patient is instructed not to suppress the natural urges and go through the normal routine. Before taking the *Nasya Karma* he should not have any food. Subsequently, the patient is taken to a comfortable room, which is without dust, extreme breeze or sunlight. *Bahyasnehana* in the form of *mrudu Abhyanga* is performed to the *shiras* first and then over the *gala, kapola, lalata*, and *karna*. After *snehana*, a mild *swedana* is performed to the part of the body above the shoulders. The eyes of the patient should be taken care of by closing them with a band of cloth. ### Pradhana Karma {#sec3-13} Once the *poorva karma* is completed, the patient is made to lie down on the table in the supine position with legs slightly raised. Eyes should be covered with a cloth. With the help of tepid medicine, *panitapa sweda* is performed over the parts of the body above the shoulder, excluding the patient\'s eyes. The head of the patient is then highly raised and medicine is poured in each nostril one after the other. The other nostril should be closed while administering the medicine in one nostril. The medicine should be slowly instilled in an uninterrupted manner called *'Avicchinna dhara'*. The patient is advised to inhale the medicine slowly and forcefully. The same procedure is repeated in both the nostrils. Care should be taken not to shake the head during the procedure. *Tapasweda* can be repeated conveniently. Aftera administration of the medicine, the patient is strictly advised not to swallow the medicine, but to spit it out. The spitting can be done after the smell and taste of the medicine disappears from the throat. Next, the patient is allowed to relax in the same posture for 100 *matra kalas* (30 -- 32 seconds), without going to sleep. ### Paschat karma {#sec3-14} *Pradhana karma* is followed by *dhoomapana, gandoosha*, and *kavala graha*. The patient is advised to follow certain rules and regimen. Criteria for assessment of the study {#sec2-12} ------------------------------------ The improvements in the patients were assessed on the basis of relief in the signs and symptoms of the disease. To analyze the efficacy of the drug, scores were given for each symptom. According to the severity of the symptoms, the grading was given, as mentioned herewith: ### Scoring pattern {#sec3-15} The improvement is documented through statistical significance. The subjective and objective parameters are assessed by means of interrogation and by ascertaining the signs and symptoms before and after the treatment. The response of the drug is assessed weekly through interrogation, signs, and symptoms. ### Statistical analysis {#sec3-16} For assessing the improvement of symptomatic relief and to analyze it statistically, the observations were recorded before and after the treatment. The mean, percentage, SD, SE, and t-value (paired t-test) were calculated from the observations recorded. Criteria for assessment of the overall therapy {#sec2-13} ---------------------------------------------- ### Complete relief {#sec3-17} One hundred percent relief in the complaints of patients, along with elevation of shoulder joint up to 180° and flexion and abduction of the joint up to 90°. ### Marked improvement {#sec3-18} More than 75% relief in the complaints as well as significant improvement in the elevation of joint up to 135°, and flexion and abduction up to 60°. ### Moderate improvement {#sec3-19} More than 50% relief in the complaints along with improvements in elevation of joint up to 90° and flexion and abduction of joints up to 30°. ### Improvement {#sec3-20} Twenty-five to fifty percent relief in the complaints. ### Unchanged {#sec3-21} Patients with less than 25% relief in their complaints were regarded as unchanged. Observations {#sec1-4} ============ The observations made of the 18 patients with *Apabahuka* were as follows: Maximum number of patients were obtained in the age group of 31 -- 40 years, that is, 33.33%, followed by 26.66% patients in the age group of 41 -- 50 years, and 20% patients in the age groups of 51 -- 60 years and 20 -- 30 years each. Most of the patients were male (60%). Most of the patients, that is, 47%, were from the Hindu community, followed by 29% from the Muslim community, and 24% from the Christian community. Most of the patients (38%) were laborers, followed by housewives (25%); the maximum number of patients (40%) were from the lower socioeconomic status group; maximum number of patients (58%) were nonvegetarian, followed by 42% who were vegetarian. Results {#sec1-5} ======= The drug *Laghumasha taila Nasya* provided a moderately significant effect (*P* \< 0.01) on the symptom *Bahupraspandita hara* (53.33%) and a mildly significant effect (*P* \< 0.05) on *Shoola* (26.66%) \[[Table 1](#T1){ref-type="table"}\]. On *atopa* the therapy showed 60% relief, followed by 37.5% relief on wasting of muscles, and 30% relief on *stambha* \[[Table 2](#T2){ref-type="table"}\]. The therapy was mildly significant on both *Asthivaha* and *Majjavaha Srotodushti* (*P* \< 0.05) and moderately significant on *Medovaha srotodushti* (*P* \< 0.02) \[[Table 3](#T3){ref-type="table"}\]. Most of the patients, that is, 53.33% were moderately improved, followed by 26.66% improved, and 6.60% were markedly improved after completion of the therapy \[[Table 4](#T4){ref-type="table"}\]. ###### Effect of the therapy on main symptoms in 15 patients with *Apabahuka*  ###### Effect of the therapy on associated symptoms of *Apabahuka* in 15 patients  ###### Effect of the therapy on *srotodushti* in 15 patients of *Apabahuka*  ###### Overall effect of the therapy  Discussion {#sec1-6} ========== The present drug formulation *Laghumasha taila* contains drugs like *Kapikacchu, Bala, Shatavari, Sita, Punarnava, Saindhava, Jingini, Sarshapa taila*, and *Masha*. Mode of action {#sec2-14} -------------- ### Laghu masha taila {#sec3-22} *Kapikacchu(Mucuna prurita* Hook) : Different varieties are available, with their potent action as *Vata* hara and qualities such as, *snidha, madhura*, and *ushna. Dravya* is well known for its anti-parkinsonism effect (*Kampavata hara*) as it contains dopamine, and its seeds are rich in protein (Kerala or Tamilnadu germplasm), hence, it is utilized internally as a taila, which tones the muscle and acts as a nervine tonic, which is the most important requirement in *Apabahuka*. *Bala (Sida cordifolia* Linn) : It is generally considered as a nervine stimulant or nervine tonic. A better term can be given as a nervine stimulant. The term *'Bala'* is applied because of its *balya* property of *moola*. In the *Laghu masha taila*, this serves the purpose of generating sufficient energy to the muscle tissue, and also by its effective supporting factors such as *madhura rasa* and *madhura vipaka* as a *Vata hara*. *Shatavari (Asparagus racemosus* Willd): This fasciculated tuberous root is utilized in different ailments, as it has the *Vata hara* property. The absorption level of this drug through the *taila* during *nasya karma* is found to be excellent. *Sita* Serves the function of enhancing the energy of the other *dravyas* and nourishes the *mastishka*. *Purnarnava (Boerhavia diffusa* Linn) : *'Dhatu punarnavatwam,'* a drug that brings new tissues in the body, helps in preventing the degeneration of tissues. In other words, it achieves the regeneration of *sapta dhatus* with its specific activities on muscle tissues. *Saindhava* During *nasya*, the *taila* used has a fixed oil base. Therefore, the ideology of formulating the taila for its easiest absorption in the procedure of *Nasya karma* has been balanced by the addition of *Saindhava lavana. Saindhava lavana* has the potent action of facilitating easy absorption of the *taila* through its effective properties. *Jingini (Odina* wodier): By its *madhura rasa* and *ushna virya* acts as *Vata hara*, but *katu vipaka* helps in the easy digestion of the taila through the *nasya karma*. *Sarshapa taila (Brassica comprestris* Linn): Acts as a base for the dravyas of the *Laghu masha taila*, and is helpful through its easy absorption activity due to the *teekshna, katu*, and *ushna* properties. *Masha kwatha (Phaseolus mungo* Linn): A potent *dhatu vardhana dravya*, is supportive as a *Vata hara* with its dominant *madhura rasa* and *ushnadi gunas*. By observing the above-mentioned ingredients and their actions, it is evident that *Kapikacchu, Bala, Shatavari*, and *Masha* are the main ingredients which give the *balya bruhmana* effects. *Sita* in a combination that acts as an energy enhancer by virtue of its *madhuratwa* (glucose). *Punarnava* with its *shotha hara* and *Rasayana karmas* rejuvenates the brain functions, and *Saindhava lavana* acts a *Kapha vilayana kari* and *sroto mukha vishodhana*. The *Katu taila* is the main base ingredient for the other drugs (oil soluble). *Katu taila* is a *yoga vahi* and carries all essential ingredients into the system by virtue of its *teekshna, sukshma*, and *ashukari guna*. Mode of action of the *Nasya karma* {#sec2-15} ----------------------------------- *Nasya karma* is one of the therapeutic procedures of the *pancha karma*, wherein the drug is administered through the nasal route. This is one of the pancha karma procedures that not only alleviates the vitiated *doshas*, but also causes complete eradication of the vitiated *dosha* and the disease. The same is applicable to the *nasya karma* also. The *Nasya karma* especially exerts its effects on the *urdhvajatrugata pradesha*. Acharya Vagbhata has stated that, the *'Nasa hi shirasodwaram,'* that is, the nose, is the easiest and closest opening for conveying the potency of medicines to the cranial cavity. He is the first person to narrate the mode of action of drugs by *Nasya karma*. The drugs administered will reach the *Shringataka marma* and spread through the opening of the *shiras* of the eyes, ears, throat, and so on, to the head. Acharya Sushruta opines that the *Shringhataka marma*, is a *sira marma*, situated at the site of the union of the *siras*, supplying to the nose, ear, eye, and tongue. Acharya Charaka, while explaining the indication for *nasya* in *siddhisthana*, has emphasized that the *nasya* drug usually acts through absorption by the *Shringataka marma*. After absorption of the drug, it acts on the diseases of *Skanda, Amsa*, and *Greeva* and the *doshas* are expelled from the *shira pradesha*. The absorption of the drugs is carried out in three media. They are; 1\. By general blood circulation, after absorption through the mucous membrane 2\. By direct pooling into the venous sinuses of the brain via the inferior ophthalmic veins 3\. By direct absorption into the cerebrospinal fluid Apart from the small emissary veins entering the cavernous sinuses of the brain, a pair of venous branches emerging from the alae nasi will drain into the facial vein. These ophthalmic veins on the other hand also drain into the cavernous sinuses of the meninges, and in addition, neither the facial vein nor the ophthalmic veins have any valves. Therefore, there are more chances of the blood draining from the facial vein into the cavernous sinus in the lowered head position. The nasal cavity directly opens into the frontal, maxillary, and sphenoidal air sinuses. The epithelial layer is also continuous throughout the length. The momentary retention of the drug in the nasopharynx and the suction, causes oozing of the drug material into the air sinuses. These sites have rich blood vessels entering the brain and meninges through the existing foramens in the bones. Therefore, there are better chances of drug transportation via this path. The *shringataka marma* has been explained by recent authors as the middle cephalic fossa of the skull consisting of paranasal sinuses, meningial vessels, and nerves. One can see the truth of the narration made by Vagbhata here --- the drug administered enters the paranasal sinuses,\[[@ref7]\] that is, the *Shringataka*, where the ophthalmic vein and the other veins spread out. The sphenoidal sinuses are in close relation with the intracranial structures. Thus, there may be a so far undetected root between the air sinuses and the cavernous sinuses, establishing the transudation of fluids as a whole. The mentioning of the *Shringataka* in this context seems to be more reasonable. As the procedure of *nasya* itself involves massaging and fomenting over maybe the *marmas* existing on the face and head, this also helps in the alleviation of *marmaksobha* and *Vatashamana*. The action of *nasya karma* depends upon the *dravya* used in it. Based on these, it is divided into *shodhana, shamana*, and *Brumhana*. In case of the *shamana nasya*, it alleviates the *dosha* and helps in reducing the *kshobha* of the *marma* and *indriya* caused by the vitiated *dosha. Brihmana nasya* provides nourishment to the *shiroindriya* and other organs and alleviates the vitiated *Vata*. Hence, it is useful in *Vatajanya* ailments. The following paragraph explains why Ayurveda has mentioned medicated sneha dravya (*siddha sneha*) in a majority of the *nasya karmas*. The nose is a highly vascular structure and its mucous membrane provides a good absorbing surface. Hence, *siddha sneha*, on their administration, spread along the nasal mucous membrane. An active principle along with *sneha* gets absorbed inside the olfactory and respiratory mucosa and from there is carried to different places. *Sneha* provides nourishment to the nasal structures and other organs of the head also. The networks of nasal blood and lymph vessels have many communications with those of the subdural and subarachnoid spaces. This fact is one of the important factors contributing to the extension of the mentioned drugs from the nose into the cranial cavity. The myelin sheath is the first covering of the nerve fiber. Neurolemma being the second. The myelin sheath is composed of lipid material. The blood--brain barrier is highly permeable for lipid substances and substances that are fat-soluble. Therefore, these substances can pass easily through the blood--brain barrier and can exert their actions. Certain lipids are used for providing energy to the nervous tissue. The lipid contents of the *'Laghumasha taila'* may pass through the blood-brain barrier easily due to their transport. Some of the active principles may reach certain levels in the nervous system where they can exert their *Vataghna* property. *Laghumasha taila* provides nourishment to the nervous system and helps in removing the irritation. It may act as an anti-inflammatory agent also. On its nasal administration, it reaches different *shirogata indriya* and causes *Vatashamana* and *Brumhana*. To conclude, *nasya karma* helps in *Apabahuka* by its *Vatashmana* and *Brumhana karma*. In other words, the *Laghumasha taila* acts as an anti-inflammatory nutritive and provides nourishment to the nerves. Conclusion {#sec1-7} ========== The following conclusions can be drawn from the observations of the present study: Strenuous physical work and direct injury are the predisposing factors in the manifestation of the disease *Apabahuka*Maximum incidence of this disease was seen in the age group of 30--40 years*\>Laghumasha taila* having a *Brimhana* effect, when used as *Marsha nasya*, brought out a moderate significant result in *Bahupraspandita hara* and a mild significant relief in *Shoola*The size of the sample was small to draw a generalized conclusion. Therefore, the therapy can be tried in a large sample for an appropriate duration, to observe its proper efficacy.

Belavić M, Sotošek Tokmadžić V, Brozović Krijan A, et al. *Ther Clin Risk Manag*. 2018;14:741--751. In this article, the author Josip Žunić did not meet the criteria for authorship and was erroneously included in the authors list. The authors wish to apologize for this error.

Introduction {#sec1-1} ============ Hyperhomocysteinemia is a risk factor for coronary atherosclerotic vascular disease, stroke, and venous thrombosis. It has been associated with Alzheimer\'s disease and vascular dementia.\[[@CIT1]--[@CIT4]\] Although its pathophysiological mechanisms are complex and not fully understood, much evidence suggests that hyperhomocysteinemia induces vascular and brain damage because of the highly reactive thiol group in homocysteine, which is readily oxidized, leading to the formation of homocysteine, homocysteine mixed disulfides and homocysteine thiolactone. During these oxidative processes, several reactive species are generated.\[[@CIT2]\] The methionine cycle is responsible for the formation of homocysteine in the body. Homocysteine is toxic sulfur containing intermediate amino acid, which results from intracellular conversion of methionine to cysteine, which can induce neuronal dysfunction and cell death. Homocysteine has now been implicated in increased oxidative stress, DNA damage, triggering of apoptosis and excitotoxicity, all important mechanisms in neurodegeneration.\[[@CIT5]\] The brain may be particularly vulnerable to high levels of homocysteine in the blood because it lacks two major metabolic pathway for its elimination - betain remethylation and transsulfuration.\[[@CIT6]\] Homocysteine is proposed to cause oxidative stress related neurotoxicity\[[@CIT7]\] and natural antioxidants play a protective role in hyperhomocysteinemia;\[[@CIT8]\] however, there is a paucity of experimental as well as clinical studies which indicates the protective role of medicinal plants in hyperhomocysteinemia. *Embelia ribes* Burm, commonly known as 'Vidanga', is a large woody climbing shrub that belongs to the family, *Myrsinaceae*, which is widely distributed in India, Sri Lanka, Malaysia and South China.\[[@CIT9]\] It is highly valued in Ayurveda as a powerful anthelmintic.\[[@CIT10]\] In a preliminary study, Tripathi\[[@CIT11]\] has demonstrated the blood glucose lowering activity of the decoction of the *Embelia ribes* fruits in glucose-fed albino rabbits. Bhandari *et al.*\[[@CIT12][@CIT13]\] have reported the diabetic dyslipidemic and antioxidant activity of *Embelia ribes* Burm in streptozotocin-induced diabetic rats, using gliclazide as a positive control drug. Recently, Bhandari *et al.*\[[@CIT14]\] have reported the cardioprotective activity of the aqueous extract of *Embelia ribes,* in isoproterenol induced myocardial infarction in albino rats. However, till date, antihyperhomocysteinemic activity has not been carried out on the aqueous extract of *Embelia ribes*. Therefore, the present study was undertaken to determine the antihyperhomocysteinemic and antihyperlipidemic activity of the aqueous extract of *Embelia ribes* in albino rats. Materials and Methods {#sec1-2} ===================== Drugs and chemicals {#sec2-1} ------------------- Methionine and folic acid were obtained from Central Drug House, Bombay. All the other chemicals that were used were of analytical grade. Double distilled water was used for all biochemical assays. Preparation of aqueous extract of Embelia ribes Burm {#sec2-2} ---------------------------------------------------- Dried fruits of *Embelia ribes* Burm were purchased from the local market, New Delhi, India, in October 2006 and the botanical authentification was carried out by the Department of Botany, Faculty of Science, Hamdard University, New Delhi, India (voucher specimen no. UB 2). The dried and coarsely powdered drug (100 g) was packed in a soxhlet apparatus and was subjected to extraction with water for 72 hours. The filtrate was evaporated under a vacuum drier (Narang Scientific Works Pvt. Ltd., New Delhi, India) and the brown mass residue that was obtained was stored at 4°C, for further use. The average yield of the aqueous extract of *Embelia ribes* was approximately 5.26%. For experimental study, the weighed amount of aqueous extract of *Embelia ribes* (100 and 200 mg/kg) was dissolved in 1% Tween 80 in distilled water. Standardization of extract {#sec2-3} -------------------------- Preliminary phytochemical screening of the aqueous extract of dried fruits of *Embelia ribes* was carried out for the detection of phytoconstituents, using standard chemical tests. Alkaloids, carbohydrates, phenolic compounds, flavonoids, proteins and saponins were detected in the extract. HPTLC fingerprints of the aqueous extract was established using CAMAG HPTLC (WinCAT software, version 2.2) and benzene: ethyl acetate (6:4) as the solvent system, which showed the presence of seven spots (R~f~ values: 0.32, 0.34, 0.42, 0.45, 0.52, 0.65 and 0.78) at 520 nm wavelength. Animals {#sec2-4} ------- Male adult Wistar albino rats, weighing 200-250 g, procured from the Central Animal House Facility, Hamdard University, New Delhi, were used. The Institutional Animal Ethics Committee (IAEC) approved the present research. Animals were acclimatized under standard laboratory conditions at 25° ± 2°C, 50 ± 15% RH and normal photoperiod (12 h light : dark cycle) for seven days. The animals were fed with commercial rat pellet diet and water *ad libitum*. Methionine-induced hyperhomocysteinemia in rats {#sec2-5} ----------------------------------------------- The animals were randomly divided into seven groups of ten animals each and treated as follows: Group I served as vehicle control and received only 1% tween 80 in distilled water (2 ml/kg, p.o.) for 30 days. Group II served as pathogenic control and received methionine (1 g/kg, p.o.)\[[@CIT15]\] for 30 days. Groups III and IV were administered aqueous extract of *Embelia ribes* at doses of 100 and 200 mg/kg respectively and co-treated with methionine (1 g/kg, p.o.) for 30 days. Group V served as positive control and received folic acid (100 mg/kg, p.o.)\[[@CIT16]\] and co-treated with methionine (1 g/kg, p.o.) for 30 days. Groups VI and VII were administered aqueous extract of *Embelia ribes* alone at doses of 100 and 200 mg/kg respectively, for 30 days. The blood samples were collected by the retro-orbital plexus using micro-capillary technique from all the groups of rats that were made to fast overnight. Serum was separated for biochemical estimations. In serum, homocysteine levels were estimated using the Fluorescence Polarization Immunoassay (FPIA) method described by Primus *et al.* (1988). UV spectrophotometric method of analysis was used for the estimation of LDH (Lum and Gambino, 1974), total cholesterol (Demacher and Hijamaus, 1980), triglycerides (Foster and Dunn, 1973) and HDL-C (Burstein *et al.*, 1970). Commercial diagnostic kits from SPAN Diagnostics, Udhna, Surat, India were used for cholesterol and triglycerides estimation. Lactate dehydrogenase (LDH) catalyses the oxidation of lactate to pyruvate, accompanied by a simultaneous reduction of NAD to NADH. Lactate dehydrogenase (LDH) activity in serum is proportional to the increase in absorbance, due to the reduction of NAD. Lactate dehydrogenase (LDH) and HDL-C levels were estimated using commercial diagnostic kits from Reckon Diagnostics Pvt. Ltd. Baroda, India. LDL-C and VLDL-C levels were calculated from the formula of Friedward *et al.* (1972) as given below: The animals were sacrificed by cervical dislocation and brain tissues were dissected for biochemical estimation and for histopathological studies. Homogenate (10%) of whole brain tissue in ice cold KCl (0.15 M) was used for the assay of the malondialdehyde, according to the method of Ohkawa *et al.* (1979). In phosphate buffer (0.1 M, pH 7.0), it was used for the assay of glutathione (GSH) content (Sedlak and Lindsay, 1968). Lipid peroxides (LPO) was measured by estimating thiobarbituric acid reactive substances (TBARS) i.e. malondialdehyde (MDA), and glutathione assay was based on the reaction with DTNB i.e. DTNB (5,5\'-dithiobis- (2-nitrobenzoic acid)) is reduced by -SH group to form one mole of 2-nitro-5-mercaptobenzoic acid (yellow color). Histopathological studies {#sec2-6} ------------------------- At the end of the experiment, whole brain tissues from all the groups were subjected to histopathological studies. The tissues were fixed in formalin (10%), routinely processed and embedded in paraffin wax. Paraffin sections (5µm thick) were cut on glass slides and stained with hematoxylin and eosin (H & E), after dewaxing, and examined under a light microscope. Statistical analysis {#sec2-7} -------------------- The results are expressed as mean±SEM. Statistical differences between means were determined by one-way analysis of variance ([anova]{.smallcaps}), followed by Dunnett t-test. Values of *P* \< 0.01 were considered significant. Results {#sec1-3} ======= Preliminary phytochemical screening of the aqueous extract of dried fruits of *Embelia ribes* was carried out and alkaloids, carbohydrates, phenolic compounds, flavonoids, proteins and saponins were detected in the extract. HPTLC fingerprints of aqueous extract showed the presence of seven spots (R~f~ values: 0.32, 0.34, 0.42, 0.45, 0.52, 0.65 and 0.78) at 520 nm wavelength. Significant (*P* \< 0.01) increase in the serum homocysteine and lactate dehydrogenase (LDH) levels were observed in pathogenic control (i.e. group II) rats, as compared to vehicle control rats (group I). Aqueous extract of *Embelia ribes* (group III and IV) significantly (*P* \< 0.01) lowered the methionine-induced elevations of serum homocysteine and LDH levels, as compared to pathogenic control rats (group II). The results were comparable to those of folic acid \[[Table 1](#T0001){ref-type="table"}\]. ###### Effect of aqueous extract of *Embelia ribes* on serum homocysteine and LDH levels in methionine-induced hyperhomocysteinemic rats *Group* *Homocysteine (µg/mL)* *LDH (IU/L)* ---------------------------------------------------------- ------------------------------------------------ ------------------------------------------------ Vehicle control 8.52 ± 0.19 28.30 ± 0.81 Methionine treated 22.65 ± 0.34[a](#T000Fn1){ref-type="table-fn"} 59.36 ± 0.69[a](#T000Fn1){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) + Methionine 16.08 ± 0.03[b](#T000Fn3){ref-type="table-fn"} 42.40 ± 0.42[b](#T000Fn3){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) + Methionine 16.08 ± 0.05[b](#T000Fn3){ref-type="table-fn"} 39.03 ± 0.44[b](#T000Fn3){ref-type="table-fn"} Folic Acid (100 mg/kg) + Methionine 14.72 ± 0.03[b](#T000Fn3){ref-type="table-fn"} 34.50 ± 0.36[b](#T000Fn3){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) 9.07 ± 1.00[c](#T000Fn2){ref-type="table-fn"} 29.84 ± 0.56[c](#T000Fn2){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) 9.07 ± 0.10[c](#T000Fn2){ref-type="table-fn"} 30.59 ± 1.10[c](#T000Fn2){ref-type="table-fn"} Values are mean ± SEM (n=10); *P*\<0.01 *P*\>0.05 as compared to vehicle control group *P*\<0.01 as compared to pathogenic control group Serum total cholesterol, triglycerides, LDL-C, VLDL-C levels were significantly (*P* \< 0.01) increased along with a significant (*P* \< 0.01) decrease in HDL-C levels in pathogenic control (i.e. group II) rats, as compared to vehicle control (i.e. group I) rats. With repeated administration of aqueous extract of *Embelia ribes* (group III and IV) and folic acid (group V) treatment, the above mentioned parameters were regained significantly (*P* \< 0.01), as compared to pathogenic control (i.e. group II) rats \[[Tables 2](#T0002){ref-type="table"} and [3](#T0003){ref-type="table"}\]. ###### Effect of aqueous extract of *Embelia ribes* on serum total cholesterol, triglycerides and HDL-C levels in methionine-induced hyperhomocysteinemic rats *Groups* *Total Cholesterol (mg/dl)* *Triglycerides (mg/dl)* *HDL-C (mg/dl)* ---------------------------------------------------------- ------------------------------------------------- ------------------------------------------------- ------------------------------------------------ Vehicle control 100.60 ± 0.87 87.15 ± 1.31 39.58 ± 0.57 Methionine treated 194.21 ± 1.65[a](#T000Fn4){ref-type="table-fn"} 179.52 ± 2.15[a](#T000Fn4){ref-type="table-fn"} 14.23 ± 0.44[a](#T000Fn4){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) + Methionine 167.66 ± 1.20[b](#T000Fn6){ref-type="table-fn"} 142.42 ± 1.52[b](#T000Fn6){ref-type="table-fn"} 21.16 ± 0.40[b](#T000Fn6){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) + Methionine 157.56 ± 1.79[b](#T000Fn6){ref-type="table-fn"} 130.63 ± 2.08[b](#T000Fn6){ref-type="table-fn"} 23.97 ± 0.51[b](#T000Fn6){ref-type="table-fn"} Folic Acid (100 mg/kg) + Methionine 147.57 ± 1.51[b](#T000Fn6){ref-type="table-fn"} 119.23 ± 1.72[b](#T000Fn6){ref-type="table-fn"} 30.46 ± 1.10[b](#T000Fn6){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) 99.57 ± 0.60[c](#T000Fn5){ref-type="table-fn"} 86.96 ± 0.68[c](#T000Fn5){ref-type="table-fn"} 39.74 ± 0.19[c](#T000Fn5){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) 97.87 ± 1.20[c](#T000Fn5){ref-type="table-fn"} 85.02 ± 1.37[c](#T000Fn5){ref-type="table-fn"} 39.15 ± 0.37[c](#T000Fn5){ref-type="table-fn"} Values are mean ± SEM (n=10); *P*\<0.01 *P*\>0.05 as compared to vehicle control group *P*\<0.01 as compared to pathogenic control group ###### Effect of aqueous extract of *Embelia ribes* on serum LDL-C, VLDL-C levels and atherosclerotic index in methionine-induced hyperhomocysteinemic rats *Groups* *LDL-C (mg/dl)* *VLDL-C (mg/dl)* *Atherosclerotic Index (cholesterol/HDL-C)* *Atherosclerotic Index (LDL-C/HDL-C)* ---------------------------------------------------------- ----------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------- Vehicle control 43.59±0.56 17.43±0.26 2.54±0.02 1.10±0.02 Methionine treated 144.08±1.71[a](#T000Fn7){ref-type="table-fn"} 35.90±0.43[a](#T000Fn7){ref-type="table-fn"} 13.69±0.41[a](#T000Fn7){ref-type="table-fn"} 10.16±0.33[a](#T000Fn7){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) + Methionine 118.01±0.68[b](#T000Fn9){ref-type="table-fn"} 28.48±0.30[b](#T000Fn9){ref-type="table-fn"} 7.93±0.11[b](#T000Fn9){ref-type="table-fn"} 5.58±0.10[b](#T000Fn9){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) + Methionine 107.46±0.87[b](#T000Fn9){ref-type="table-fn"} 26.12±0.42[b](#T000Fn9){ref-type="table-fn"} 6.58±0.07[b](#T000Fn9){ref-type="table-fn"} 4.49±0.06[b](#T000Fn9){ref-type="table-fn"} Folic Acid (100 mg/kg) + Methionine 93.46±0.61[b](#T000Fn9){ref-type="table-fn"} 23.84±0.34[b](#T000Fn9){ref-type="table-fn"} 4.89±0.12[b](#T000Fn9){ref-type="table-fn"} 3.10±0.10[b](#T000Fn9){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) 42.45±0.28[c](#T000Fn8){ref-type="table-fn"} 17.39±0.14[c](#T000Fn8){ref-type="table-fn"} 2.50±0.01[c](#T000Fn8){ref-type="table-fn"} 1.07±0.01[c](#T000Fn9){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) 41.71±0.56[c](#T000Fn8){ref-type="table-fn"} 17.01±0.27[c](#T000Fn8){ref-type="table-fn"} 2.50±0.01[c](#T000Fn8){ref-type="table-fn"} 1.06±0.01[c](#T000Fn8){ref-type="table-fn"} Values are mean±SEM (n=10); *P*\<0.01 *P*\>0.05 as compared to vehicle control group *P*\<0.01 as compared to pathogenic control group Methionine treatment significantly (*P* \< 0.01) increased the LPO levels and decreased the GSH levels in brain homogenates in pathogenic control (i.e. group II) rats, as compared to vehicle control (i.e. group I) rats. Aqueous extract of *Embelia ribes* (group III and IV) and folic acid (group V) treatment in methionine-induced hyperhomocysteinemic rats significantly (*P* \< 0.01) decreased the LPO levels and increased the GSH levels in brain homogenates, as compared to pathogenic control (i.e. group II) rats \[[Table 4](#T0004){ref-type="table"}\]. ###### Effect of aqueous extract of *Embelia ribes* on LPO and GSH levels in brain in methionine-induced hyperhomocysteinemic rats *Groups* *LPO (nmol MDA/mg protein)* *GSH (µmole of phosphorous liberated/min/mg protein)* ---------------------------------------------------------- ---------------------------------------------- ------------------------------------------------------- Vehicle control 1.75±0.01 13.63±0.33 Methionine treated 6.87±0.12[a](#T000Fn10){ref-type="table-fn"} 8.93±0.47[a](#T000Fn10){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) + Methionine 4.40±0.09[b](#T000Fn12){ref-type="table-fn"} 10.70±0.31[b](#T000Fn12){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) + Methionine 3.71±0.06[b](#T000Fn12){ref-type="table-fn"} 11.02±0.17[b](#T000Fn12){ref-type="table-fn"} Folic Acid (100 mg/kg) + Methionine 3.21±0.04[b](#T000Fn12){ref-type="table-fn"} 12.84±0.14[b](#T000Fn12){ref-type="table-fn"} Aqueous *Embelia ribes* extract (100 mg/kg) 1.78±0.04[c](#T000Fn11){ref-type="table-fn"} 14.52±0.20[c](#T000Fn11){ref-type="table-fn"} Aqueous *Embelia ribes* extract (200 mg/kg) 1.86±0.03[c](#T000Fn11){ref-type="table-fn"} 14.61±0.54[c](#T000Fn11){ref-type="table-fn"} Values are mean±SEM (n=10); *P*\<0.01 *P*\>0.05 as compared to vehicle control group *P*\<0.01 as compared to pathogenic control group Photomicrograph of vehicle control group showed normal fibrillary background. The neuronal cell morphology and tissue architecture was maintained \[[Figure 1A](#F0001){ref-type="fig"}\]. Photomicrograph of pathogenic control group showed coagulative necrosis and vacuolar changes \[[Figure 1B](#F0001){ref-type="fig"}\]. Photomicrographs of aqueous extract of *Embelia ribes* (100 and 200 mg/kg) treated groups showed normal fibrillary background and absence of degenerative changes or necrosis. The neuronal cell morphology and tissue architecture was retained \[[Figures 1C and 1D](#F0001){ref-type="fig"}\]. Photomicrographs of folic acid (100 mg/kg) treated group showed normal fibrillary background, without any pathological symptoms \[[Figure 1E](#F0001){ref-type="fig"}\]. Photomicrographs of the aqueous extract of *Embelia ribes* (100 and 200 mg/kg) *per se* groups showed normal fibrillary background and neuronal cell morphology \[[Figures 1F and 1G](#F0001){ref-type="fig"}\]. {#F0001} Discussion {#sec1-4} ========== Hyperhomocysteinemia has recently emerged as an independent risk factor for the development of coronary, cerebrovascular and peripheral arterial occlusive disease.\[[@CIT17]\] Elevated homocysteine promotes atherosclerosis through increased oxidative stress, impaired endothelial function and induction of thrombosis. The role of oxidative stress in neurodegeneration has been intensively studied. Oxidative stress was one important mechanism for homocysteine toxicity in neuronal cells.\[[@CIT18]\] Antioxidant treatment restores several toxic effects of homocysteine.\[[@CIT19]\] Bhandari *et al.*\[[@CIT12][@CIT13]\] reported the diabetic dyslipidemic and antioxidant activity of *Embelia ribes* Burm in streptozotocin-induced diabetes in rats, using gliclazide as a positive control drug. In the present study, we examined the homocysteine and lipid lowering potential of the aqueous extract of *Embelia ribes* (100 and 200 mg/kg, p.o.) in methionine-induced hyperhomocysteinemia and hyperlipidemia in rats. Several potential mechanisms underlying the deleterious effect of homocysteine in the brain have been proposed, which include oxidative stress, alterations in DNA methylation and activation of the excitotoxic NMDA receptors.\[[@CIT5]\] Hyperhomocysteinemia may promote the generation of reactive oxygen species (ROS) such as H~2~O~2~ and hydroxyl radicals via the auto-oxidation of sulfhydryl (-SH) group\[[@CIT20]\] or by decreasing the intracellular levels of GSH that are involved in the elimination of free radicals. Homocysteine, a thiol containing amino acid derived from demethylation of dietary methionine, may generate partially reduced ROS that are able to stimulate the lipid peroxidation involved in the atherosclerotic process. Thus, an imbalance in dietary methionine may contribute to the development of atherosclerosis by increasing homocysteine levels.\[[@CIT21]\] The data in our present study showed that methionine (1 g/kg, p.o.) treatment for 30 days in pathogenic control group (Group II) rats significantly (*P* \< 0.01) elevated the levels of serum homocysteine, LDH, total cholesterol, triglycerides, LDL-C and VLDL-C and atherosclerotic index values. Further, there was a decrease in serum HDL-C levels, with decrease in GSH content and increase in LPO levels in brain homogenates. Atherogenic index indicates the deposition of foam cells or plaque or fatty infiltration or lipids in heart, coronaries, aorta, liver and kidneys. The higher the atherogenic index, the higher is the risk of the above organs for oxidative damage.\[[@CIT22]\] Free radicals generated by hyperhomocysteinemia, initiate lipid peroxidation of the membrane bound polyunsaturated fatty acids, leading to the impairment of the structural and functional integrity of the membrane.\[[@CIT23]\] This concurs with the present findings, wherein the levels of LPO were found to be significantly (*P* \< 0.01) increased in the animals subjected to methionine treatment. Due to this increased lipid peroxidation, GSH levels are lowered.\[[@CIT24]\] In the present study, elevated levels of homocysteine, LDH, total cholesterol, triglycerides, LDL-C and VLDL-C in serum and LPO in brain homogenates were reduced significantly (*P* \< 0.01) after treatment with aqueous extract of *Embelia ribes*. Further, there was a decrease in the atherogenic index values, suggesting antihyperhomocysteinemic and antihyperlipidemic potential of *Embelia ribes*. Further, the levels of HDL-C in serum and GSH in brain homogenates were increased significantly (*P* \< 0.01), thereby, enhancing the endogenous antioxidant levels. And also, the results of test drug were comparable to folic acid, a standard positive control. Biochemical assay of various parameters in serum and brain tissues of the animals revealed that aqueous extract of *Embelia ribes* in both the doses favorably modified various biochemical markers in methionine-induced hyperhomocysteinemic rats significantly (*P* \< 0.01), as compared to pathogenic hyperhomocysteinemic rats. Chemically, *Embelia ribes* is reported to contain embelin, quercitol (polyphenol), tannins and alkaloids,\[[@CIT25]\] which may contribute to its antioxidant activity. In the present study, on standardization of the aqueous extract of *Embelia ribes,* it was found to contain alkaloids, carbohydrates, flavonoids, phenolic compounds, proteins and saponins. It can thus be concluded that the antioxidant effect of the aqueous extract of *Embelia ribes* can be due to the content of alkaloids, flavonoids, phenolic compounds and saponins. Lastly, the light microscopic observations of the brain tissues of the aqueous extract of *Embelia ribes* treated animals exhibited near normal pattern, thereby further supporting its role as a promising neuroprotective agent. Conclusion {#sec1-5} ========== The present results provide clear evidence that aqueous extract of *Embelia ribes* treatment enhances the antioxidant defense against methionine-induced hyperhomocysteinemia, hyperlipidemia and oxidative stress in brain. On the basis of above findings, it can be assumed that aqueous extract of *Embelia ribes* could be a potential source for a novel discovery for central nervous system disorders. The research project was supported by the project grant to Dr. Uma Bhandari by All India Council of Technical Education, New Delhi, India.

1. Introduction {#sec1-sensors-18-02955} =============== One of the most important and promising applications for structural health monitoring (SHM) systems is the aeronautics industry. Global aviation is growing rapidly promising even increased prospects for growth in the future. As discussed in \[[@B1-sensors-18-02955]\] the world commercial aircraft fleet increased on average by 1.8% during 2010 reaching more than 25,000 new aircraft in operation. In addition, there are many old aircrafts that are still operating in the global air space \[[@B2-sensors-18-02955],[@B3-sensors-18-02955]\]. It is important to mention that aged aircraft structures are predominantly made of aluminum and consequently monitoring the integrity of those metallic structures constitutes an increased demand for the aeronautics industry. Accordingly, new SHM methods may considerably reduce the maintenance cost and enable much more structural safety. Driven by new services in the area of structural analysis, SHM is developing in order to adapt academic work to practical SHM systems. Hence, NDE (Non-Destructive Evaluation) methods have extensively been proposed in recent years, as these methods allow the application of various types of structures and consequently the identification of various types of damage. In SHM, physical or mathematical models are very useful for dynamic structural analysis, however, most of the real applications present some type of nonlinearity originated from geometric features, type of material, or boundary conditions, which leads to complex and expensive models. Owing to that, many authors have avoided working on structural modeling making use of techniques which allow the damage identification directly from the structural responses obtained from sensors signals. On this subject, the Electromechanical Impedance (EMI) technique plays an important role within the NDE methods. The EMI technique uses low-cost, small, and lightweight piezoelectric (PZT-lead zirconate titanate) transducer glued onto the structure \[[@B4-sensors-18-02955]\]. This technique is well-known in the literature \[[@B5-sensors-18-02955],[@B6-sensors-18-02955],[@B7-sensors-18-02955],[@B8-sensors-18-02955],[@B9-sensors-18-02955],[@B10-sensors-18-02955],[@B11-sensors-18-02955],[@B12-sensors-18-02955]\]. In the past, different SHM methods, based on neural networks (NN), had been widely investigated in order to evaluate structural conditions. Many SHM applications were proposed focusing on the multilayer perceptron and backpropagation algorithms \[[@B13-sensors-18-02955],[@B14-sensors-18-02955],[@B15-sensors-18-02955]\]. Subsequently, new classes of NN such as probabilistic neural network (PNN) and fuzzy ARTMAP network (FAN) took place. These methods have been successfully shown on various structures. For example, methods based on PPN applied to damage identification in SHM were addressed in \[[@B16-sensors-18-02955],[@B17-sensors-18-02955],[@B18-sensors-18-02955]\]. In the same way, FAN methods were investigated in \[[@B19-sensors-18-02955],[@B20-sensors-18-02955],[@B21-sensors-18-02955],[@B22-sensors-18-02955],[@B23-sensors-18-02955],[@B24-sensors-18-02955],[@B25-sensors-18-02955],[@B26-sensors-18-02955],[@B27-sensors-18-02955]\]. More recently, the convolutional neural network (CNN) has exploded in popularity and real-world applications. The CNN simply provides a new class of NN which uses the concept of deep learning. CNN is one of the most recent major breakthroughs in the area of computer vision, speech recognition, biomedical systems, and natural language processing \[[@B28-sensors-18-02955],[@B29-sensors-18-02955]\]. Unlike an ordinary NN, the layers of CNN can arrange neurons in three dimensions: width, height, and depth. Accordingly, some CNN applications have successfully emerged in the SHM field focusing mostly on vibration analysis as summarized next. Many CNN applications in SHM systems have focused on vibration analysis for monitoring faults on rotating machinery. For example, a fast and accurate motor condition monitoring and early fault detection system using 1D-CNN was proposed in \[[@B30-sensors-18-02955]\]. Similar approaches were also addressed in \[[@B31-sensors-18-02955],[@B32-sensors-18-02955]\]. Likewise, in reference \[[@B33-sensors-18-02955]\] was proposed a method to address fault diagnosis based on CNN directly on raw vibration signals. The authors mentioned that the model works well in noisy environments and performs well when the working load changes. Similarly, a 1D-CNN vibration-based method was applied to damage detection and localization in real-time from the raw acceleration signals \[[@B34-sensors-18-02955]\]. The method was applied to large-scale test structures. In \[[@B35-sensors-18-02955]\], the authors proposed to incorporate sensor fusion by taking advantages of the CNN structure to achieve higher and more robust diagnosis accuracy. They analyzed both temporal and spatial information of the raw data from multiple sensors for the training process of the CNN. They pointed out that their method, compared with traditional approaches which use manual feature extraction, results in superior diagnosis performance. In \[[@B36-sensors-18-02955]\], the authors proposed a CNN-based approach (LiftingNet) to learn features adaptively from raw mechanical data without prior knowledge. The authors highlighted that the advantages in applications are the ability to classify mechanical data sampled under different rotating speeds and achieving high classification accuracy with considerable noise present. Although all those above approaches obtained good results, none of them focused on using PZT-EMI-based method to identify structural damage. In \[[@B37-sensors-18-02955]\], the authors proposed a wireless-sensor-networks-based method which takes advantage of an individual training 1D CNN for each wireless sensor in the network in a format where each CNN is assigned to process the locally-available data only, eliminating the need for data transmission and synchronization. That method operates directly on the raw ambient vibration condition signals without any filtering or preprocessing. In the same way, in reference \[[@B38-sensors-18-02955]\], the authors proposed an enhanced CNN-based approach that requires only two measurement sets regardless of the size of the structure in order to overcome the limitation of training CNNs which predominantly requires a significant amount of measurements especially if applied to large-scale systems. They pointed out that their method was able to successfully estimate the actual amount of damage for the nine damage scenarios of the benchmark study. Recently a single CNN application emerged in the SHM field focusing on video processing \[[@B39-sensors-18-02955]\]. Therein, the authors proposed to analyze individual video frames for inspection of crack in a nuclear power plant via CNN and Naive Bayes Data Fusion. They pointed out that their framework achieves 98.3 of hit rate. Despite the good results, they mentioned that one disadvantage is that CNN needs substantial training data (e.g., more than 100,000 samples) to make the training converge and prevent overfitting. Another disadvantage is that the computations of CNN rely heavily on GPU (graphics processing unit). Conclusively, the SHM literature shows clearly that there are no details for the combination of PZT-EMI-based method and CNN when applied to monitor structures, underscoring the novelty of the approach presented here Unlike existing studies, the major contribution of this work consists of a novel strategy for damage detection via the combination of the EMI-PZT-based technique and the CNN algorithm. The developed methodology was experimentally tested based on the EMI technique. The validation of the methodology was carried out in an aluminum plate which contains three attached PZT patches. The damage scenarios were simulated by gluing a small metallic nut at three different positions. The results, therefore, showed that it can identify various structural conditions with accuracy, reliability, and efficiency. In summary, the main contributions of this paper are:We developed a novel method suitable for mechanical data analysis. A method that takes advantage of the combination of the EMI-PZT-based method along with CNN.A way of converting PZT response based on the EMI technique to a RGB frame constitutes a novel approach;Frames were computed through a wide range of frequency instead of choosing only the best range in which the EMI presents higher sensitivity. This issue provides an important advantage because that task is very difficult;An unpublished frame dataset encompassing a total of four types of structural conditions for each PZT is introduced;An enhanced method which requires only a small dataset for training the CNN without using GPU. Furthermore, only three epochs are needed to yield 100% of hit rate. The remainder of the paper is organized as follows. Firstly, the main theoretical fundamentals are addressed. Secondly, the developed method, highlighting the combination of the EMI-PZT along and the CNN algorithm, is presented. Next, the results followed by a comparison with other SHM approaches are presented. Finally, the paper concludes by highlighting remarks on the developed approach. 2. Theoretical Fundamentals {#sec2-sensors-18-02955} =========================== 2.1. Structural Health Monitoring Systems {#sec2dot1-sensors-18-02955} ----------------------------------------- Structural health monitoring (SHM) systems have become a crucial element in maintenance and inspection activities in the industry, with special emphasis on aeronautical engineering, aerospace, civil, maritime and other related fields. Owing to the high level of safety required, the aeronautical industry has demanded high investments in order to guarantee an adequate operating condition in aircrafts. According to \[[@B40-sensors-18-02955]\], SHM systems could significantly reduce maintenance costs, as the damage could be detected in early stage, accounting for 27% of the cost of its life cycle. In SHM, the damage is characterized by changes in the dynamic response of the structure due to variations in stiffness, mass, energy dissipation, mechanical impedance and/or geometric properties of the structure \[[@B41-sensors-18-02955]\]. Hence, the concentration of various damages in a structure can lead to failures compromising the operation of the entire system. In general, the term "integrity" is the condition of the structure that allows its proper operation with satisfactory performance. In this context, structural integrity is the borderline condition between safety and failure of structural components \[[@B42-sensors-18-02955]\]. SHM systems are characterized by their ability to detect, locate, quantify, and estimate the life of the structure according to the occurred damage \[[@B43-sensors-18-02955]\]. However, according to \[[@B7-sensors-18-02955]\] when incorporating smart materials (PZT, magnetostrictive strain, shape memory alloys, etc.) into the detection system, three more levels should be considered: self-diagnosis of structural damage, structural self-repair, and a simultaneous system of control and monitoring. In SHM, NDE (nondestructive evaluation) methods have extensively been proposed in recent years, as these methods allow the application of various types of structures and consequently the identification of various types of damage. NDE methods have been applied based on different techniques such as: acoustic emission, Eddy current, radiography, thermography, shearography, Lamb waves, and electromechanical impedance \[[@B42-sensors-18-02955]\]. Wherein, the electromechanical impedance (EMI) technique plays an important role due to this technique makes use of a low-cost piezoelectric transducer (PZT) attached to the monitored structure \[[@B4-sensors-18-02955]\]. In this technique, several structural responses are collected to evaluate the structure considering its dynamic condition through a forced excitation via PZT patches. It is remarkable that the same PZT is also used as a sensor to collect structural responses for further processing. Considering the use of the EMI technique, piezoelectric materials play important roles due to these materials can be used as passive and/or active elements. These materials cover a large range of frequency (from a few Hz up to GHz). Low-frequency applications are covered mainly by the polycrystalline materials (ceramics, polymers or composites). In turn, crystals and thin films are the most used in high-frequency applications \[[@B44-sensors-18-02955]\]. PZT ceramics have the following advantages: good electromechanical coupling, good stability, high stiffness, linear response to low-cost electric field \[[@B45-sensors-18-02955]\]. Among the various types of piezoelectric materials, PZTs have shown very efficiently, being able to convert about 80% of the mechanical energy into electric energy \[[@B45-sensors-18-02955]\]. From a practical point of view of applying the EMI technique in SHM systems, the PZT transducers are glued into the monitored structure by high stiffness adhesive glue based on cyanoacrylate or an epoxy resin. From that, a coupling is established between the structure and the transducer PZT enabling to monitor variations of the mechanical impedance of the structure by measuring the electrical impedance of the PZT \[[@B4-sensors-18-02955]\]. Hence, exciting the PZT using a sinusoidal source V~X~ (with amplitude V~P~ and angular frequency (*ω*)) will produce a current I with amplitude I~P~ and phase Ψ. The electrical impedance of the PZT (Z~E~(*ω*)) is given as follows \[[@B4-sensors-18-02955]\]:$$Z_{E}\left( \omega \right) = \frac{Vx}{I} = \frac{1}{j\omega a}{({{\overline{\mathsf{\varepsilon}}}_{33}}^{T} - \frac{Z\left( \omega \right)}{Z\left( \omega \right) + Z_{a}\left( \omega \right)}d_{3x}^{2}{\hat{Y}}_{xx}^{E})}^{- 1}$$ where Z~a~(*ω*) and Z(*ω*) represent the mechanical impedances for the transducer and monitored structure, respectively. In Equation (1), ${{\overline{\mathsf{\varepsilon}}}_{33}}^{T},~{\hat{Y}}_{xx}^{E},~d_{3x}^{2}$, and j represent dielectric constant, Young's modulus, electric field constant, geometric constant and imaginary unit respectively. Note from Equation (1) that any variation in terms of the structural impedance will cause changes in the electrical impedance of the PZT patch and this, in turn, causes changes in the EMI signatures. Extra details of how PZT impedance is related to the structural condition via the EMI technique can be explored in the following references \[[@B4-sensors-18-02955],[@B7-sensors-18-02955],[@B46-sensors-18-02955],[@B47-sensors-18-02955],[@B48-sensors-18-02955],[@B49-sensors-18-02955]\]. 2.2. The Convolutional Neural Network {#sec2dot2-sensors-18-02955} ------------------------------------- The convolutional neural network (CNN) is a deep linear network inspired by the functioning of the visual cortex of mammals. Its first version was proposed by \[[@B50-sensors-18-02955]\] and was conceived inspired by the work of \[[@B51-sensors-18-02955]\]. Posteriorly, authors proposed an enhanced CNN architecture by incorporating processes of supervised learning through the backpropagation method \[[@B52-sensors-18-02955]\]. In reference \[[@B53-sensors-18-02955]\] was proposed the LeNet network, which can be considered the first architecture to present all features of the current CNN. Following Google's involvement in the competition promoted by ImageNet, the largest database of image classification, CNN has become the state-of-the-art for image classification \[[@B54-sensors-18-02955]\]. This made CNN popularity increase and, consequently, the amount of published work grew up proportionally. The main trend in the modeling of CNN is towards the use of ever deeper networks \[[@B54-sensors-18-02955]\]. The fundamental difference between an "ordinary" neural network and a CNN consists of the fact that CNN uses the convolution operation instead of the multiplication of the array of neurons in at least one of its layers \[[@B55-sensors-18-02955]\]. In the image processing, where the image is a two-dimensional matrix, the convolution operation is very useful for edge detection, image smoothing, attribute extraction, among other features. As a consequence, the convolution operation reduces the size of the original image due to the difference in the filter size. However, this reduction can be overcome by using the well-known zero padding technique. There are three important distinctive features on CNN compared to other Neural Network (NN): shared weights, spatial/temporal subsampling, and local receptor fields \[[@B53-sensors-18-02955]\]. The shared weight enables the network to learn only a smaller set of filters that can be applied to all the regions of the image, instead of learning specific weights for each region of the image, increasing the power of generalization of the network \[[@B56-sensors-18-02955]\]. The subsampling procedure in the CNN is usually conceived in the pooling layer (downsampling). This concept was first introduced by \[[@B52-sensors-18-02955]\]. For that, the max pooling computation is done for an image region followed by creating an array of these maximums. Thus, it eliminates non-maximum values, reducing both the size of the data representation and the computation required for the next layers \[[@B55-sensors-18-02955]\]. The third distinctive feature is the existence of local receptor fields. In the classical NN, each input value of each layer is completely connected to the input values of the previous layer (fully connected). Hence, the NN needs to perform several multiplications to find the connected neuron activation, requiring a great computational power mainly for images that have many connected neurons. On contrary, as in natural images, the adjacent pixels tend to be more strongly correlated than the distant pixels, the CNN is architected for that each filter learns on only one subregion of the data received from the previous layer \[[@B56-sensors-18-02955]\]. This allows increasingly complex patterns to be modeled from combinations of simple local operations \[[@B55-sensors-18-02955]\]. In addition to these important properties, other computational resources are used to avoid overfitting and training time of CNN. For example, the dropout consists of randomly removing half of the neurons from the hidden layers at each iteration of the training procedure. This technique also gives the network the ability to learn more robust parameters, since a neuron cannot depend on the specific presence of other neurons. In summary, CNN networks are composed of convolution layers, which involve the convolution process and the pooling process, in addition to using the concept of local receiver fields to optimize the image processing; layer normalization, which involves the dropout process and other processes used to improve network performance; and the fully connected layer responsible for sorting. [Figure 1](#sensors-18-02955-f001){ref-type="fig"} shows a general architecture for the CNN. The first part of the network consists of the convolution (C1, C2, etc.) and subsampling (S1, S2, etc.) layers. Basically, these layers are responsible for extracting the network features. The second part of the network consists of the normalization and fully connected layers. This block is used as images classifier after the image has passed through the feature extraction block. The data entries of each hidden layer form a set of feature maps obtained by processing the data in the previous layer. The feature maps do not require the preprocessing of the image, which is a process that usually requires higher computational power, playing a fundamental role in the advantage of the use of this type of network in image processing. Extra details about CNN and deep learning are shown in previous studies learning \[[@B50-sensors-18-02955],[@B51-sensors-18-02955],[@B52-sensors-18-02955],[@B53-sensors-18-02955],[@B54-sensors-18-02955],[@B55-sensors-18-02955],[@B56-sensors-18-02955],[@B57-sensors-18-02955],[@B58-sensors-18-02955]\]. 3. Developed Method {#sec3-sensors-18-02955} =================== The [Figure 2](#sensors-18-02955-f002){ref-type="fig"} shows the developed framework for the methodology based on the EMI-CNN applied to identify structural damage. The methodology consists of three phases as described in the following subsections. In phase 1, impedance signals are obtained based on the EMI principle. For this, three PZTs (called PZT\#1, PZT\#2 and PZT\#3) considering four different structural conditions (Healthy (H), Damage 1 (D1), Damage 2 (D2) and Damage 3 (D3) were considered. Further details about the experimental set up are presented in the next subsection. In phase 2, Euclidean distances (ED) were computed from the structural response signals in order to form frames. Those frames were used to form a dataset for both the training and test phases. In phase 3, the dataset was used as inputs for the CNN. Each CNN is responsible for recognizing four different structural conditions: H, D1, D2, and D3. 3.1. Phase 1: Acquisition of the EMI Signals {#sec3dot1-sensors-18-02955} -------------------------------------------- In order to obtain the structural response signals, we developed a method based on the EMI technique. EMI requires that the structure is excited through a PZT at low amplitude considering over a wide frequency range to produce a forced excitation of the structure \[[@B4-sensors-18-02955]\]. Each PZT acts as actuator and sensor at the same time. In our example, an aluminum plate of size 400 mm × 250 mm × 5 mm was suspended in both tips using fishing lines in order to simulate free-free boundary conditions. Three piezoelectric diaphragms (called PZT\#1, PZT\#2 and PZT\#3) with diameters of 12 mm were used, that had active elements of type P-7 PZT ceramics (Murata Electronics). These diaphragms were bonded (using 3M Scotch-Weld Epoxy Adhesives DP460 Off-White) to the plate at three different positions ([Figure 3](#sensors-18-02955-f003){ref-type="fig"}). Subsequently, a chirp signal sweeping from 20 kHz to 110 kHz with amplitude of 3 V was used to excite the set PZT/structure. Although many authors consider that the real part of EMI in a frequency range from 20 kHz up to 40 kHz constitutes the best set in terms of damage sensibility (for example \[[@B6-sensors-18-02955],[@B7-sensors-18-02955]\]), the frequency band of the EMI signature for higher sensitivity and repeatability depends on several features, such as geometry, mass, boundary conditions and other structural features \[[@B5-sensors-18-02955]\]. Also, studies show that the structure suffers less interference of global conditions in higher frequencies vibration modes \[[@B10-sensors-18-02955]\], which justify the chosen frequency range. Another important remark regarding the excitation signal is that its variation in terms of amplitude does not affect the EMI-signatures \[[@B59-sensors-18-02955]\]. The acquisition system (DAQ) was developed in LabVIEW software and used here to excite and obtain the structure responses \[[@B8-sensors-18-02955]\]. This system is pictured in [Figure 4](#sensors-18-02955-f004){ref-type="fig"}. The resistor R was set to 1 kΩ, in order to limit the electric current through the PZT patch. Using that system, a set of measurements for the pristine structural condition was performed. These measures were stored to form the Baseline (B) set. Each PZT response signal was separately sampled at a rate of 1 MS/s. At a different time, a new set of measurements, considering the same structural condition, was carried out to form a new data set for the Healthy (H) condition. Next, three damage cases were separately simulated by gluing (using 3M Scotch-Weld Epoxy Adhesives DP460 Off-White) a metallic nut of about 10 g (diameter of 12 mm and height of 7 mm) at three different positions in the structure ([Figure 2](#sensors-18-02955-f002){ref-type="fig"} and [Figure 3](#sensors-18-02955-f003){ref-type="fig"}), being only one damage per time (named D1, D2 and D3). Hence considering D1, the PZT\#1 was separately excited and its own response is obtained individually, as proposed in references \[[@B4-sensors-18-02955],[@B5-sensors-18-02955],[@B6-sensors-18-02955],[@B7-sensors-18-02955],[@B8-sensors-18-02955]\]. Afterward, the same procedure is applied to PZT\#2 followed by PZT\#3. From this approach, the response signals are obtained separately for each PZT patch, thereby allowing the proposed method to work on each response signal separately. Posteriorly, the nut was removed and bonded at the position D2. The response signals for PZT\#1, PZT\#2 and PZT\#3 were separately obtained. Finally, the same procedure was carried out for D3. In a total, there were 1080 EMI signatures (60 for each structural condition). The time interval between two consecutive samples was 30 s. The environmental temperature of the room was kept constant to 22 °C throughout the experiment. The EMI signals were used to form RGB frames. 3.2. Phase 2: Formation of the Frames {#sec3dot2-sensors-18-02955} ------------------------------------- As stated earlier, the SHM literature shows clearly that there is not a combination of PZT-based methods and CNN due to the difficulty in obtaining images/videos from the PZT responses. As a consequence, there is a lack of using CNN along with the EMI technique applied to monitor structures. To overcome that, this paper introduces an innovative way of forming frames from PZT-EMI signatures, as explored in detail next. The RGB frame formation process is composed of eight steps, as follows ([Figure 5](#sensors-18-02955-f005){ref-type="fig"}):**Step 1:** The matrix containing the raw EMI data, sampled by the LabVIEW acquisition software, is read;**Step 2:** As the proposed method uses only the real part of the EMI, those samples are retrieved from the matrix into an array;**Step 3:** The EMI signatures (baseline and unknown conditions) are divided into equal parts (10 parts for each signal);**Step 4:** Those parts are used to compute Euclidian distances and generate a new array;**Step 5:** That new array is transformed into a square matrix;**Step 6:** Those obtained values (inside the array) are normalized by the maximum mean;**Step 7:** Using the *colormap function* (MATLAB), the normalized matrix is mapped to a colored matrix (RGB);**Step 8:** The generated image is then saved as a JPEG image. The image will be used as an input to the CNN preprocessing block (Figure 9). Next, the most important steps are further detailed from a practical point of view. Firstly, the real parts of the EMI are divided into several parts as illustrated in [Figure 6](#sensors-18-02955-f006){ref-type="fig"} (Step 3). For example, [Figure 6](#sensors-18-02955-f006){ref-type="fig"} shows two EMI signatures for the baseline (top) and unknown (bottom) conditions. Each signal was equally divided in three parts forming six parts in a total. Those parts were named as B1, B2 and B3 for the baseline signature and U1, U2 and U3 for the unknown condition. Using MATLAB^®^, Euclidean Distances (ED) were computed from the EMI parts, as follows (Step 4):$${Ed}\left( {B1,B1} \right) = \sqrt{\sum\limits_{j = 1}^{n}\left( {B1_{j} - B1_{j}} \right)^{2}}$$ $${Ed}\left( {B1,U1} \right) = \sqrt{\sum\limits_{j = 1}^{n}\left( {B1_{j} - U1_{j}} \right)^{2}}$$ where, B1 and U1 are the baseline and unknown structural conditions, respectively. This procedure was repeated among all parts in order to form an ED-matrix. Considering the example case, [Figure 7](#sensors-18-02955-f007){ref-type="fig"} sums up all possible combinations of the ED into an ED-matrix (Step 5). It is important to highlight that the principal diagonal of the ED-matrix is zero because the method computes EDs for the same part of the signals there. This matrix is formed for each PZT-EMI signature and this will be used to form a frame. In this paper, the baseline signature is always used in the first part of the ED-matrices. Each element of ED-matrix was transformed into a RGB (red, green, and blue) scale in order to form a RGB frame with three dimensions (width, height, and depth). This procedure was easily run in the developed MATLAB software (Step 7). [Figure 8](#sensors-18-02955-f008){ref-type="fig"} shows its correspondent RGB frame for the previous example ([Figure 7](#sensors-18-02955-f007){ref-type="fig"}). Each obtained frame has a width, height, and depth of 895, 656 and 3, respectively. As observed in [Figure 8](#sensors-18-02955-f008){ref-type="fig"}, the obtained frame presents regular symmetry over and under the principal diagonal. Once the structural condition varies, the frame colors will turn accordingly. As a consequence, each frame will be subtlety different for each structural condition and such differences will be perceived by the CNN algorithm. It is important to mention that during the frame assembly, we form a corresponding frame for each PZT-EMI signature along with its respective baseline signature. Furthermore, the developed methodology assembles frames through a wide frequency range instead of choosing only the best range in which the EMI technique presents higher sensitivity, as is the case in standard EMI approaches. This is an advantage because it eliminates the difficult task of searching for the most sensitive frequencies \[[@B60-sensors-18-02955]\]. From the assembled frames a frame dataset with 720 frames formed from the EMI-PZT signals, encompassing a total of 4 types of structural conditions for each PZT is formed. [Table 1](#sensors-18-02955-t001){ref-type="table"} shows how the dataset is distributed for PZTs \#1 and \#2. The distribution for PZT\#3 is similar. This dataset is used as input to feed the CNN algorithm. 3.3. Phase 3: CNN-Based Damage Detection Method {#sec3dot3-sensors-18-02955} ----------------------------------------------- As aforementioned, the CNN forms a new class of neural networks (NN) which uses the concept of deep learning \[[@B50-sensors-18-02955],[@B51-sensors-18-02955],[@B52-sensors-18-02955],[@B53-sensors-18-02955],[@B54-sensors-18-02955],[@B55-sensors-18-02955],[@B56-sensors-18-02955],[@B57-sensors-18-02955],[@B58-sensors-18-02955]\]. The CNN takes advantage of the fact that the input consists of images/videos and they constrain the architecture in a more sensible way. Unlike an ordinary NN, the layers of a CNN have neurons arranged in three dimensions: width, height, and depth. According to \[[@B57-sensors-18-02955]\], the CNN architecture was designed to ensure some degree of shift, scale, and distortion invariance. Further, each unit in a layer is organized in planes which all units share the same set of weights. The set of outputs of the unit in a given plane is called a feature map. Hence, a full convolutional layer is composed of several feature maps with different weight vectors. As a consequence, several features can be extracted at each location in the image \[[@B57-sensors-18-02955]\]. A sequential implementation of the feature maps consists in scanning the image with a single unit that has a local receptive field and stores the states of this unit at the corresponding position on the feature map. The kernel (filter) of the convolution process is used to connect weights used by the units into the feature maps \[[@B57-sensors-18-02955]\]. It is fair to say that the recent success of the CNN architecture can be largely attributed to the strong emphasis on modeling multiple levels of abstractions. In order to evaluate structural conditions, this approach proposes a framework for the CNN as shown in [Figure 9](#sensors-18-02955-f009){ref-type="fig"}. The method uses one CNN architecture like that for each PZT sensor. The CNN is fed with the obtained frames computed from the impedance signatures under various structural conditions ([Table 1](#sensors-18-02955-t001){ref-type="table"}). The preprocessing block is the first step to be considered. This block consists of two steps of image processing. The first step is to read and convert the RGB image to grayscale. Besides, the image is resized from 875 × 656 × 3 pixels to 128 × 128 × 1 pixels in order to reduce the processing time for the CNN. A second step towards finalizing the preprocessing block consists of converting the grayscale image into a feature vector by flattening the image to an array. This array contains all the pixels of the image and it is structured by adding the first row of pixels from the image to an empty array, then, the second row of pixels is added to the end of that array and so on, until the last row of the image. Posteriorly, the array type is changed to float to enable performing of standardization. This process is important because some machine learning algorithms may present low performance when there are large variations in the used data. Finally, the array is normalized with Gaussian distribution with zero-mean and unit-variance. A brief explanation of the most significant characteristics of the CNN architecture shown in [Figure 9](#sensors-18-02955-f009){ref-type="fig"} is stated next. Firstly, the grayscale image \[128 × 128 × 1\] was applied to the first Conv module. This module is composed of 32 filters (kernel size of 3 × 3), resulting in a volume such as \[126 × 126 × 1\]. It is important to mention that this Conv block applies the zero-padding algorithm aiming to avoid losses. Other Conv modules can be understood similarly except they do not take advantage of the zero-padding algorithm. Next, the Maxpooling size was set to 2 × 2 whilst the Drop Out was set to 0.5 in order to reduce the possibility of over-fitting and improve the generalization of the CNN. In order to optimize the weights of the training algorithm, this approach used the popular RMS (Root Mean Square) backpropagation algorithm wherein the weights were changed according to the gradient descent direction of an error. The Soft Max block outputted four structural conditions: H, D1, D2, and D3. 4. Experimental Results {#sec4-sensors-18-02955} ======================= In order to evaluate the developed methodology, this section presents the results obtained considering the experimental set up described above. Firstly, structural response signals were obtained from the various structural conditions and positions of damage on the structure, through PZTs using the above-mentioned acquisition system. Sample signals are presented in [Figure 10](#sensors-18-02955-f010){ref-type="fig"}. For brevity, only the real part of the impedance for PZT\#2 is shown. Signatures are shown for four different structural conditions: healthy (H), damage 1 (D1), damage 2 (D2) and damage 3 (D3). As observed, the damage insertion will cause changes in the electrical impedance of the PZT and this in turn causes changes in the EMI signatures. Those changes happen in both magnitude and frequency. For the majority of the cases, the structural change causes only subtle variations in the EMI signatures needing methods more precise that are able to identify such variations automatically. Secondly, the obtained structural response signals were divided, in ten parts for each signal, as shown in [Figure 6](#sensors-18-02955-f006){ref-type="fig"}. Next, Euclidean distances (ED) were computed from the split signals, as in Equations (2) and (3) and as following those values are put onto the ED-matrix ([Figure 7](#sensors-18-02955-f007){ref-type="fig"}). From the formed ED-matrix, each ED value is transformed to the RGB frame (see procedures presented in [Figure 5](#sensors-18-02955-f005){ref-type="fig"}). [Figure 11](#sensors-18-02955-f011){ref-type="fig"} illustrates a set of frames formed from the EMI signatures for PZT\#2. The frame showed in [Figure 11](#sensors-18-02955-f011){ref-type="fig"}a is formed by computing ED for signatures for the baseline with healthy (H) structural conditions. Similarly, [Figure 11](#sensors-18-02955-f011){ref-type="fig"}b--d present the formed frames considering the baseline with D1, baseline with D2 and baseline with D3, respectively. As observed in [Figure 11](#sensors-18-02955-f011){ref-type="fig"}, a substantial difference between the frames for healthy and damaged conditions is perceptible, mainly from the second diagonal. This diagonal is only presented for the healthy condition. There are also subtle differences, almost imperceptible by human eyes if we analyze the frames brightness. In contrast, taking into account only the frames for the damaged structural conditions, such differences are visually misperceived demanding a very precise algorithm to overcome that. In this sense, this approach applies the CNN algorithm to bring up those subtle differences in order to provide precise and reliable damage detection, as shown next. It is important to highlight that the obtained frames are used to form a dataset for training and testing procedures, which are used as input to the CNN algorithm ([Table 1](#sensors-18-02955-t001){ref-type="table"}). Thirdly, the CNN block is fed to the aforementioned dataset ([Table 1](#sensors-18-02955-t001){ref-type="table"}). Both training and testing phases were carried out on a Laptop running Windows 8. The Laptop has an Intel Core i5-3320 M with 8 GB of RAM. It is important to point out that this approach did not take advantage of any dedicated GPU. Keras along with Theano backend libraries were used to run the training and test. Those libraries were specially developed in Python for deep learning applications. The batch size was set to 8 and, after running 3 epochs the training procedure successfully converged. Three CNN blocks were designed, corresponding one for each PZT sensor. [Figure 12](#sensors-18-02955-f012){ref-type="fig"} shows the feature maps for the 1st CNN layer after applying 32 kernels onto a correspondent frame for PZT\#2, considering D1 and H structural conditions. Analyzing [Figure 12](#sensors-18-02955-f012){ref-type="fig"}, we can realize that there are substantial differences among the feature maps for D1 and H. Such differences are primordial to guarantee the suitability of the developed methodology. In order to extract the most relevant features of the frame, each frame is passed by several blocks: Conv, Maxpooling, ReLu, Drop Out and so on ([Figure 9](#sensors-18-02955-f009){ref-type="fig"}). [Figure 13](#sensors-18-02955-f013){ref-type="fig"} depicts the output (feature maps) for the third Conv block (7th CNN layer) in the framework ([Figure 9](#sensors-18-02955-f009){ref-type="fig"}), after applying 64 kernels onto PZT\#2 frames considering H, D1, D2 and D3 structural conditions. Investigating the results presented in [Figure 13](#sensors-18-02955-f013){ref-type="fig"} it is possible to see how PZT\#2 perceives each structural condition. Further, it is clear that each frame presents outstanding distinctive features for each structural condition compared with the results presented in [Figure 11](#sensors-18-02955-f011){ref-type="fig"}, therefore, making this methodology very promising in SHM. Considering that one CNN is designed for each PZT sensor and the training and testing phases have been carried out, the CNN successfully converged after running three epochs. [Table 2](#sensors-18-02955-t002){ref-type="table"} shows separately the results for each PZT. The results show that the method was effectively able to identify various structural conditions with 100% accuracy. It is important to highlight that this result was obtained using only a small dataset for training the CNN ([Table 1](#sensors-18-02955-t001){ref-type="table"}) without using any type of GPU. This may provide an excellent and reliable solution for industrial applications where the availability of structural response signals to form the training set is generally scarce. As stated earlier, the developed method successfully converged after running 3 epochs. This issue is further investigated in [Figure 14](#sensors-18-02955-f014){ref-type="fig"}. For that, the method was evaluated varying the number of epochs from 1 to 60 and, the accuracy and loss rates (for PZT\#2), for both training and validation phases, were computed and presented in [Figure 14](#sensors-18-02955-f014){ref-type="fig"}a,b, respectively. From the results, we can see that during the validation phase the accuracy rate was always constant and equal to 100%. On the other hand, during the training procedure, this rate shows significant variations. However, there is a small plateau for the third epoch. A similar analysis can be done for the loss rate showed in [Figure 14](#sensors-18-02955-f014){ref-type="fig"}b. The method results in a loss rate of zero for three epochs. Based on these results, we henceforth set the number of epochs to three. It is fair to mention that the number of epoch has a straight relation to the training times as shown in [Figure 14](#sensors-18-02955-f014){ref-type="fig"}c. Analyzing [Figure 14](#sensors-18-02955-f014){ref-type="fig"}c we can realize that when using three epochs, the training time is about 120 s. 5. Comparison with Other State-of-the-Art Solutions {#sec5-sensors-18-02955} =================================================== In order to evaluate the performance of the developed method, [Table 3](#sensors-18-02955-t003){ref-type="table"} shows the success rates for testing phase as a comparison of different methods, running in the same conditions. Methods based on probabilistic neural network (PNN) \[[@B24-sensors-18-02955]\], simplified fuzzy ARTMAP network (SFAN) \[[@B22-sensors-18-02955],[@B24-sensors-18-02955]\], Savitzky--Golay (SG), Savitzky--Golay with first derivative (SGFD) and Savitzky--Golay with second derivative (SGSD) were considered \[[@B27-sensors-18-02955]\]. The SFAN, SG, SGFD and SGSD methods used setup parameters as follows: ρ = 0.78, α = 0.25 and β = 1 \[[@B24-sensors-18-02955],[@B27-sensors-18-02955]\]. For the PNN, the spread constant (σ) was set to 0.1 \[[@B24-sensors-18-02955],[@B27-sensors-18-02955]\]. Analyzing [Table 3](#sensors-18-02955-t003){ref-type="table"}, we can realize that the method enhanced the success rates for all PZTs sensor. For example, PZT\#1 yielded an improvement of 17% and 6% compared with the SFAN-SGSD and SFAN-SGFD methods, respectively. Therefore, the enhancement of this approach over existing approaches is undoubted. Training and testing times are stated next. As aforementioned, the training time is directly related to the number of epochs. Hence, [Table 4](#sensors-18-02955-t004){ref-type="table"} shows a time consumption comparison for three different methods, considering the results for PZT\#1. All methods were run, under the same conditions, on a laptop (stated earlier). As observed in [Table 4](#sensors-18-02955-t004){ref-type="table"}, the SFAN-based method showed the best performance in terms of both training and testing times. The PNN method obtained the second place with a subtle difference in relation to SFAN. The CNN-based method results in a longer time for both training and test. This is because the processing images consist of a time-consuming task as recurrently shown in the literature. Further analysis about time consumption will be stated in the next subsection. Advantages and Drawbacks ------------------------ The feasibility of the above approach is validated based on EMI-measurement datasets. The method results in an accuracy rate of 100% for all tested scenarios. Therefore, the main advantages of the developed method can be summarized into four points. Firstly, a new way of converting PZT response to RGB frames along with the CNN-based method represents a new approach to structural health monitoring. Based on the results, the method has direct implications in terms of diminishing the percentage of false alarms whilst the damage detection is being performed. Secondly, the major achievement in applying the method is the ability to classify structural damage with higher accuracy compared with the state-of-the-art approaches \[[@B22-sensors-18-02955],[@B24-sensors-18-02955],[@B27-sensors-18-02955]\]. This is possible because the CNN applies several banks of filters in order to extract the best features that represent different structural conditions, in each frame. It potentially has direct application in the composite materials industry especially when applied to identify small damage and its progression as discussed in \[[@B22-sensors-18-02955],[@B24-sensors-18-02955],[@B61-sensors-18-02955]\]. Thirdly, it is important to quote that this method, proves to be more reliable to detect both internal and non-visual damage compared with a method based on only video/image processing \[[@B39-sensors-18-02955]\]. Furthermore, the method presents another important advantage compared with \[[@B39-sensors-18-02955]\] because it does not require a GPU and can be run in an ordinary laptop, a direct consequence of the small dataset used to train the CNN. Fourthly, the developed method forms frames through a wide range of frequency instead of choosing only the best range in which the EMI presents higher sensitivity. This issue comprises an important advantage because that task is very difficult as pointed out in \[[@B62-sensors-18-02955]\]. Despite the advantages, improvements of the developed method still need to be investigated. The major problem with the current approach is the time consumption issue. As presented in [Table 4](#sensors-18-02955-t004){ref-type="table"}, the CNN-based method used substantially more time compared with the methods addressed in \[[@B22-sensors-18-02955],[@B24-sensors-18-02955],[@B27-sensors-18-02955]\]. On the other hand, it is important to mention that if we essentially consider that the majority of the industrial applications run the training phase offline and, that the demanded test time is 7.93 s for processing 96 frames, thus it would take a meaningfully time of 83 ms for processing one single frame. This time seems to be impeding for real-time applications; however, in practice, the procedure of acquisition of the EMI signatures can be carried out in an even longer time frame (e.g., over minutes). Therefore, the method can be perfectly considered for many SHM applications running on real-time using an ordinary laptop. It is important to remark that SHM techniques based on PZT transducers are highly influenced by environmental conditions, noise \[[@B33-sensors-18-02955],[@B36-sensors-18-02955],[@B63-sensors-18-02955]\] and especially temperature, during structural damage detection procedure. There are several approaches to compensate temperature effects on PZT-based SHM systems and most of them make corrections in the EMI signatures \[[@B49-sensors-18-02955],[@B64-sensors-18-02955],[@B65-sensors-18-02955]\]. So, although the proposed method does not consider temperature variations, under such conditions it will only requires a preprocessing of EMI signatures before using them, which does not change the proposed method. To date, we can only guarantee the capabilities of the method for damage detection and size/type estimation in terms of a rather large size as defined here. Smaller damage sizes will form future investigations. However, the simulated damage represents an only negligible increment of structural mass when compared to the total mass of the structure, which is compatible with real damage. Furthermore, future research will be undertaken to evaluate the accuracy of the developed method for randomly initiated defects and to establish the outcomes from having two or more areas of damage at the same time. Another interesting point to be addressed is the evaluation of different type, position, and size of damage \[[@B66-sensors-18-02955],[@B67-sensors-18-02955],[@B68-sensors-18-02955]\]. 6. Conclusions and Future Work {#sec6-sensors-18-02955} ============================== This paper has introduced an exploration of the suitability of a CNN-based method applied to monitor structural damage in aluminum structures. Accordingly, we developed a method which takes advantage of the combination of the EMI-PZT-based method and CNN. This methodology presents a new approach for SHM. Additionally to the fact of that CNN-based method was developed here for the first time, this approach consists of a reliable and innovative way of converting PZT response based on the EMI technique to the RGB frame. Based on the results, the CNN-based method shows significant enhancement in terms of the overall success rate whilst the structural damage detection is carried out. As a result, a hit rate of 100% was obtained running only three epochs, which outperforms current approaches. Furthermore, the method runs only a small dataset for training the CNN without using any type of dedicated GPU. To conclude, the method identified the damage scenarios with higher accuracy, therefore, rendering this approach in a promising and useful contribution in the SHM area. Future work will focus on the evaluation of the sensitivity of the developed methodology to identify the progression of the structural damage in structures made of composite materials. Those materials present a higher damping coefficient compared with aluminum, demanding a more accurate method such as shown throughout the paper. Future goals, motivated by the outcomes presented in this paper, will focus on features other than the success rate for measuring the classifier efficiency such as the Kappa coefficient. Further research will be undertaken in evaluation the CNN configuration such as: image size, training dataset size, and the number of kernels to optimize the required time consumption. The authors are grateful to D.J. Inman of the University of Michigan (Aerospace Department) for valuable comments in which they improve the quality of the article. For M.A.d.O. and J.V.F. conceived and designed the experiments; A.V.M. performed the experiments; M.A.d.O. and A.V.M. wrote the paper. This work has been funded by the PROPES-IFMT (Grants 099-2017 and 069-2018) and CNPq (Grant 310726/2016-6). The authors declare no conflicts of interest. {#sensors-18-02955-f001} {#sensors-18-02955-f002} ![Representation of the general diagram for the acquisition system (dimensions in millimeters) \[[@B27-sensors-18-02955]\].](sensors-18-02955-g003){#sensors-18-02955-f003} ![Experimental set up including: aluminum plate containing three PZT patches, DAQ (Data Acquisition) and computer running the acquisition software \[[@B27-sensors-18-02955]\].](sensors-18-02955-g004){#sensors-18-02955-f004} {#sensors-18-02955-f005} {#sensors-18-02955-f006} {#sensors-18-02955-f007} {#sensors-18-02955-f008} {#sensors-18-02955-f009} {#sensors-18-02955-f010} {#sensors-18-02955-f011} {#sensors-18-02955-f012} {#sensors-18-02955-f013} {#sensors-18-02955-f014} sensors-18-02955-t001_Table 1 ###### Distribution of number of frames, formed from the PZT-EMI signals for PZTs \#1 and \#2, into the dataset. Structural Conditions PZT \#1 PZT \#2 ----------------------- --------- --------- ----- ---- Healthy (H) 36 24 36 24 Damage 1(D1) 36 24 36 24 Damage 2(D2) 36 24 36 24 Damage 3(D3) 36 24 36 24 Total 144 96 144 96 sensors-18-02955-t002_Table 2 ###### Results for the CNN method: training and testing phases. Sensors Training Accuracy Testing Accuracy --------- ------------------- ------------------ PZT \#1 98% 100% PZT \#2 100% 100% PZT \#3 100% 100% sensors-18-02955-t003_Table 3 ###### Comparison of the CNN-based method with other NN approaches: Success rates obtained for the testing phase. Methods PZT\#1 PZT\#2 PZT \#3 ------------------------------------------------------------- -------- --------- --------- SFAN-SGSD \[[@B27-sensors-18-02955]\] 83.33% 100.00% 98.95% SFAN-SGFD \[[@B27-sensors-18-02955]\] 94.79% 85.41% 88.54% SFAN-SG \[[@B27-sensors-18-02955]\] 83.33% 100.00% 98.95% SFAN-ED \[[@B22-sensors-18-02955],[@B24-sensors-18-02955]\] 61.41% 98.95% 77.08% PNN-SGSD \[[@B27-sensors-18-02955]\] 75.00% 100.00% 98.95% PNN-SGFD \[[@B27-sensors-18-02955]\] 50.00% 75.00% 85.41% PNN-SG \[[@B27-sensors-18-02955]\] 75.00% 100.00% 98.95% sensors-18-02955-t004_Table 4 ###### Comparison among consumption times for: CNN, PNN, and SFAN. Methods Training Time (s) Testing Time (s) ---------------------------------------------------------------------------------- ------------------- ------------------ CNN 121.10 7.9300 SFAN \[[@B22-sensors-18-02955],[@B24-sensors-18-02955],[@B27-sensors-18-02955]\] 0.1265 0.0079 PNN \[[@B24-sensors-18-02955]\] 1.6724 0.6742

Introduction ============ Even though red blood cell (RBC) transfusion is a common intervention in the critical care setting, there is a paucity of data regarding its impact on tissue metabolism. The aim of this study was to explore the effect of RBC transfusion on microdialysis-assessed interstitial fluid metabolic parameters in septic patients. Methods ======= We conducted an observational, clinical study in a 25-bed, medical-surgical ICU of a university hospital. We analyzed the effect of transfusion of either 1 or 2 RBC units on interstitial fluid metabolic activity by means of a microdialysis (MD) catheter inserted in the subcutaneous adipose tissue of the upper thigh. Samples were collected before (T0) and after (T1a and T1b; spaced out by 4 hours) transfusion. Lactate, pyruvate, glycerol and glucose concentrations were measured with a bedside analyzer and the lactate/pyruvate (LP) ratio was calculated automatically. Results ======= We enrolled 37 patients with severe sepsis/septic shock requiring RBC transfusion. After transfusion, the mean arterial pressure increased from 79 ± 9 to 82 ± 10 (T1a vs. T0: *P*\< 0.05) and 83 ± 10 mmHg (T1b vs. T0: *P*\< 0.001). Besides a nonstatistically significant drop in arterial partial oxygen pressure, we observed no change in arterial blood gases and vital signs. Overall, RBC transfusion did not alter any of the MD-assessed parameters (that is, lactate, pyruvate, glycerol and glucose) or blood lactate, but it decreased the tissue LP ratio from (T0) 18.80 (interquartile range (IQR), 14.85 to 27.45) to (T1a) 17.80 (IQR, 14.35 to 25.20) (*P*\< 0.05) and (T1b) 17.90 (IQR, 14.45 to 22.75) (*P*\< 0.001). The post-transfusion changes in LP ratio at T1a (*r*= -0.42; 95% CI, -0.66 to -0.098; *P*= 0.01) and T1b (*r*= -0.68; 95% CI, -0.82 to -0.44; *P*\< 0.001) were significantly correlated with the pre-transfusion LP ratio but not with baseline demographic characteristics, vital signs, severity scores, hemoglobin level and blood lactate. Finally, 39.0% of the transfused RBC units were leukoreduced and their median storage time was 16 days (IQR, 11 to 24). RBC storage time and leukocyte reduction had no influence on the tissue metabolic response to transfusion. Conclusion ========== Tissue oxygenation is improved by red blood cell transfusion in critically ill septic patients. Monitoring of the tissue LP ratio by microdialysis may represent a useful method for individual clinical management.

Introduction ============ Lambert--Eaton myasthenic syndrome (LEMS) is a rare but well-known paraneoplastic disorder characterized by muscle weakness and fatigability predominantly involving the proximal lower limbs. Recently, measurement of serum anti-P/Q-type voltage-gated calcium channel (VGCC) antibody level has been used, in addition to clinical manifestations and nerve conduction studies, to diagnose LEMS. Patients with LEMS have mostly small cell lung cancer (SCLC); other subtypes of lung cancer are extremely rare. We present a rare case of LEMS associated with pulmonary adenocarcinoma, chronic rheumatoid arthritis (RA) and Sjögren syndrome (Sjs) and provide findings of the resected specimen immunostained with P/Q-type VGCC. Case presentation ================= The patient was a 75-year-old Japanese man with progressive muscle weakness of his bilateral lower extremities and gait disturbance. He was referred to the Department of Neurology of our hospital for workup. His neurological examination showed muscle weakness and atrophy of his lower extremities, depressed deep tendon reflexes and autonomic dysfunction, that is, dry mouth and orthostatic hypotension. His results for manual muscle strength testing increased from 3 out of 5 to 4-- out of 5 for his bilateral lower extremities after a sustained 30-second contraction. His sensation was intact to light touch, and pinprick and Babinski's sign were down-going bilaterally. A provocative nerve conduction study on the ulnar nerve showed no reduction after repetitive stimulation and tended to increase at 15Hz of high-frequency stimulation (Figure [1](#F1){ref-type="fig"}). Serum P/Q-type VGCC antibody level was elevated to 31.8pmol/L (cut-off point \<20.0pmol/L \[[@B1]\]). Taken together with clinical findings, we made a diagnosis of LEMS. A chest abdominal computed tomography (CT) to screen for malignant lesions revealed an abnormal shadow in the right pulmonary lower lobe. He was then referred to our department (Thoracic Surgery in Respiratory Disease Center) for diagnosis and treatment of lung tumor. {#F1} The lung tumor with spiculation was located in Segment 9b, with a maximum diameter of 30mm (Figure [2](#F2){ref-type="fig"}). There was no lymphadenopathy or pleural effusion. A CT of the patient's chest showed a very bilateral emphysematous lung. No abnormality was seen in his abdomen by CT or in the brain by enhanced magnetic resonance imaging (MRI). A \[^18^F\]-fluorodeoxyglucose positron emission tomography showed increased uptake of fluorodeoxyglucose in the mass region and right hilar region (maximum standardized uptake values were 9.6 and 3.3, respectively). Although a bronchoscopic examination did not reveal malignancy, primary lung cancer was strongly suspected. {#F2} The patient had worked as a carpenter before the onset of muscle weakness. However, as his symptoms worsened, he required assistance in daily life (performance status 2). He was admitted to our ward by wheelchair. He had a history of chronic RA medicated by oral prednisolone therapy. He also complained of thirst for many months. A full blood examination and biochemical examination were almost within the normal range. Carcinoembryonic antigen was slightly elevated to 6.0ng/mL, whereas other tumor markers for lung cancer (squamous cell carcinoma-related antigen, sialylated Lewis X-i antigen, cytokeratin fragment, neuron-specific enolase and progastrin-releasing peptide) were within normal ranges. On screening for autoimmune disease and collagen disease, serum CH~50~ was slightly elevated to 46.7U/mL (normal range, 30 to 45), and rheumatoid factor was within the normal range (10.4IU/mL; normal range, 0 to 20). On screening for other collagen diseases, anti-nuclear antibody was positive at the titer of 1:640 and anti-SSA/Ro antibody was elevated to \>500U/mL (normal range, 0--100). At this time it was clear that the patient also had Sjs. He had a history of cigarette smoking (40 cigarettes per day for 40 years). The preoperative spirometry result was, %VC: 63.9% and FEV~1.0%~ 76.0%; and the blood gas analysis result was pCO~2~ 38.9mmHg and pO~2~ 83.1mmHg (room air). An operation was performed for a definite diagnosis and treatment of the tumor. An intraoperative frozen diagnosis obtained by tumor biopsy revealed lung adenocarcinoma, and he underwent right lower lobe lobectomy and lymph node dissection with video-assisted thoracoscopic surgery. The operation took 230 minutes, with a blood loss of 200mL. In the postoperative course, because of prolonged air leakage from the remaining right pulmonary lobes, tube drainage lasted for 9 days. Muscle weakness gradually improved on approximately postoperative day 5, and he could walk without assistance. Serum P/Q-type VGCC antibody level was still elevated to 30.6pmol/L on postoperative day 7. He underwent daily rehabilitation for discharge. Approximately 2 months after the operation, he had aspiration pneumonia with respiratory failure. Intubation and respiratory management were performed in the intensive care unit. He remains ventilator dependent at 4 months postoperation. Postoperative provocative nerve conduction studies could not be performed. His tumor was composed of a proliferation of infiltrating atypical cells with cribriform or papillary structures. The tumor measured 30mm in size and the diagnosis was moderately-differentiated (G2) mixed-type adenocarcinoma (papillary and acinar adenocarcinoma) (Figure [3](#F3){ref-type="fig"}). Lymph nodes metastases in the lung parenchyma were noted. Pathological T2aN1M0 (Stage IIa: TNM staging 7th edition) was diagnosed. Immunohistochemistry revealed that most of the cancer cells were positive for P/Q-type VGCC (Sigma-Aldrich®; St. Louis, MO, USA) (Figure [4](#F4){ref-type="fig"}). {#F3} {#F4} Discussion ========== LEMS is one of the representative paraneoplastic syndromes and an autoimmune disease of the neuromuscular junction. Because of its scarcity, the exact incidence of LEMS is unknown. The prevalence of as few as 400 cases in the USA in 1994 \[[@B2]\] and of 2 to 3 cases per million inhabitants in the Netherlands in 2004 \[[@B3]\] has been reported. LEMS is known to be associated with malignant neoplasm, especially SCLC. Approximately 50 to 75% of patients with LEMS have SCLC \[[@B2]-[@B7]\]. LEMS is also thought to be a complication of organ-specific autoimmune diseases, such as thyroid disease, vitiligo, pernicious anemia, coeliac disease, and juvenile-onset diabetes mellitus. However, systemic lupus erythematosus, Sjs and RA associated with LEMS is rare; only a few cases are reported in the English literature \[[@B4],[@B8]\]. Clinical manifestation of LEMS is usually characterized by proximal muscle weakness affecting the legs more than the arms, fatigability and dysautonomia. Malignant neoplasm usually occurs within the first 2 years after the onset of LEMS and in almost all patients after 4 years \[[@B5]\]. Therefore, once LEMS is diagnosed or strongly suspected, screening for malignant neoplasm, especially SCLC (for example, using chest CT or tumor markers: neuron-specific enolase and progastrin-releasing peptide), is recommended. Moreover, careful observation and screening should be required for at least 2 years \[[@B4]-[@B6]\]. The treatment of SCLC controls its mortality and often results in normalization of both electrodiagnostic findings and clinical symptoms of LEMS \[[@B5],[@B6]\]. The diagnosis of LEMS is established by clinical manifestation and by characteristic findings on nerve conduction studies. The nerve conduction study findings indicate reduced amplitude of compound muscle action potential that increases by over 100% after maximum voluntary activation or 20 to 50Hz of nerve stimulation, and incremental response (waxing) of compound muscle action potential on the rapid rate of repetitive stimulation \[[@B5],[@B6]\]. In the 1990s, Motomura and co-workers established a new immunoassay of antibodies to VGCC for the diagnosis of LEMS \[[@B1]\]. VGCCs are situated on the presynaptic nerve terminal and have a central role in the control of neurotransmitter release \[[@B1],[@B9]\]. Antibodies to VGCC inhibit transmitter (acetylcholine) release at presynaptic nerve terminals. It has been assumed that SCLC expresses functional calcium channels and that autoantibodies to these channels may cross-react with similar VGCCs \[[@B1],[@B5],[@B6],[@B9]\]. Motomura \[[@B1]\] reported 85% of patients with LEMS to be positive for VGCC and suggests that the titer of anti-P/Q-type VGCC antibody in particular occupies an important position in the diagnosis of LEMS; the remaining 15% of patients were seronegative for VGCC \[[@B9]\]. In this subgroup, SCLC is less common and the exact explanation of seronegative LEMS is still unknown. Thus, a negative antibody titer of VGCC is not necessarily an indicator of the absence of LEMS \[[@B9]\]. LEMS is rarely associated with other subtypes of lung cancer like adenocarcinoma. A PubMed literature search (keywords, 'Lambert-Eaton Myasthenic Syndrome' and 'lung adenocarcinoma') found only two publications in the English literature \[[@B7],[@B10]\]. In Japan, although five cases have been reported to date, there are only two reports of LEMS associated with lung adenocarcinoma in Japanese research with an English abstract \[[@B11],[@B12]\]. The clinical features of these five cases, including our case report, are summarized in Table [1](#T1){ref-type="table"}. The patients (all men) ranged from 32 to 75 years of age (mean age, 57 ± 16.0 years). Adenocarcinomas arose in the right lung in all cases. Coexistent collagen diseases were recognized in two cases, including our case (dermatomyositis, RA and Sjs). Three out of these five cases could undergo lung resection. Almost all LEMS cases are related to SCLC, making them unsuitable for resection. For cases not associated with SCLC, resection is a possibility. Therefore, once LEMS is strongly suspected, screening for malignancy and early detection of lung carcinoma and its histological type is very important. Of note, the histological differentiation of all demonstrated cases was poor to moderate. ###### Characteristics of patients with Lambert--Eaton myasthenic syndrome associated with lung adenocarcinoma **Case no.** **Year** **First author** **Age** **Sex** **Site** **Histological grade** **Coexistent collagen disease** **Treatment** **Outcome** ----------------------- ---------- --------------------- --------- --------- ---------- ------------------------ --------------------------------- ------------------------- ----------------------- 1 1987 Ramos-Yeo \[[@B7]\] 56 M RLL Poor None Steroid, plasmapheresis Death at 2 years (due to infection) 2^a^ 1989 Sumitomo \[[@B11]\] 58 M RML Poor None Lobectomy, Surviving at 2 years adjuvant chemotherapy 3^a^ 1996 Okudera \[[@B12]\] 32 M RUL Poor None Chemotherapy Death at 5 months (due to DIC) 4 2008 Milanez \[[@B10]\] 66 M RUL Moderate DM Lobectomy Death at 16 days (due to sepsis) 5 2012 This case report 75 M RLL Moderate RA, Sjs Lobectomy Surviving at 4 months a: English abstract only; *DIC* disseminated intravascular coagulation, *DM* dermatomyositis, *RA*, rheumatoid arthritis, *RLL* right lower lobe, *RML* right middle lobe, *RUL* right upper lobe, *Sjs* Sjögren syndrome. We examined P/Q-type VGCC expression immunohistochemically using resected lung tumor sections. Most of the neoplastic cells were stained positive, and the area consisting of poorly-differentiated neoplastic cells tended to show stronger signals. As far as we know, no report has demonstrated immunohistochemical expression of VGCC in neoplastic cells. Conclusions =========== Taking together the results of immunostaining and the fact that our patient also had collagen diseases (RA and Sjs), autoimmune function may be related to the onset of LEMS and, in the case of lung adenocarcinoma, lower-differentiated cases appear to develop P/Q-type VGCC. However, the actual mechanism by which anti-P/Q-type VGCC antibody causes LEMS is unclear because the experimental model of this rare disease has not been established \[[@B6]\]*.* Further molecular biological studies are required. Clinically, physicians must manage patients with muscle weakness while considering LEMS as a differential diagnosis. Recognition and awareness of this rare syndrome are essential for a better prognosis and quality of life for these patients. Consent ======= Written informed consent was obtained from the patient of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interest ================== The authors declare that they have no competing interests. Authors' contributions ====================== HA was responsible for the conception and writing of the manuscript. KH, KK and TN participated in the collection of data and patient care. HK, KO and AN provided collection of data, patient care and editing of the manuscript. KI, MT, TK and MM participated in final revision of the manuscript and guidance. All authors read and approved the final manuscript. Acknowledgements ================ We are grateful to Dr. Masakatsu Motomura (Department of Clinical Neuroscience and Neurology Graduate School of Biomedical Science, Nagasaki University) for analyzing the serum levels of the anti-P/Q-type VGCC antibody titer and useful discussion.

1. Introduction =============== Mucosal melanomas are a rare clinical entity, in the literature the incidence is described with 1% to 2% of all melanomas and 2 to 2.6 per 1,000,000 persons/year.^\[[@R1]--[@R3]\]^ Melanomas arising from mucosal surfaces have a different profile of risk factors (eg, no exposure to ultraviolet radiation) and other genetic mutations than cutaneous melanomas, especially KIT-mutations are more frequent in mucosal melanomas.^\[[@R4]\]^ Mucosal melanomas have a poor prognosis which is much worse than that of cutaneous melanomas.^\[[@R1]\]^ It remains uncertain whether the poorer prognosis is due to the usually more progressed disease at initial diagnosis or to the biologically more aggressive growth. Prognostic factors are not well established thus far.^\[[@R5]\]^ Therefore, we have retrospectively analyzed 75 patients with mucosal melanomas at different locations of the primary tumor in regard to their prognostic factors. Furthermore, we summarized our experiences using new immunologic and targeted therapies. 2. Patients und methods ======================= 2.1. Patients ------------- Patients of our Department including the years 1993 to 2015 with primary mucosal melanomas were recorded in a database, their history was regularly updated. The patients were divided in 3 groups in regard to the location of the primary tumor: head/neck, anorectal, and female genital tract (FGT). Since the American Joint Committee on Cancer-classification^\[[@R6]\]^ for cutaneous melanoma is not established for mucosal melanoma, we implemented 3 groups for a clinical tumor grading according to the Mucosal Melanoma Staging System published by Iversen and Robins^\[[@R7]\]^ in 1980 and proposed by Thoelke et al: I -- local tumor, II -- regional lymph node metastasis, and III -- distant metastasis.^\[[@R8],[@R9]\]^ The follow-up, adjuvant and palliative therapy, was done according to the recommendations for patients with cutaneous melanomas.^\[[@R10]\]^ One patient with a KIT Exon 11 L576P Mutation was treated with imatinib, this case has already been published as a case report.^\[[@R11]\]^ The median follow-up time was 32 months, with a minimum of 2 and a maximum of 231 months. Mutation analysis was performed partly in the context of scientific research,^\[[@R12]\]^ others within clinical trials and routine clinical treatment. Sixty-two patients were screened for KIT and 57 patients for BRAF-mutations by various methods: Sanger sequencing for the KIT-gene and on 5 patients for the BRAF-gene, further analysis of the BRAF-gene was performed via melting curve analysis for 29 and pyrosequencing for 23 patients. The ethics committee of the Hannover Medical School provided IRB approval for the retrospective data collection of melanoma patients (vote no. 1612--2012). 2.2. Statistical analysis ------------------------- The Programs Statistica 8 (Statsoft), GraphPad Prism version 5.01 for Windows, GraphPad Software, and EpiInfo 3.5.3 (Centers for Disease Control and Prevention) were used for the statistical analysis. The evaluation included the usual descriptive statistics (mean, median, and percentages) and survival analysis with the Kaplan--Meier estimate. The Log-Rank-test was used for the calculation of significance for the overall and relapse-free survival between the groups. The influence of various prognostic factors was tested on the basis of the Cox proportional hazard model. The initial group comparison (Table [1](#T1){ref-type="table"}) for nominal data was done with the Chi-square test or Fisher exact test, respectively, for the comparison of multiple groups with ordinal or metric data by use of the Kruskal--Wallis test. A *P*-value \< 0.05 was considered significant. ###### Characterization of the patients in regard to localization.  3. Results ========== 3.1. Clinical parameters ------------------------ We identified 75 patients with mucosal melanomas, 32 melanomas were localized in the head/neck area, 24 melanomas were situated in the FGT, and 19 melanomas were diagnosed in the anorectal region, respectively (Table [1](#T1){ref-type="table"}). The median age at the time of 1st diagnosis was 66 years, with 57% female and 43% male patients. The overall higher percentage of females is due to patients with a melanoma in the FGT, tumors in the head/neck region occurred more often in males, whereas anorectal melanomas were evenly distributed between females and males (Table [1](#T1){ref-type="table"}). After initial diagnosis the total excision of the mucosal melanoma could be achieved in 64/75 patients (85%). Total lymphadenectomy of the regional lymph nodes was performed in 43/75 patients (57%), 20 of which had metastasis at time of initial diagnosis, 8 following a positive sentinel lymph node (SLN), and 15 as an elective procedure (Table [1](#T1){ref-type="table"}). A total of 34/75 patients received an adjuvant therapy. Adjuvant radiotherapy was applied in 20 patients, 17 of them with the primary tumor in the head/neck region, 2 with anorectal, and 1 with FGT tumors. Adjuvant interferon alpha with a dosage of 3 × 3 million units per week for 24 month was administered to 21 patients, 5 of them also had succeeding adjuvant radiotherapy. 3.2. Prognosis -------------- A recurrence of the disease was recorded in 48/61 (79%) of the patients where an initial complete excision had been realized. The median time until the 1st recurrence was 14 month (Table [1](#T1){ref-type="table"}). An initial local recurrence or in transit metastasis was recorded in 18/48 patients, regional lymph node metastasis in 8/48 patients, and distant metastasis in 22/48 patients. During follow-up 48/75 (64%) patients died of the melanoma, 1/75 (1.3%) patients of others causes. The median overall survival through all groups was 32 months (Table [1](#T1){ref-type="table"}). 3.3. Prognostic factors in the univariate analysis for the overall and recurrence-free survival ----------------------------------------------------------------------------------------------- The 5-year survival rate was 26.3%, 22.0% in the head/neck group, 10.6% anorectal, and 33.2% in the FGT group. Patients with the melanoma in the head/neck region and anorectal region displayed a significant worse overall survival than patients with melanomas in the FGT (*P* = 0.04, Table [2](#T2){ref-type="table"}, Fig. [1](#F1){ref-type="fig"}B). ###### Univariate analysis of the prognostic factors (log rank test).  {#F1} The probability for recurrence-free survival after 5 years was 13.8% overall 3 groups, 8.1% with mucosal melanoma in the head/neck region, and 23.8% in the FGT, a 5-year follow-up period was not reached for anorectal tumors (Fig. [1](#F1){ref-type="fig"}A). Again a significant advantage could be demonstrated for the group with melanomas in the FGT for the recurrence-free survival versus the patients with anorectal and head/neck tumors (*P* = 0.03, Table [2](#T2){ref-type="table"}). For patients under 60 years of age at the time of 1st diagnosis we saw a trend for an advantage in overall and recurrence-free survival (*P* = 0.06, Table [2](#T2){ref-type="table"}, Fig. [1](#F1){ref-type="fig"}E). Patients in disease stage I at the time of 1st diagnosis had significant advantage in terms of overall survival (*P* = 0.04, Fig. [1](#F1){ref-type="fig"}D, Table [2](#T2){ref-type="table"}) and a highly significant for the recurrence-free survival (*P* \< 0.001, Table [2](#T2){ref-type="table"}) in comparison with patients with stages II and III at the time of the initial diagnosis. In regard to gender no significance for overall and recurrence-free survival could be demonstrated. Because of the long period of observation we divided the patients in 3 groups, 1993 to 2005, 2006 to 2009, and 2010 to 2015, the comparison showed no significant difference between the groups for recurrence-free survival and overall survival (Fig. [1](#F1){ref-type="fig"}F). In the group of head/neck mucosal melanomas, in which the major part of the adjuvant radiation took place, a significant advantage (*P* = 0.02) toward the prevention of local recurrences was shown (Fig. [2](#F2){ref-type="fig"}). {#F2} As expected the multivariate analysis (Table [3](#T3){ref-type="table"}) showed a high statistical significance for the tumor-stage at time of the initial diagnosis in terms of recurrence-free survival (*P* \< 0.001). Also age younger than 60 was correlated with a significant recurrence-free survival benefit (*P* = 0.03). In contrast the localization of the primary did not reach any independent prognostic significance. ###### Multivariate analysis of the prognostic factors (Cox proportional hazard).  3.4. KIT/BRAF mutations ----------------------- A molecular analysis of the KIT-gene was performed in 62 of the patients studied. A total of 7/62 (11.3%) of the patients had a KIT mutation, 5 in exon 11 (once each 579del, K550N, W557R, twice L576P), 1 in exon 13 (K642E), and 1 in exon 18 of the KIT-gene (I841V). The analysis for 36 patients was done in our own laboratory (exons 9, 11, 13, 17, and 18), 6 of them showed a KIT-mutation. These results were already described in detail.^\[[@R12]\]^ Within the TEAM-study (Tasigna Efficacy in Advanced Melanoma) the KIT-exons 9, 11, 13, and 17 were analyzed in 6 further patients, all 6 patients did not show a mutation. Another 20 analyses were done during routine diagnostics employing Sanger-Sequencing of the exons 9, 11, and 13, which revealed a K642E-mutation in 1 patient. In regard to localization of the primary tumor KIT-mutations were shown in 2/27 (7.4%) patients in the head/neck region, 2/16 (12.5%) anorectal, and 3/19 (15.8%) in the FGT. A BRAF V600E Mutation was demonstrated in 2/57 (3.5%) of the patients, one each in the anorectal and head/neck-group. 3.5. Targeted therapy and therapy with checkpoint inhibitors ------------------------------------------------------------ Four patients were treated with targeted therapies, patients 1 to 3 with imatinib due to a KIT-mutation in exon 11 or 13. Patient 1 achieved a short-term partial remission (PR), patient 2 a short-term stable disease (SD) (Table [4](#T4){ref-type="table"}). ###### Therapy with targeted therapies and checkpoint inhibitors.  Patient 4 (Table [4](#T4){ref-type="table"}) is a male patient with a metastasized anorectal melanoma harboring a BRAF mutation. Therapy with ipilimumab yielded in a PR for 6 month, subsequent vemurafenib-therapy resulted in a PR for another 5 month. Reexposition first with ipilimumab then with vemurafenib did not lead to any further tumor control. Besides these patients 6 other patients were treated with ipilimumab (Table [4](#T4){ref-type="table"}), one of those had an SD for 4.5 months. Seven patients received programmed disease-1 (PD-1)-checkpoint inhibitors (Table [4](#T4){ref-type="table"}), in 2 patients a PR lasting 366+ and 240+ days was achieved. 4. Discussion ============= Mucosal melanoma is disease of advanced age, among our patients the median age over all 3 groups was 66 years corresponding well to the spectrum described in the literature ranging from 60 to 75 years.^\[[@R8],[@R13]--[@R17]\]^ The gender distribution with 57% female patients was consistent with other observations and can be explained by the patients with melanoma in the FGT.^\[[@R15],[@R18]\]^ As described in the literature male patients suffered more often (69%) from mucosal melanomas in the head/neck region.^\[[@R16]\]^ Anorectal melanomas showed a fairly balanced gender distribution with 53% male patients in our study in contrast to the literature where mostly a higher ratio of male patients is described.^\[[@R16],[@R19]\]^ An SLN biopsy is particularly possible if lymph drainage into peripheral lymph stations is expected, for example, distal the linea dentata for anal melanomas^\[[@R20]\]^ or vulvar melanomas in the FGT.^\[[@R21]\]^ In individual cases SLN biopsy is described even for mucosal melanomas in the head/neck region.^\[[@R19]\]^ Contrary to cutaneous melanomas the SLN is not established as a prognostic factor in mucosal melanoma. For having a poor prognosis in respect of distant metastasis a complete regional lymph node dissection following a positive SLN is discussed controversially.^\[[@R20],[@R21]\]^ During follow-up 79% of the patients had a recurrence, especially mucosal melanomas in the head/neck region demonstrated a short recurrence-free survival time with a median of 10 months. In our study 64% of the patients died during follow-up, the 5-year survival rate was 26.3%, which is slightly lower than indicated in the literature with 5-year survival rates of 32.4%,^\[[@R22]\]^ 34%,^\[[@R15]\]^ and up to 55.8%.^\[[@R23]\]^ In line with expectations, the tumor-grade is an important prognostic factor as described in the literature.^\[[@R17],[@R22]\]^ In our study, the age with a threshold value of 60 years was an additional important prognostic factor. In the literature age is described as a prognostic factor for either mucosal melanomas^\[[@R14],[@R22]\]^ and skin melanomas.^\[[@R24]\]^ A better prognosis for tumors in the FGT became apparent in our univariate analysis but could not be confirmed in the multivariate analysis. Mehra et al^\[[@R23]\]^ could also show a better prognosis for vulvar melanomas in comparison to other localizations of the primary tumor. Local recurrence occurred notably in patients with the primary tumor in the head/neck region, which had not undergone adjuvant radiotherapy, among the adjuvant group the local tumor control was significantly better. Some studies already have shown that adjuvant radiation of mucosal melanomas in the head/neck region can reduce the risk of local recurrence from 33%--83% to 0%--56%.^\[[@R19]\]^ Another study could demonstrate that at least 54 Gray should be applied to have a positive effect.^\[[@R25]\]^ For KIT-mutations we could show a higher rate in mucosal melanomas in the FGT (15.8%) in comparison to the other 2 groups, head/neck (7.4%) and anorectal (12.5%). This is consistent with a recent publication that outlined the highest rate of KIT-mutations for vulvovaginal melanomas (35%) followed by anorectal melanomas (25%) and mucosal melanomas in the head/neck region (10%).^\[[@R26]\]^ In our patient collection, the treatment with imatinib showed 1 short-term SD and 1 short-term PR in 2 of 3 patients with KIT-mutations in exon 11 and 13, respectively (Table [4](#T4){ref-type="table"}). In the literature, several case series describe an objective response in 16% to 30% of the patients treated imatinib, the median time for recurrence-free survival was about 3 months, with major benefit for patients with mutations in exon 11 and 13.^\[[@R27]--[@R29]\]^ In a case series with 7 patients with mucosal melanomas and KIT-mutations in exon 11 or 13 sunitinib showed a response in 3 cases.^\[[@R30]\]^ A reason for the relative poor response to KIT-inhibitors could be a concurrent NRAS-mutation, which are described in patients with KIT-mutations.^\[[@R29]\]^ This results in activation of the pathway downstream of KIT, thus it is advisable to rule out NRAS-mutations before using a KIT-inhibitor. BRAF-mutations are described in mucosal melanoma. In our cohort 3.5% of the patients examined displayed a BRAF-mutation, in the literature the values differ from 3.6%,^\[[@R31]\]^ 6%^\[[@R32]\]^ up to 11.1%,^\[[@R33]\]^ and 16.5%.^\[[@R34]\]^ Little is published on the response of targeted therapies in BRAF positive patients with mucosal melanomas, the response in our patient suggests that this is possible. For the use of ipilimumab for the treatment of metastatic mucosal melanoma there are case studies mostly on pretreated patients. In a series of 30 patients (76% of which with the dosing of 3 mg/kg bodyweight, 24% with 10 mg per/kg) 1 patient responded with a complete remission, 1 patient with a PR, and 5 patients with an SD.^\[[@R35]\]^ In a series of 71 patients in the Italian "early access program" a response rate of 12.5% and a 36% rate of SD was observed.^\[[@R36]\]^ On the use of PD-1 inhibitors in mucosal melanomas there are individual case reports.^\[[@R37]--[@R39]\]^ A pooled analysis of mucosal melanomas in a variety of trials using nivolumab (n = 86), nivolumab plus ipilimumab (n = 35), or ipilimumab (n = 36) provided evidence for an effect of nivolumab in mucosal melanomas.^\[[@R40]\]^ The median progression-free survival and response rates were 2.96 months and 23.2% for nivolumab, 5.85 months and 37.2% for nivolumab plus ipilimumab, and 2.69 months and 8.3% for ipilimumab, respectively. This corresponds well with our data showing a long-lasting response in 2/7 patients treated with PD-1 inhibitors. 5. Conclusions ============== In our study, mucosal melanomas displayed a poor prognosis with metastasis often being already present at the time of initial diagnosis. We could confirm the recommendation for an adjuvant radiation of the primary tumor region on patients with head/neck mucosal melanomas in order to significantly lower the risk of local recurrence. In case of metastazation considered inoperable targeted therapies and immunotherapies with checkpoint inhibitors can be considered. PD-1 inhibitors or their combination with ipilimumab appear to show the highest response rates and longest progression-free survival. In case of a KIT mutation, additional analysis of NRAS is recommended before treating with a KIT-inhibitor. In the rare event of a BRAF V600 mutation, targeted therapy analogous to cutaneous melanomas is recommended. The limitations of this study are the small number of patients and the long period of patient acquisition, in particular the low number of patients with targeted and immunotherapy treatment. Therefore, no conclusions with regard to therapy standards can be achieved. Abbreviations: FGT = female genital tract, PD-1 = programmed disease-1, PR = partial remission, SD = stable disease, SLN = sentinel lymph node, SLNB = sentinel lymph node biopsy. The authors have no conflicts of interest to disclose.

**Funding information** No funding information provided. A woman aged in her 80s presented at our hospital complaining of sudden‐onset abdominal and right hip pain. An abdominal ultrasound revealed protruded small intestine and fluid in the right groin area (Fig. [1A](#ams2547-fig-0001){ref-type="fig"}, asterisk). Computed tomography (CT) established the diagnosis of obturator hernia (Fig. [1B](#ams2547-fig-0001){ref-type="fig"}, asterisk). She underwent emergency intestinal resection and was discharged without complications 10 days after the surgery. {#ams2547-fig-0001} As the obturator foramen is located deep inside the body, it is difficult to palpate the protruded intestine. The obturator hernia is often misdiagnosed as hip osteoarthritis or hip fracture.[^1^](#ams2547-bib-0001){ref-type="ref"} Computed tomography is the gold standard for the diagnosis of the obturator hernia,[^2^](#ams2547-bib-0002){ref-type="ref"} however, it can be time‐consuming, and delaying diagnosis can cause intestinal necrosis. However, because obturator hernias are more common among thin elderly women whose pelvic floor muscles are atrophic, obturator foramen can be evaluated by ultrasound with high‐frequency, small‐part linear transducers.[^3^](#ams2547-bib-0003){ref-type="ref"} In this case, ultrasound could visualize the pectineus (Fig. [1](#ams2547-fig-0001){ref-type="fig"}, dagger) and the obturator internus muscle (Fig. [1](#ams2547-fig-0001){ref-type="fig"}, double dagger). Ultrasound can be easily carried out in an emergency room and is less invasive than CT. We can establish the diagnosis of obturator hernia faster than CT, and that leads to early intervention, such as surgery. Disclosure {#ams2547-sec-0002} ========== Approval of the research protocol: N/A. Informed consent: N/A. Registry and the registration no. of the study/trial: N/A. Animal studies: N/A. Conflict of interest: None.

1. Introduction {#sec1-molecules-21-00911} =============== Agarwood is the dark resinous and aromatic wood from genus *Aquilaria* trees belonging to the family Thymelaeaceae \[[@B1-molecules-21-00911],[@B2-molecules-21-00911],[@B3-molecules-21-00911]\], and is created as a tree response to various form of injury, including natural injuries, such as lightning strikes, animal grazing, insect attacks or microbial invasion, or artificial injuries, such as cutting, nailing, holing, fire, chemical wounding, and deliberate fungal inoculation \[[@B3-molecules-21-00911],[@B4-molecules-21-00911],[@B5-molecules-21-00911]\]. Agarwood not only plays an important role in Traditional Chinese Medicine, but also has been used for centuries as incense in Buddhist, Hindu and Islamic ceremonies \[[@B1-molecules-21-00911],[@B2-molecules-21-00911],[@B3-molecules-21-00911]\]. Unfortunately, in natural forests, only 7%--10% of the agarwood trees contain resinous material, and wild agarwood is rare due to its exhaustive exploitation. Nowadays, agarwood in trade mostly comes from cultivated *Aquilaria* trees induced by artificial methods, of which the traditional artificial holing is the most common and popular one. As far as we know, nineteen accepted species have been reported up to date, growing from southeast Asia to the Malay Archipelago \[[@B1-molecules-21-00911],[@B2-molecules-21-00911]\]. However, only the heartwood of *A. crassna*, *A. malaccensis*, *A. sinensis* and *A. filarial* have been commercially exploited \[[@B1-molecules-21-00911]\]. 2-(2-Phenylethyl)chromone derivatives, of which about one hundred different structures have been reported, are considered to be one of the characteristic and most abundant constituents responsible for the quality of agarwood \[[@B1-molecules-21-00911],[@B3-molecules-21-00911],[@B4-molecules-21-00911],[@B6-molecules-21-00911],[@B7-molecules-21-00911],[@B8-molecules-21-00911],[@B9-molecules-21-00911]\]. These compounds were divided into four types according to the various chromone moiety skeletons, in which the benzyl moiety is the same. In these four types, both 2-(2-phenylethyl)chromones of the flidersia type (FTPECs) and 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones (THPECs) were widely distributed and accumulated most in agarwood, while only four 5,6-epoxy-2-(2-phenylethyl)chromones (EPECs) and three 5,6:7,8-diepoxy-2-(2-phenylethyl) chromones (DEPECs) with one or two epoxy substituents on the chromone moiety respectively, have been reported from 2005 to the present \[[@B7-molecules-21-00911],[@B10-molecules-21-00911],[@B11-molecules-21-00911]\]. Based on the above reports, 2-(2-phenylethyl)chromones were considered as the necessary diagnostic components to evaluate the quality of agarwood. Mei et al. summarized the MS characterization of the FTPECs, which was helpful for the analysis and characterization of FTPECs in agarwood by GC-MS \[[@B3-molecules-21-00911]\]. Xia et al. reported that 29 FTPECs were detected from agarwood by supercritical fluid chromatography in combination with mass spectrometry (SFC-MS) then obtained further detailed structural information by using tandem mass spectrometry (MS/MS) \[[@B12-molecules-21-00911]\]. Li et al. identified and quantified eight characteristic THPECs in Chinese eaglewood, and analyzed the MS fragmentation behavior of THPECs by HPLC/DAD/MS \[[@B13-molecules-21-00911]\]. Lancaster et al. detected diagnostic ions at *m/z* 319.118 (FTPECs) or 349.129 (FTPECs) and the occurrence of ten or more of the other target chromone ions by real time time-of-flight mass spectrometry (DART-TOFMS) to infer agarwood \[[@B5-molecules-21-00911]\]. Espinoza et al. analyzed the diagnostic chromones ions by using DART-TOFMS followed by discriminant analysis to differentiate wild agarwood from cultivated samples \[[@B2-molecules-21-00911]\]. However, the MS characterization of EPECs and DEPECs have not been reported yet. In this paper, the MS characterization and fragmentation behavior of EPECs and DEPECs was described, and the fragmentation regularity of mass spectra (MS) used to distinguish and identify the four different types of 2-(2-phenylethyl)chromones was concluded for the first time. Nowadays, artificial holing agarwood from *A. crassna* tree plays an extremely important role in the market. In this report, we detected and analyzed 2-(2-phenylethyl)chromones in three agarwood samples (from *A. crassna*) with different holing times by using HPLC/DAD/ESI/MS^2^, and revealed the variation of relative contents of different types of 2-(2-phenylethyl)chromones in agarwood after two, four and five years of artificial holing. This could be referenced for the variation of 2-(2-phenylethyl)chromones, also identification and quality evaluation of agarwood with different formation time. 2. Results and Discussion {#sec2-molecules-21-00911} ========================= 2.1. Characteristic Fragmentation Behavior of Reference Compounds ***F1**--**F31*** {#sec2dot1-molecules-21-00911} ----------------------------------------------------------------------------------- In this study, the different fragmentation behavior of four types of chromones according to their MS spectra was summarized by the reference compounds ([Table 1](#molecules-21-00911-t001){ref-type="table"} and [Figure 1](#molecules-21-00911-f001){ref-type="fig"}). The characteristic fragment ions produced by four types of chromones due to their structural difference on the basic skeleton of chromone moiety were used as the basis for identification. For THPECs (compounds **F1**--**F4**), the characteristic fragmentations were the loss of a molecule of H~2~O (\[M + H--18\]^+^) and a successive loss of another H~2~O molecule (\[M + H--18--18\]^+^) \[[@B13-molecules-21-00911]\]. For EPECs (compounds **F5**--**F7**), the characteristic fragmentations were loss of a molecule of H~2~O (\[M + H--18\]^+^), followed by loss of a molecule of CO (\[M + H--18--28\]^+^). For DEPECs **F8**--**F10**, the characteristic fragmentation was the loss of a molecule of CO (\[M + H--28\]^+^). For twenty-one FTPECs in the reference compounds, their characteristic fragmentations were benzyl ions and/or chromone moiety ions. In addition, the benzyl ions observed in the MS spectrum of these four types chromones were the same, and the typical ions included *m/z* 91 \[C~7~H~7~\]^+^, 107 \[C~7~H~6~ + OH\]^+^, 121 \[C~7~H~6~ + OCH~3~\]^+^, 137 \[C~7~H~5~ + OH + OCH~3~\]^+^, 151 \[C~7~H~5~ + 2OCH~3~\]^+^. [Figure 2](#molecules-21-00911-f002){ref-type="fig"} presents examples of the MS^2^ fragmentation behavior from each type of chromone. 2.2. The Method Deduced for Identification of Four Types of 2-(2-Phenylethyl)chromone Derivatives {#sec2dot2-molecules-21-00911} ------------------------------------------------------------------------------------------------- (1)The type of 2-(2-phenylethyl)chromone could be determined by the characteristic fragment ions as discussed above and listed in [Figure 3](#molecules-21-00911-f003){ref-type="fig"}. The present of the ions \[M + H--18\]^+^ and \[M + H--18--18\]^+^ was proposed to be THPECs. When both ions \[M + H--18\]^+^ and\[M + H--28\]^+^ were observed in the spectrum, EPECs was deduced. If only \[M + H--28\]^+^ was detected, it would be DEPECs. FTPECs only gave the peaks of benzyl ions and/or chromone moiety ions in the MS spectrum. It should be noticed that the \[M + H--18\]^+^ ion may appear as a pseudo-characteristic ion during identification, when the CH~2~-CH~2~ group between chromone moiety and phenyl moiety was substituted by a hydroxyl group (-OH) \[[@B1-molecules-21-00911]\].(2)The four types of 2-(2-phenylethyl)chromone derivatives in agarwood have different basic skeletons (the epoxy group was assigned as a part of the basic skeletons), but substituted with similar substituent groups (mainly hydroxyl, methoxyl group and/ or chlorine atom). The molecular weights of the basic skeletons of THPECs, EPECs, DEPECs and FTPECs were 254, 268, 282 and 250, respectively. Thus, the number of different substituents of the structure could be deduced by Formula (1), where "MW" meant the molecular weight, "a" meant the number of methoxy groups, "b" represented the number of hydroxyl groups, and "c" meant the number of chlorine atom. $${MW} - (30a + 16b + 34c) = \left\{ \begin{array}{l} {254~\left( {THPECs} \right)} \\ {268~\left( {EPECs} \right)} \\ {282~\left( {DEPECs} \right)} \\ {250~\left( {FTPECs} \right)} \\ \end{array} \right.$$(3)The four types of chromones presented the same characteristic benzyl ion, while it could not be observed in some compounds. The benzyl moiety without substituent group was 91 \[C~7~H~7~\]^+^, therefore, the number of hydroxyl and/or methoxyl groups substituted on the benzyl moiety could be deduced according to Formula (2), where "MW~bm~" means the molecular weight of benzyl ion, "a~bm~" means the number of methoxy groups on the benzyl moiety, "b~bm~" represents the number of hydroxyl groups on the benzyl moiety. For example, according to Formula (2), the hydroxyl- or methoxyl-substituted benzyl moiety provided characteristic ions at *m/z* 107 \[C~7~H~6~ + OH\]^+^, 121 \[C~7~H~6~ + OCH~3~\]^+^, 137 \[C~7~H~5~ + OH + OCH~3~\]^+^, 151 \[C~7~H~5~ + 2OCH~3~\]^+^:(4)Except for FTPECs, the fragment ion of the chromone moiety was rarely observed in the MS^2^ spectra of THPECs, EPECs and DEPECs, nevertheless the number of substituent groups of the chromone moiety could still be calculated by subtracting the number of substituents on the benzyl moiety deduced by Formula (2) from the number of substituents on the whole structure deduced by Formula (1).For FTPECs with hydroxyl-substituted benzyl moieties, according to \[[@B3-molecules-21-00911],[@B6-molecules-21-00911]\], if one of characteristic fragment ions at *m/z* 161 \[C~10~H~8~O~2~ + H\], 177 \[C~10~H~7~O~2~ + OH + H\], 191 \[C~10~H~7~O~2~ + OCH~3~ + H\], 193 \[C~10~H~6~O~2~ + 2OH + H\], 207 \[C~10~H~6~O~2~ + OCH~3~ + OH + H\], 221 \[C~10~H~6~O~2~ + 2OCH~3~ + H\] from different substituted chromone moieties was observed, a 4′/2′-OH substituent on the benzyl moiety was deduced. Otherwise, if none of them was observed, a hydroxyl group substituted at the 3′ position of the benzyl moiety was deduced, whether an ion of chromone moiety at *m/z* 160, 176, 190, 192, 206, 220 appeared or not. The above mentioned MS characterization has been confirmed by the FTPEC reference compounds in [Table 1](#molecules-21-00911-t001){ref-type="table"}. The observed fragment ions of the chromone moieties of **F11**, **F15**, **F18**, **F22** and **F24** with 4′/2′-OH on the benzyl moiety were *m/z* 177, 177, 161, 191 and 161, respectively, which were not observed for the **F12**, **F17**, **F20**, **F23** and **F27** with 3′-OH on the benzyl moiety, while only a fragment ion at *m/z* 220 of **F17** was observed. These chromone moiety ions could be used to suggest the position of hydroxyl groups on the benzyl moieties.(5)Following the above procedure, a compound could be characterized as one of the four types of 2-(2-phenylethyl)chromones, as well as calculated the numbers of substituent groups of the chromone moiety and benzyl moiety. Furthermore, the position of substituent groups could be deduced sometimes. Besides, if more than one structure were suggested for a single peak, the comparison of retention time and MS spectra with reference compounds was performed. The compound was identified when the data were the same to the reference compound, otherwise, it might be a new compound. 2.3. Identification of 2-(2-Phenylethyl)chromones According to MS Characterization {#sec2dot3-molecules-21-00911} ---------------------------------------------------------------------------------- In the HPLC chromatograms of the ether extract of three agarwood samples (S1, S2, S3) detected at UV 254 nm, a total of fifty-six compounds (four types of chromones) were detected ([Figure 3](#molecules-21-00911-f003){ref-type="fig"}). By comparing their retention time and MS spectra with reference compounds, twenty-six of them were identified, and thirty compounds were tentative identified. The identified or characterized results of THPECs, EPECs and DEPECs were listed in [Table 2](#molecules-21-00911-t002){ref-type="table"}, and the retention time of THPECs, EPECs and DEPECs were in the range of 12\~30 min, 18\~34 min, 21\~43 min, respectively. The results of FTPECs were showed in [Table 3](#molecules-21-00911-t003){ref-type="table"}, and the retention time of FTPECs were in the range of 34\~80 min, except for compound **20** (26.1 min). ### 2.3.1. Structural Analysis of THPECs {#sec2dot3dot1-molecules-21-00911} Taking compounds **1** and **3** for example, the characteristic fragment ions at *m/z* 331(\[M + H--18\]^+^) and 313 (\[M + H--18--18\]^+^), indicated both were THPECs. The molecular weight (MW) of these two compounds were determined as 348 based on the protonated precursor ion at *m/z* 349. According to formula (1) (MW − (30a + 16b +34c) = 254), a, b and c were calculated as 1, 4 and 0, respectively, which meant compounds **1** and **3** had four hydroxyl and one methoxyl substituent groups on the whole compound. Furthermore, the benzyl ion at *m/z* 121 was found in compound **1**, which meant one methoxy substitution occurred on its benzyl moiety according to Formula (2), thus, the other four hydroxyl substitutions occurred on its chromone moiety. Neither benzyl ion nor chromone moiety ions were observed in compound **3**, thus the positions of substituent groups were uncertain. By searching the literature, we found six THPECs with the MW = 348 were reported, including four of them possessing one methoxy substitution occurring on the benzyl moiety, and four hydroxyl substitutions occurring on their chromone moiety, which matched the above deduced structural characterization of compound **1**, so compound **1** was tentative identified as one of the four reported compounds. The positions of the substituent groups of compound **3** were uncertain, so it was tentative identified as one of the six reported compounds while different from compound **1**. By comparing the retention time and MS spectra with the reference compounds, compound **1** was identified as **F3**, and further proved that the above identification method was reasonable. According to above method of identification, compounds **1**--**7** were deduced as THPECs. Except for compound **5** (*m/z* 317), the other six compounds, with \[M + H\]^+^ ions of *m/z* 319, 303, 367, 337, have been reported as THPECs. Compound **5** was identified as a new compound. ### 2.3.2. Structural Analysis of EPECs {#sec2dot3dot2-molecules-21-00911} Taking compounds **10** and **11** for example, according to the characteristic fragment ions of *m/z* 313 (\[M + H--18\]^+^) and 285 (\[M + H--18--28\]^+^), they were identified as EPECs. The protonated precursor ions at *m/z* 331 meant their molecular weight (MW) was 330. According to the Formula (1), a, b and c were calculated as 1, 2 and 0, respectively, which meant they had two hydroxyl and one methoxyl substitutions on the whole compound. The benzyl ion at *m/z* 121 indicated one methoxy substitution occurred on its benzyl moiety according to Formula (2), therefore, the two hydroxyl groups substitutions occurred on the chromone moiety. Subsequently, by literature searching, only one EPEC with MW = 330 was reported, which meant that one of them would be a new compound. By comparing the retention time and MS spectra with reference compound, compound **11** was identified as **F6**, which proved that the above identification method was feasible, and meanwhile compound **10** should be a new compound. By the same method, compounds **8**--**12** were deduced as EPECs. Except for compound **9** (which \[M + H\]^+^ ion is *m/z* 285), all the other compounds, which \[M + H\]^+^ ions were at *m/z* 347 or 301, have been reported. By comparing retention time and MS spectra with reference compounds, compounds **8** and **12** were identified as reference compounds **F5** and **F7**, respectively. Compounds **9** and **10** were proposed to be new compounds as listed in [Table 2](#molecules-21-00911-t002){ref-type="table"}, because only four EPECs have been reported until now \[[@B7-molecules-21-00911],[@B14-molecules-21-00911]\]. ### 2.3.3. Structural Analysis of DEPECs {#sec2dot3dot3-molecules-21-00911} According to the above method of identification, compounds **13**--**19** were assigned as DEPECs. By comparing the retention time and MS spectra with the reference compounds, compounds **16**, **18**, and **19** were identified as the reference compounds **F8**\~**F10**, respectively. The other DEPECs were proposed to be new compounds, as only a total of three DEPECs (**F8**\~**F10**) have been reported until now \[[@B10-molecules-21-00911]\]. The results are shown in [Table 2](#molecules-21-00911-t002){ref-type="table"}. ### 2.3.4. Structural Analysis of FTPECs {#sec2dot3dot4-molecules-21-00911} Taking compounds **22**\~**24**, **27** and **30** for example, only benzyl ions (*m/z* 137 and 121) and chromone moiety ion (*m/z* 177) were detected, leading to identification of these compounds as FTPECs. All of them showed protonated precursor ions at *m/z* 313, which meant their molecular weight (MW) was 312. According to Formula (1), the a, b and c values were calculated as 1, 2 and 0, respectively, which meant they have two hydroxyl and one methoxyl substituent group on the whole compound. Then, according to Formula (2), the benzyl ion at *m/z* 137 was found in compounds **22** and **23**, which meant one methoxyl and one hydroxyl substitution occurred on their benzyl moiety, while the benzyl ion at *m/z* 121 was detected in compounds **24**, **27** and **30**, which meant one methoxy substitution occurred on their benzyl moiety. Consequently, one hydroxyl substitution occurred on the chromone moiety of compounds **22** and **23**, and two hydroxyl substitutions occurred on the chromone moiety of compounds **24**, **27** and **30**. In particular a chromone moiety ion (*m/z* 177) was detected in compound **22**, which indicated the hydroxyl group should be substituted at the 4′/2′ position of the benzyl moiety. Five FTPECs with MW = 312 have been traced from the literature, so the five FTPECs were tentatively identified as reported compounds. By comparing the retention time and MS spectra with reference compounds, compounds **22**, **24** and **27** were identified as **F12**, **F13** and **F16**, respectively, and the results proved that the above identification method was reasonable. According to the method of identification, compounds **20**--**56** were deduced as FTPECs ([Table 3](#molecules-21-00911-t003){ref-type="table"}), among which nineteen compounds were identified according to the reference compounds. Finally, fifty-six 2-(2-phenylethyl)chromone derivatives were identified or characterized from the three samples according to the fragmentation behavior and the comparison of retention time and MS spectra with reference compounds ([Table 1](#molecules-21-00911-t001){ref-type="table"}). A total of 37 (seven DEPECs, four EPECs, three THPECs and 23 FTPECs), 43 (five EPECs, five THPECs and 33 FTPECs), and 29 (one DEPEC, two EPECs, three THPECs and 23 FTPECs) 2-(2-phenylethyl)chromones were identified or characterized in S1, S2 and S3, respectively. The respective relative content of 2-(2-phenylethyl)chromone derivatives was 66.42%, 81.39% and 79.20% in S1, S2 and S3 ([Table 4](#molecules-21-00911-t004){ref-type="table"}). Based on the above data, it was found that the number and relative content of DEPECs (7/8.01%) from S1 was the highest among the three samples, while they were merely traces in S2 and S3 ([Table 4](#molecules-21-00911-t004){ref-type="table"}, [Figure 4](#molecules-21-00911-f004){ref-type="fig"}). The relative content of EPECs showed a downtrend from S1 to S3 (12.58%, 8.07% and 2.96%, respectively), while the relative content of THPECs and FTPECs showed an uptrend from S1 to S3 (1.58%/1.73%/2.47%, 44.25%/71.59%/73.56%, respectively). It was noticeable that both the total number and relative content of DEPECs, and EPECs decreased obviously (20.59%, 8.07% and 3.17%, respectively) from S1 to S3, and this was consistent with that observed in the HPLC chromatograms ([Figure 3](#molecules-21-00911-f003){ref-type="fig"}), in which the height of peaks for the DEPECs, EPECs (before 43 min) showed a remarkable decreasing trend. This finding was in agreement with the proposed biosynthetic pathways of the four types of chromones ([Figure 4](#molecules-21-00911-f004){ref-type="fig"}) \[[@B11-molecules-21-00911]\], which showed transformations among them during different agarwood formation time. DEPECs have been recognized as precursors, and only accumulated at the early stage of agarwood formation. Then, the next group was EPECs, where the low occurrence rate also suggested they were early intermediates during the agarwood formation. The following group was the highly oxidized THPECs, and the last presented group were FTPECs, both of which were widely distributed in agarwood. In total, twenty-one 2-(2-phenylethyl)chromones (two EPECs, two THPECs and 17 FTPECs) were detected in all three samples, while ten (six DEPECs, one THPEC and three FTPECs), twelve (one EPEC, three THPECs and eight FTPECs), and one (THPEC) 2-(2-phenylethyl)chromone were only found in S1, S2, and S3, respectively. Furthermore, we found that the relative content of six FTPECs, 6,8-dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone, 6-methoxy-7-hydroxy-2-\[2-(4-methoxy)-phenylethyl\]chromone, 6-hydroxy-2-(2-phenylethyl)chromone, 6,7-dimethoxy-2-(2-phenylethyl) chromone, 2-\[2-(4-methoxy)phenylethyl\]chromone, and 2-(2-phenylethyl)chromone, which are reported as the main constituents of agarwood, showed a uptrend from S1 to S3. This finding suggested that the relative content of the six 2-(2-phenylethyl)chromones was the significant factor to evaluate the formation time of agarwood. 3. Experimental Section {#sec3-molecules-21-00911} ======================= 3.1. Chemicals and Materials {#sec3dot1-molecules-21-00911} ---------------------------- HPLC-grade acetonitrile (ACN) and methanol were supplied by Tedia (Fairfield, CR, USA). Chromatographic grade absolute formic acid was purchased from Roe Scientific Inc. (Shanxi, HPLC, China). Water was purified using a Milli-Q Plus185 system from Millipore (Milford, MA, USA). The times of artificial holing into the trunk of *A. crassna* tree were Aug of 2011 (S1), 2009 (S2) and 2008 (S3), respectively, so the agarwood formation times of S1--S3 were 2 years, 4 years and 5 years, respectively ([Figure 5](#molecules-21-00911-f005){ref-type="fig"}). The samples were collected by Dr. Haofu Dai from Guangnan Province of Vietnam at 14 August 2013. Voucher specimens were deposited at Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. Thirty-one references compounds were isolated and purified from agarwood in our previous work \[[@B11-molecules-21-00911],[@B14-molecules-21-00911],[@B15-molecules-21-00911],[@B16-molecules-21-00911],[@B17-molecules-21-00911]\]. Their structures were identified by spectroscopic methods (MS and NMR), and they are listed in [Table 1](#molecules-21-00911-t001){ref-type="table"} and [Figure 1](#molecules-21-00911-f001){ref-type="fig"}. The purity of each compound was determined to be higher than 97% by high-performance liquid chromatography (HPLC). 3.2. Preparation of Sample Solutions and Reference Compound Solutions {#sec3dot2-molecules-21-00911} --------------------------------------------------------------------- In this study, the crushed agarwood (100g, dry weight) was soaked in Et~2~O (3 × 200 mL) and extracted by ultrasonication (3 × 15 min). The Et~2~O extracts were filtered and evaporated to get brownish yellow oil. The yields of S1, S2 and S3 were 0.56%, 0.46%, and 2.08%, respectively. Then each extract oil sample was diluted to 1 mg/mL with methanol in a volumetric flask. The solution was filtered through 0.45 µm membranes and stored at 4 °C until use. Accurately weighed samples of thirty-one reference compounds were dissolved in methanol (1.0 mg/mL), respectively, and filtered through 0.45 µm membranes to get the standard solutions. All standard solutions were stored at 4 °C until use. 3.3. HPLC Chromatographic Condition {#sec3dot3-molecules-21-00911} ----------------------------------- A Dionex 3000 series HPLC instrument composed of a diode array detector, a vacuum degasser, a quaternary pump and an auto-sampler was used (Bruker Daltonics Inc., Bremen, HPLC/DAD/VD/QP/AS, Germany). The chromatographic separation was carried out on a Dionex-Acclaim 120 C~18~column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile (A) and water-acetic acid (99.5:0.5, *v*/*v*) (B). The gradient elution program was as follows: 25%--55% A in 0--60 min, 55%--80% A in 60--80 min, 80%--100% A in 80--90 min, and 100% A in 90--95 min. The flow rate was 0.4 mL/min, the column temperature was maintained at 26 °C, and the injection volume was 20 µL. The detection wavelength was set at 254 nm for all the tested compounds. 3.4. MS Spectrometry {#sec3dot4-molecules-21-00911} -------------------- MS experiments were performed using an electrospray ionization tandem mass spectrometry system (Amazon SL, Bruker Daltonics Inc., Bremen, Germany) mainly in positive-ion mode. Helium gas was used as the collision gas and high-purity nitrogen gas as the nebulizer and drying gas at flow rates of 0.4 L/min and 6.0 L/min, respectively. The ESI source conditions were as follows: capillary voltage, −4000 V (position); end plate voltage, −500 V (position); drying gas temperature of 250 °C; and nebulizer pressure 15 psi. Scan spectra from *m/z* 70 to 2200. All the mass data was processed using Bruker Compass Data Analysis 4.0 software (Bruker Daltonics: Germany, 2009). 4. Conclusions {#sec4-molecules-21-00911} ============== HPLC/ESI-MS/MS is a powerful tool to identify or characterize 2-(2-phenylethyl)chromone derivatives in agarwood. Thirty-one reference compounds including four types of chromones were analyzed by ESI-MS/MS, thus the characteristic fragmentation behavior of DEPECs and EPECs and the methods to distinguish the four types of chromones by their MS characteristic fragmentations were described for the first time, on the basis of the MS spectra of the thirty-one reference compounds. A total of fifty-six 2-(2-phenylethyl)chromone derivatives comprising seven DEPECs, five EPECs, seven THPECs and thirty-seven FTPECs were identified or characterized by their fragmentation pathways and characteristic fragment ions. It was found, with the increase of artificial holing time of agarwood (two years to five years), the total relative content of DEPECs and EPECs showed a downtrend, while the relative content of THPECs and FTPECs showed an increasing trend, which was consistent with the biosynthetic pathway of the formation of agarwood. The relative content of six FTPECs went upward from S1 to S3. This finding could be useful to distinguish agarwood samples with different formation times, and further studies on more agarwood samples with different formation times are needed to validate these conjectures. Supported by Special Fund for Agro-scientific Research in the Public Interest (201303117), Special Fund for The Young Talents's Science and Technology Project of Hainan Association for Science and Technology (HASI201628), Special Fund for Key Research Project of Hainan Province in 2016 (ZDYF2016210), and Major Technology Project of Hainan Province (ZDKJ2016004). **Sample Availability:** Not available. The list authors contributed to this work as follows: J.L. Yang performed the HPLC/MS/MS and prepared the manuscript. W.H. Dong, F.D. Kong and G. Liao contributed to the revision of this manuscript. G. Liao and W. Li provided the reference compounds, and J. Wang dealt with the picture. The whole research was performed based on the planning of H.F. Dai and W.L. Mei. All authors approved the final version of the manuscript. The authors declare no conflict of interest. {#molecules-21-00911-f001} {#molecules-21-00911-f002} {#molecules-21-00911-f003} {#molecules-21-00911-f004} {#molecules-21-00911-f005} molecules-21-00911-t001_Table 1 ###### Characterization of thirty-one reference compounds by HPLC/ESI-MS/MS. Type No. t~R~/min Fragment Ions (*m/z*) Name -------- --------- ---------- ----------------------- ------ ----- ----- ----- ----- ------------------------------------------------------------------------------------------------------------------------ THPECs **F1** 12.9 349 331 313 137 *(5S\*,6R\*,7S\*)-5,6,7-Trihydroxy-2-\[2-(3-hydroxy-4-methoxyphenyl)ethyl\]-5,6,7,8-tetrahydrochromone* **F2** 13.2 349 331 313 137 *(5S\*,6R\*,7R\*)-5,6,7-Trihydroxy-2-\[2-(3-hydroxy-4-methoxyphenyl)ethyl\]-5,6,7,8-tetrahydrochromone* **F3** 15.2 349 331 313 121 *5α,6β,7α,8β-Tetrahydroxy-2-\[2-(4-methoxyphenyl)-ethyl\]-5,6,7,8-tetrahydrochromone* **F4** 17.3 383 365 347 137 *(5S\*,6R\*,7S\*,8R\*)-8-Chloro-5,6,7-trihydroxy-2-\[2-(3-hydroxy-4-methoxyphenyl)ethyl\]-5,6,7,8-tetrahydro-chromone* EPECs **F5** 18.3 347 329 301 137 *5,6-Edroxy-7,8-dihydroxy-2-\[2-(3-hydroxy-4-methoxy)phenylethyl\]-5,6,7,8-tetrahydrochromone* **F6** 33.1 331 313 285 121 *5,6-Edroxy-7,8-dihydroxy-2-\[2-(4-methoxy)phenyl-ethyl\]-5,6,7,8-tetrahydrochromone* **F7** 33.8 301 283 255 91 *5,6-Edroxy-7,8-dihydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone* DEPECs **F8** 24.4 329 301 137 *5,6:7,8-Diedroxy-2-\[2-(3-hydroxy-4-methoxy)phenyl-ethyl\]-5,6,7,8-tetrahydrochromone* **F9** 42.6 313 285 121 *Oxidoagarochromones B* **F10** 43.5 283 255 91 *Oxidoagarochromones A* FTPECs **F11** 34.7 283 177 107 *6-Hydroxy-2-\[2-(4-hydroxy)phenylethyl\]chromone* **F12** 36.6 313 137 *6-Hydroxy-2-\[2-(3-hydroxy-4-methoxy)phenylethyl\]-chromone* **F13** 38.3 313 192 121 *6,8-Dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* **F14** 39.5 283 192 91 *6,8-Dihydroxy-2-(2-phenylethyl)chromone* **F15** 41.5 283 177 107 *6-Hydroxy-2-\[2-(2-hydroxy)phenylethyl\]chromone* **F16** 42.4 313 121 *6,7-Dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* **F17** 43.2 357 220 137 *6,7-Dimethoxy-2-\[2-(3-hydroxy-4-methoxy)phenyl-ethyl\]chromone* **F18** 47.5 297 161 137 *2-\[2-(3-Methoxy-4-hydroxy)phenylethyl\]chromone* **F19** 49.8 267 176 91 *8-Hydroxy-2-(2-phenylethyl)chromone* **F20** 50.4 296 137 *2-\[2-(3-Hydroxy-4-methoxy)phenylethyl\]chromone* **F21** 51.2 327 121 *6-Methoxy-7-hydroxy-2-\[2-(4-methoxy)phenylethyl\]-chromone* **F22** 52.3 327 191 137 *6-Methoxy-2-\[2-(3-methoxy-4-hydroxy)phenylethyl\]-chromone* **F23** 54.3 327 137 *6-Methoxy-2-\[2-(3-hydroxy-4-methoxy)phenylethyl\]-chromone* **F24** 55.2 267 161 107 *2-\[2-(2-Hydroxy)phenylethyl\]chromone* **F25** 56.5 297 121 *6-Hydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* **F26** 58.3 267 176 91 *6-Hydroxy-2-(2-phenylethyl)chromone* **F27** 59.1 343 137 *5-Hydroxy-6-methoxy-2-\[2-(3-hydroxy-4-methoxy)-phenylethyl\]chromone* **F28** 64.9 311 220 91 *6,7-Dimethoxy-2-(2-phenylethyl)chromone* **F29** 72.0 281 121 *2-\[2-(4-Methoxy)phenylethyl\]chromone* **F30** 73.8 251 160 91 *2-(2-Phenylethyl)chromone* **F31** 78.7 326 121 *5-Hydroxy-6-methoxy-2-\[2-(4-methoxy)phenylethyl\]-chromone* CM: chromone moiety; BM: benzyl moiety. molecules-21-00911-t002_Table 2 ###### The MS characterization and identified or characterized results of DEPECs, EPECs and THPECs from S1 to S3. No. t~R~ (min) \[M + H\]^+^ (*m/z*) (+)Fragment Ions (*m/z*) OCH~3~ OH Identification RFC RC (%) -------- ------------ ---------------------- -------------------------- -------- ----- ---------------- ----- -------- --- --------------------------------------------------------------------------------------------------------- --------- ------ ------ ------ THPECs **1** 15.9 349 331 313 121 1 4 *5α,6β,7α,8β-Tetrahydroxy-2-\[2-(4-methoxyphenyl)ethyl\]-5,6,7,8-tetrahydrochromone* **F3** 0.51 **2** 16.0 319 301 283 4 *(5S,6S,7R)-5,6,7-Trihydroxy-2-\[2-(2-hydroxyphenyl)-ethyl\]-5,6,7,8-tetrahydrochromone or isomers* 0.15 **3** 16.7 349 331 313 1 4 *Isomer of **F3*** 0.50 **4** 23.7 303 285 267 3 *rel-(5R,6S,7R)-5,6,7-Trihydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone* 0.09 **5** 24.2 317 299 281 121 1 2 *New compound (two hydroxy on CM, one methoxy on BM)* 0.08 **6** 29.4 367 349 331 121 1 3 *rel-(5R,6S,7S,8R)-8-Chloro-5,6,7-trihydroxy-2-\[2-(4-methoxyphenyl)ethyl\]-5,6,7,8-tetrahydrochromone* 0.65 0.65 1.16 **7** 30.1 337 319 301 3 *(5S,6S,7S,8R)-8-Chloro-2-(2-phenylethyl)-5,6,7-trihydroxy-5,6,7,8-tetrahydrochromone or new compound* 0.43 0.76 0.80 EPECs **8** 18.5 347 329 301 137 1 3 *5,6-Edroxy-7,8-dihydroxy-2-\[2-(3-hydroxy-4-methoxy)-phenylethyl\]-5,6,7,8-tetrahydrochromone* **F5** 1.13 0.12 **9** 23.3 285 267 239 1 *New compound (one hydroxy on CM)* 0.09 **10** 25.0 331 313 285 121 1 2 *New compound (isomer of F6)* 0.94 0.15 **11** 33.0 331 313 285 121 1 2 *5,6-Edroxy-7,8-dihydroxy-2-\[2-(4-methoxy)phenylethyl\]-5,6,7,8-tetrahydrochromone* **F6** 7.25 4.95 2.11 **12** 33.7 301 283 255 2 *5,6-Edroxy-7,8-dihydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone* **F7** 3.26 2.76 0.85 DEPECs **13** 21.8 329 301 137 1 1 *New compound (isomer of **F8**)* 1.00 **14** 23.0 329 301 193 137 1 1 *New compound (isomer of **F8**)* 0.50 **15** 23.3 329 301 137 1 1 *New compound (isomer of **F8**)* 0.28 **16** 24.2 329 301 137 1 1 *5,6:7,8-Diedroxy-2-\[2-(3-hydroxy-4-methoxy)phenylethyl\]-5,6,7,8-tetrahydrochromone* **F8** 0.47 **17** 24.6 329 301 137 1 1 *New compound (isomer of **F8**)* 0.02 **18** 42.0 313 285 121 1 *Oxidoagarochromones B* **F9** 4.85 **19** 42.9 283 255 *Oxidoagarochromones A* **F10** 0.89 0.21 CM: chromone moiety; BM: benzyl moiety; RFC: Reference compound; RC: Relative content. molecules-21-00911-t003_Table 3 ###### The MS characterization and identified or characterized results of FTPECs from S1 to S3 by HPLC/DAD/ESI/MS^2^. No. t~R~ (min) \[M + H\]^+^ (*m/z*) Fragment Ions (*m/z*) CM BM Identification RFC RC (%) -------- ------------ ---------------------- ----------------------- ----- ------ ---------------- ----- ----------- -------------------------------------------------------------------------------------------------------- --------- ------ ------ ------- **20** 26.1 329 137 2 1 3′- *6,8-Dihydroxy-2-\[2-(3-hydroxy-4-methoxyl)phenylethyl\]-chromone* 0.28 **21** 34.2 283 177 107 6- 4′- *6-Hydroxy-2-\[2-(4-hydroxy)phenylethyl\]chromone* **F11** 8.6 0.03 **22** 35.0 313 177 137 1 1 4′or 2′- *6-Hydroxy-2-\[2-(3-methoxyl-4-hydroxy)phenylethyl\]-chromone* 0.26 0.26 **23** 36.8 313 137 6- 4′- 3′- *6-Hydroxy-2-\[2-(3-hydroxy-4-methoxyl)phenylethyl\]-chromone* **F12** 0.66 0.23 0.41 **24** 38.4 313 121 6,8- 4′- *6,8-Dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* **F13** 0.66 0.87 2.32 **25** 39.5 283 192 91 6,8- *6,8-Dihydroxy-2-(2-phenylethyl)chromone* **F14** 0.93 1.81 **26** 41.3 283 177 107 6- 2′- *6-Hydroxy-2-\[2-(2-hydroxy)phenylethyl\]chromone* **F15** 0.05 **27** 42.7 313 121 6,7- 4′- *6,7-Dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* **F16** 2.80 0.46 0.45 **28** 44.0 283 192 91 2 *New compound (isomer of **F14**)* 1.33 0.52 **29** 59.1 343 137 6- 5- 3′- 4′- *5-Hydroxy-6-methoxy-2-\[2-(3-methoxy-4-hydroxy)phenyl-ethyl\]chromone* **F27** 1.18 **30** 66.3 313 121 2 1 *5,8-Dihydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone* 1.82 **31** 68.5 283 192 91 2 *5,8-Dihydroxy-2-(2-phenylethyl)chromone* 0.78 **32** 43.1 357 220 137 6,7- 4′- 3′- *6,7-Dimethoxy-2-\[2-(3-hydroxy-4-methoxyl)phenylethyl\]-chromone* **F17** 0.46 **33** 46.8 267 161 107 1 *2-\[2-(4-Hydroxy)phenylethyl\]chromone* 0.27 0.21 0.20 **34** 47.5 297 161 137 1 4′- *2-\[2-(3-Methoxy-4-hydroxy)phenylethyl\]chromone* **F18** 0.22 **35** 48.5 297 191 107 1 2′ or 4′- *6-Methoxy-2-\[2-(4-hydroxy)phenylethyl\]chromone or new compound* 0.16 **36** 49.2 267 176 91 8- *8-Hydroxy-2-(2-phenylethyl)chromone* **F19** 0.78 1.16 **37** 50.3 297 121 1 1 *6-Hydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone or new compound* 0.16 0.04 1.16 **38** 51.4 327 121 6- 7- 4′- *6-Methoxy-7-hydroxy-2-\[2-(4-methoxy)phenylethyl\]-chromone* **F20** 1.27 1.57 2.55 **39** 52.3 327 191 137 6- 3′- 4′- *6-Methoxy-2-\[2-(3-methoxy-4-hydroxy)phenylethyl\]-chromone* **F22** 2.88 0.85 1.88 **40** 53.0 297 206 1 1 *6-Hydroxy-7-methoxy-2-(2-phenylethyl)chromone* 1.74 2.32 **41** 54.3 327 137 6- 4′- 3′- *6-Methoxy-2-\[2-(3-hydroxy-4-methoxy)phenylethyl\]-chromone* **F23** 5.43 2.06 1.98 **42** 54.9 267 161 107 2′- *2-\[2-(2-Hydroxy)phenylethyl\]chromone* **F24** 1.06 **43** 55.8 297 121 1 1 *6-Hydroxy-2-\[2-(4-methoxy)phenylethyl\]chromone or new compound* **F25** 4.25 5.36 8.17 **44** 57.6 267 176 91 6- *6-Hydroxy-2-(2-phenylethyl)chromone* **F26** 1.42 6.64 8.17 **45** 58.7 297 191 107 1 2′ or 4′- *6-Methoxy-2-\[2-(4-hydroxy)phenylethyl\]chromone or new compound* 0.27 **46** 78.2 357 121 2 1 1 *New compound (two methoxyls and one hydroxyl on CM, one methoxyl on BM)* 1.00 **47** 78.7 327 121 6- 5- 4′- *5-Hydroxy-6-methoxy-2-\[2-(4-methoxy)phenylethyl\]-chromone* **F31** 4.31 2.79 3.47 **48** 80.3 297 206 91 1 1 *5-Hydroxy-6-methoxy-2-(2-phenylethyl)chromone* 1.36 2.58 2.23 **49** 62.5 341 121 2 1 *6,7-Dimethoxy-2-\[2-(4-methoxy)phenylethyl\]chromone* 5.67 5.06 4.43 **50** 63.3 341 151 1 2 *New compound (one methoxyl on CM, two methoxyls on BM)* 1.28 0.51 **51** 64.8 311 220 91 6,7- *6,7-Dimethoxy-2-(2-phenylethyl)chromone* **F28** 5.58 6.41 11.93 **52** 71.7 281 121 4′- *2-\[2-(4-Methoxy)phenylethyl\]chromone* **F29** 0.62 0.98 1.18 **53** 73.2 281 190 91 1 *6-Methoxy-2-(2-phenylethyl)chromone or new compound* 0.17 **54** 73.4 251 160 91 *2-(2-phenylethyl)chromone* **F30** 0.17 1.05 1.52 **55** 74.7 311 121 1 1 *6-Methoxy-2-\[2-(3-methoxy)phenylethyl\]chromone or 6-methoxy-2-\[2-(4-methoxy)phenylethyl\]chromone* 4.89 6.59 **56** 76.3 281 190 91 1 *6-Methoxy-2-(2-phenylethyl)chromone or new compound* 2.28 10.6 9.34 CM: chromone moiety; BM: benzyl moiety; RFC: Reference compound; RC: Relative content.2.3.1. Structural Analysis of THPECs molecules-21-00911-t004_Table 4 ###### The statistical results of 2-(2-phenylethyl)chromone derivatives. N(RC/%) DEPECs EPECs Total (E) THPECs FTPECs Total (all) --------- ----------- ------------ ------------- ----------- ------------- ------------- S1 7 (8.01%) 4 (12.58%) 11 (20.59%) 3 (1.58%) 23 (44.25%) 37 (66.42%) S2 0 (0%) 5 (8.07%) 5 (8.07%) 5 (1.73%) 33 (71.59%) 43 (81.39%) S3 1 (0.21%) 2 (2.96%) 3 (3.17%) 3 (2.47%) 23 (73.56%) 29 (79.20%) N: number; RC: Relative content; E: DEPECs and EPECs.

INTRODUCTION {#s1} ============ Sexual reproduction is generally accepted as a process that occurs when opposite mating partners interact physically to produce genetically variable and, ideally over time, better adapted offspring. However, the ability to reproduce sexually in the absence of a partner is known to occur in some eukaryotes, where it has been described in species as diverse as the fan worm ([@R52]), land snail ([@R7]), and is commonplace in plants ([@R40]). Fungi are no exception, and indeed display a fascinating range of sexual strategies found in self-fertile basidiomycetes and ascomycetes, as reviewed by [@R26]. These fungi are known as homothallic, in contrast to heterothallic where outcrossing is obligatory ([@R6]). Homothallism in fungi has historically been defined as the ability of a single spore to produce a sexually reproducing colony when propagated in complete isolation. This is the definition by which self-fertility was first described ([@R6]) and it is the strategy used to define the sexual status of a newly described fungal species. However, "homothallism" is an all-inclusive term that describes diverse sexual strategies. Describing a fungus as homothallic is therefore over-simplified, given that it can utilize one or more strategies to give rise to sexual progeny. The rapidly growing availability of fungal genome data ([@R49]) is allowing more intensive genetic inquiry into fungal mating and this will result in a more informed perspective on sexual reproduction, especially with respect to self-fertility. In this regard, it has become possible to define the various mechanisms underpinning homothallism from a genetic perspective and thus improve our understanding of the evolutionary outcomes of sexual reproduction as a whole. In order to consider homothallism from a genetic standpoint, it is important to understand that sexual reproduction and mating specificity in fungi is controlled largely by genes present at the mating type (*MAT1*) locus ([@R22]). The allelic variants, or idiomorphs, of this locus encode one of two classes of proteins; those that give rise to the MAT-1 phenotype- the genes at the *MAT1-1* idiomorph- and those that give rise to the MAT-2 phenotype- the genes at the *MAT1-2* idiomorph ([@R53]). In its most commonly understood form, sexual reproduction requires the expression of genes from both these idiomorphs ([@R37]). Thus, fungal mating systems can be classified based on the genic content of the *MAT1* locus. Using this designation, homothallic individuals possess genes of both idiomorphs within a single cell, enabling combined *MAT1-1* and *MAT1-2* expression by a single individual. This is in contrast to individuals of heterothallic species that possess genes from only one of the two idiomorphs. In these species, the combined expression of genes from both these idiomorphs requires the presence of two opposite mating type partners. While heterothallism represents a relatively simple and well-understood opposite mate interaction, homothallism constitutes a variety of distinct strategies that collectively allow for single individuals to sexually reproduce independently of an opposite mating partner. Although the genetic and biochemical mechanisms underlying a few of these homothallic sexual strategies have been elucidated in some (typically model) fungi, the majority remain unstudied. This review seeks to compare and contrast these mechanisms in terms of their causative molecular basis. It further aims to particularly emphasize the most recently described and unique form of homothallism known as unisexual reproduction. CONVENTIONAL CATEGORIES OF HOMOTHALLISM {#s2} ======================================= Primary homothallism {#s2a} -------------------- Primary homothallism is the classic mechanism by which self-fertility is achieved. Here, species adhere to the strict genetic requirement of the combined expression of *MAT1-1* and *MAT1-2* genes in a single genome ([@R37]; [Fig. 1](#F1){ref-type="fig"}). This is exactly the opposite of heterothallism and, as such, primary homothallic species possess all the *MAT* genes typically found in both MAT-1 and MAT-2 isolates of a closely-related heterothallic species ([@R26]). Genes from the alternate idiomorphs can either exist at a single locus (linked or fused) or be present in separate regions (unlinked) within the genome ([@R44], [@R64], [@R13]). The mechanism that governs primary homothallism has been relatively well studied in comparison to the other forms of this condition. This is most likely due to the model species *Aspergillus nidulans* exhibiting a primary homothallic sexual cycle ([@R45]). In this species, individuals derived from a single uninucleate and haploid cell are able to undergo sexual reproduction under environmentally conducive conditions ([@R55]). Genetically, primary homothallism was confirmed in *A. nidulans* when both the *MAT1-1-1* gene (possessing the conserved alpha domain) and the *MAT1-2-1* gene (possessing the conserved HMG box domain) were identified and shown to be expressed in a single genome ([@R41]). This is in contrast to heterothallic *Aspergillus* species, such as *A. parasiticus* and *A. fumigatus*, where individuals possess either the *MAT1-1-1* or the *MAT1-2-1* gene ([@R20], [@R39]). Pseudohomothallism {#s2b} ------------------ Pseudohomothallism adheres to the physiological definition of homothallism and, as such, single spores of species exhibiting this type of self-fertility are fully able to undergo independent sexual reproduction ([@R58]). However, from a cellular and molecular standpoint, the underlying mechanism is more complex than that of primary homothallism and has consequently been referred to as an example of secondary homothallism ([@R58]) or functional heterothallism ([@R10], [@R26]). In species exhibiting pseudohomothallic behaviour, self-fertility is the result of the packaging of two independent and opposite mating type nuclei within a single spore ([Fig. 1](#F1){ref-type="fig"}). This ensures that when the spore germinates, a heterokaryotic and thus self-fertile mycelium is produced ([@R9]). In this situation, a single spore actually contains the genetic complement of two opposite mating partners as two discrete nuclei. Functionally, it thus represents a heterothallic system that is able to occur within a single originating cell ([@R10]). This behaviour has been described in several species including *Neurospora tetrasperma* ([@R9]), *Podospora anserina* ([@R3]), and *Gelasinospora tetrasperma* ([@R11]). Pseudohomothallism has been intensively studied in *N. tetrasperma*, where it was first described ([@R9]). In this species, a significant majority of the resulting ascospores are larger than those produced by closely-related heterothallic species. Furthermore, while heterothallic *Neurospora* species typically produce eight ascospores, *N. tetrasperma* produces only four ([@R9]). These large ascospores, representing about 90 % of those produced ([@R46]), possess two independent nuclei: one of which expresses the MAT-1 phenotype while the other expresses the MAT-2 phenotype ([@R9]). This comes about due to a programmed meiotic spindle alignment that allows two opposite mating type nuclei derived from a single diploid nucleus to be packaged in a single ascospore. This might be considered as a form of reproductive "assurance" allowing unhindered sexual reproduction even in the absence of a suitable mating partner ([@R31]). The remaining \~10 % of the ascospores produced in *N. tetrasperma* are much smaller, possess only a single nucleus, and thus express only one of the two mating types ([@R46]). These spores have been suggested to represent a mechanism whereby outcrossing can be maintained in the species, along with the benefits attributed to functional recombination ([@R46]). Asci comprised of both the large and small spores ensure that both homothallic and heterothallic reproduction can occur, thereby maintaining the benefits associated with both. MATING TYPE SWITCHING {#s3} ===================== A second mechanism that allows for homothallic behaviour is referred to as mating type switching and it represents yet another example of secondary homothallism ([@R26]). In this mating strategy, an individual of one mating type can undergo a switch to the opposite mating type, either bidirectionally (reversibly) or unidirectionally (irreversibly). This allows a single cell to produce a colony of mixed mating type that is subsequently able to reproduce sexually ([@R26]; [Fig. 1](#F1){ref-type="fig"}). Bidirectional Mating Type Switching {#s3a} ----------------------------------- The first case of this unusual mating behaviour was described in *Saccharomyces cerevisiae,* after the species had been described as homothallic by one group of researchers ([@R62]) but heterothallic by another ([@R28], [@R29]). It was later shown that populations of this ascomycetous yeast are comprised of both self-fertile and self-sterile individuals. Once the genetic mechanism underlying this type of homothallism had been elucidated, it became apparent that every individual within the population, regardless of mating behaviour, possessed sequence of the *MAT1-1* idiomorph (termed *MAT**α***) as well as sequence of the *MAT1-2* idiomorph (termed *MAT**a***). The presence of both mating type idiomorphs in a single cell appeared to be congruent with the standard homothallic requirement of the expression of genes from both mating types in a single cell. However, it did not explain the presence of self-sterile individuals in the population that also possessed all the genes typically required for sexual reproduction. Only then was it shown that, in addition to the typical *MAT1* locus that is present in this species, there are also two MTL (mating-type like) loci that flank the *MAT1* locus ([@R34]). While the active *MAT1* locus harbours sequence of only one of the *MAT* idiomorphs, the first MTL possesses *MAT1-1* sequence and is termed *HML**α*** and the second possesses *MAT1-2* sequence and is termed *HMR**a***. These MTL, however, are transcriptionally silent due to the presence of tightly wound heterochromatin. Consequently, the only *MAT* genes expressed in a single cell are those found at the active *MAT1* locus itself ([@R48]). This implies that any one cell can either be classified as MAT-1 or MAT-2, despite possessing sequence for both mating types. The consequence is that the species undergoes a traditional heterothallic cycle. In *S. cerevisiae*, homothallic behaviour is due to a gene entirely independent of the *MAT1* locus and the associated MTL. This type of self-fertility is a result of the HO gene and its ability to initiate mating type switching events. The HO+ gene encodes an active endonuclease that recognizes the active *MAT1* locus, makes a double stranded DNA break and excises the locus ([@R33]). The flanking MTL of the opposite mating type is then used as a template from which a new *MAT1* locus can be constructed *via* gene conversion. This mechanism allows for a switch to take place from one mating type to the other due to the presence of MTL loci for both the *MAT**α*** and *MAT**a*** idiomorphs. It is, therefore, possible for a single originating cell to produce a colony of mixed mating types that is consequently able to sexually reproduce in a functionally heterothallic manner. If a cell possesses the inactive copy of the HO gene (HO-), it cannot switch and is thus considered self-sterile and not able to independently produce a sexually competent colony. The genetic mechanisms and cellular processes underlying this mechanism have been comprehensively reviewed ([@R17], [@R18]). Unidirectional Mating Type Switching {#s3b} ------------------------------------ While the mechanisms underlying the bidirectional switching as observed in *S. cerevisiae* are well-defined, those relating to unidirectional switching remain poorly understood. This is possibly because this form of switching has only been observed in a small number of fungi residing in distantly-related genera including *Chromocrea, Ceratocystis*, *Glomerella,* and *Sclerotinia* ([@R57], [@R30], [@R54], [@R42], [@R19], [@R63], [@R60]). These fungi have not been included amongst the model organisms used in fungal genetics studies and thus their sexual strategies have not been clearly elucidated. Unidirectional mating type switching has been partially investigated in *Chromocrea spinulosa*, where spore size is intricately linked to mating type and is most likely a pleiotropic expression of mating type ([@R30]). The asci of this species have eight two-celled ascospores, half of which are small and produce individuals of *l* mating type while the others are large and produce individuals of the *l+* mating type ([@R30]). When single ascospore isolates of *C. spinulosa* were originally produced in order to identify the species as heterothallic or homothallic, they segregated into both self-fertile and self-sterile individuals. The sexually competent individuals were shown to have arisen from the larger spores. Those remaining barren of sexual structures had been produced from smaller spores and could only undergo sexual reproduction when co-cultured with individuals derived from large spores ([@R30]). Interestingly, the asci produced during the self-fertile, *l+* sexual cycle of *C. spinulosa* possessed four large and four small ascospores. This is despite the absence of an interaction with the *l* mating type partner which would have produced the small ascospores in a typical heterothallic reaction ([@R30]). The most likely explanation for this phenomenon is 'unidirectional mating type switching'. In this situation, hyphae of the *l+* mating type are able to switch *via* mating type mutation to the *l* mating type. This results in a mixed mating type culture that is then able to sexually reproduce in a functionally heterothallic process ([@R30]). The mechanism is treated as unidirectional because individuals derived from small ascospores are unable to switch their mating type and subsequently remain sexually inactive unless co-incubated with a suitable partner. Unidirectional mating type switching is well-known in some species of *Ceratocystidaceae* ([@R8]), a group of important plant pathogens but one that has not received substantial attention from fungal geneticists. This sexual strategy was first recognized in the sweet potato black rot fungus *Ceratocystis fimbriata* ([@R56]). More recent studies of mating type switching in species of *Ceratocystidaceae* ([@R19], [@R63], [@R60]). [@R60] and [@R25] have shown that self-fertile isolates of *C. fimbriata* and *C. albifundus* have three genes at the *MAT1* locus, two of the *MAT1-1* idiomorph (*MAT1-1-1* and *MAT1-1-2*) and one of the *MAT1-2* idiomorph (*MAT1-2-1*). Ascospores produced during sexual reproduction segregate into those that produce self-fertile isolates and those that produce self-sterile isolates. Genetically, the switch from a self-fertile to a self-sterile mating strategy is the result of the complete deletion of that *MAT1-2-1* gene from the genome. This results in isolates with only *MAT1-1* genes ([@R63], [@R60]). How this change occurs has not been resolved, but it is thought to involve homologous recombination due to identical repeats that flank the *MAT1-2-1* gene ([@R60]). It remains necessary to determine the significance of the unidirectional mating type switching mechanism in *Ceratocystidaceae*. This is especially relevant considering that the self-sterile isolates with the *MAT1-2-1* deletion are significantly less fit than their self-fertile counterparts ([@R25]). It would thus be interesting to determine whether the unidirectional mating type switching observed in *C. fimbriata* could resemble the functionally heterothallic system seen in *Chromocrea spinulosa*, which would then preserve the self-sterile isolate despite its reduced fitness. The existence of pseudohomothallism and mating type switching systems challenges our understanding of the concept of homothallism. In populations displaying these systems, fungi are considered functionally heterothallic, despite both mating partners being derived from a single originating cell. It has been speculated that these mating systems allow for the preservation of homothallic mating under circumstances where genetically distinct opposite mating partners are not easily accessible. Additionally, the ability to outcross under conditions where functional recombination is possible is also conserved ([@R26]). Nevertheless, systems such as pseudohomothallism and mating type switching preclude simplistic classification of fungi as either homothallic or heterothallic. While it is convenient to identify fungal species as either homothallic or heterothallic, such designations are often times naïve, especially when the species in question has both types of individuals. Not surprisingly, fungal classification by mating strategy is virtually impossible, not to mention very contentious ([@R62], [@R28]). UNISEXUALITY-- AN UNUSUAL FORM OF HOMOTHALLISM {#s4} ============================================== [@R16] discovered a novel form of homothallism in *Neurospora africana*. The same phenomenon was subsequently described in three other *Neurospora* species, *N. galapagosensis*, *N. dodgei*, and *N. lineolata* ([@R4]). While typically heterothallic *Neurospora* species occur and have either the *mat A* (homologous to *MAT1-1*) or the *mat a* (homologous to *MAT1-2*) idiomorphs ([@R14], [@R51]), some are self-fertile. These species have both the *mat A* and *mat a* idiomorphs in a single genome and in this regard, they exhibit primary homothallism ([@R4]). However, only the *mat A* idiomorph has been found in the homothallic *N. africana* and related species. They consequently provide an apt example of sexual reproduction in the absence of typically essential *MAT* genes ([@R16]). Interestingly, however, when closely-related heterothallic species are transformed with the *N. africana mat A* sequence, the self-fertility is not transferred ([@R16]). This suggests that other genetic mechanisms are responsible for the homothallic behaviour and that self-fertility is not the result of a unique function of the *mat A* sequence in the species. The mating strategy in *N. africana* and species with the same behaviour has recently been termed unisexual reproduction. Its discovery and recognition represents a paradigm shift in the way we view homothallism. In all previously defined forms of homothallism (primary and secondary), the presence and expression of genes from both the *MAT1-1* and *MAT1-2* idiomorphs play an essential role in the initiation and full process of sexual reproduction. This is regardless of the origin or physical location of these genes. What is unique about unisexual reproduction is that it describes an atypical system where species are able to complete an entire sexual cycle when only genes from a single *MAT* idiomorph are expressed ([@R47]; [Fig. 1](#F1){ref-type="fig"}). A comprehensive description of the genetic and biochemical mechanisms underlying unisexual reproduction has been presented using the basidiomycetous yeast *Cryptococcus neoformans* as a model ([@R27], [@R12], [@R36]). This species, which has a well-characterized heterothallic sexual cycle, has also been shown to undergo **α**-**α** cell mating, or unisexual reproduction ([@R27]). Unisexual reproduction has been linked to the pathogenicity of the species and thus has important implications in terms of human health. Naturally occurring populations of *C. neoformans* have been shown to be highly clonal, consisting of \>99 % **α** cells ([@R24]). Before unisexual reproduction was discovered in the species, this clonality was attributed to high levels of asexual reproduction and an almost non-existent heterothallic mating system, although **α** and **a** individuals have been known to mate ([@R23]). It was subsequently observed, however, that **α** cells were able to undergo a tissue differentiation process similar to that seen during opposite sex mating and that this was the cause of the bias of **α** cells in *C. neoformans* populations ([@R59]). This system was originally referred to as "monokaryotic fruiting" and was thought to be a strictly mitotic event. It was only more recently recognized as a sexual event that relies on essential meiotic genes, and unisexual reproduction was consequently described in the species ([@R27]). The unisexual cycle in *C. neoformans* can be initiated *via* one of two pathways. One of these involves endoreplication of the entire genome of a single cell and represents the strictly homothallic version of unisexual reproduction. Alternatively, two cells of the same mating type can undergo cellular and nuclear fusion. The later pathway represents an almost heterothallic system, where outcrossing still takes place despite an identical *MAT1* locus. In both cases, the resulting diploid cell undergoes filamentation and subsequent basidium formation. Meiosis then takes place in the basidium and basidiospores are produced ([@R27]). The unisexual process relies on almost all the genes that are essential in typical bisexual mating. Thus the sexual spores are similar in both types of sexual reproduction, with the exception that unisexual reproduction produces spores of only the **α** mating type. Consequently, it has been suggested that unisexual reproduction is able to confer the benefits of sexual reproduction. This would, for example, be through allowing genetic recombination while minimizing the cost required to locate a partner, thereby efficiently producing infectious sexual spores in a seemingly clonal population ([@R43]). A unisexual cycle has also been described in the ascomycetous yeast *Candida albicans*. This species has long been thought of as an asexual, obligate diploid unable to produce sexually recombinant progeny ([@R38]). In addition to unisexual reproduction, *C. albicans* can also engage in parasexuality under suitable circumstances. The identification of a mating type-like locus ([@R21]) and the discovery of sexually-competent mating partners made it possible to characterize a parasexual cycle in the species ([@R5]). This cycle involves two diploid cells, **a**/**a** and **α**/**α**, undergoing cell and nuclear fusion resulting in an unstable tetraploid, which subsequently undergoes concerted chromosome loss until a near-diploid state is reached ([@R5]). Unisexual reproduction in *C. albicans* can be achieved *via* one of two independent pathways, both of which allow sexual reproduction between cells of the **a** mating type ([@R2]). The first involves the mutational inactivation of the *Bar1* gene. In this species, the *Bar1* gene product is a protease that is produced by **a** cells in order to degrade the production of endogenously-produced **α** pheromone ([@R50]). When this protease is defective, **a** cells are able to respond to this self-made pheromone by the activation of Ste2, the receptor for this pheromone. This leads the cell to initiate sexual reproduction as it would in the presence of a pheromone-producing **α** cell ([@R2]). The second pathway by which same-sex matings can be achieved involves ménage à trois matings. Here, **a**-**a** mating can occur due to the production of the **α** pheromone by a limited number of **α** cells in the population. In this case, **a** cells are able to recognize the exogenously-produced **α** pheromone despite the Bar1 protease activity and can initiate sexual reproduction with a second **a** cell. This form of same-sex mating is also found when a limited number of **a** cells are co-incubated with **α** cells which are then able to undergo **α**-**α** mating ([@R2]). The most recently encountered example of unisexual mating is in the filamentous ascomycete *Huntiella moniliformis* ([@R61]), a saprobic species of *Ceratocystidaceae* and previously treated in *Ceratocystis* ([@R8]). Unisexual reproduction in this species appears to resemble that in *N. africana*, where only a single mating type, MAT-2, has been identified despite an extant sexual cycle existing. A closely-related heterothallic species, *H. omanensis*, possesses individuals of the MAT-1 mating type as well as the MAT-2 mating type and exhibits a typically heterothallic sexual cycle. Surprisingly, unisexual reproduction is not seen in the latter despite the *MAT1-2* idiomorph being highly conserved between these two species ([@R61]). Unisexual reproduction is clearly an incompletely described form of homothallism. Although it is currently known in only a small number of species, this does not necessarily mean that it is an uncommon sexual strategy. When it was originally described in *N. africana*, the evolutionary persistence of the mechanism was questioned due to the negative effects associated with inbreeding. However, the discovery of unisexual reproduction in three related *Neurospora* species has emphasized the relevance of unisexual mating as an evolutionarily significant reproductive strategy in fungi ([@R15], [@R16], [@R47]). Furthermore, it has been shown in *Cryptococcus neoformans* that homothallic unisexual reproduction allows for the production of limited genotypic and subsequent phenotypic diversity *de novo*. From an evolutionary perspective, this would be beneficial because already well-adapted genotypes are maintained while allowing for a restricted level of genetic admixture to take place within a population ([@R36]). CONCLUSIONS {#s5} =========== Self-fertility allows for reproductive assurance in species across all major groups of eukaryotes. In fungi, this condition is known as homothallism and ensures that a single individual is able to undergo sexual reproduction, even when a suitable mating partner is not present in the environment. Sexual spores that are typically more environmentally resistant can thus be produced, allowing for growth and persistence in unfavourable environments ([@R1]). Sexual reproduction also allows for recombination, which in the case of homothallism could provide fungi that reproduce predominantly clonally with a means of escaping the accumulation of deleterious mutations ([@R35]). Homothallism in fungi clearly encompasses a wide variety of genetically distinct mechanisms that all result in sexually reproducing cultures from single originating cells. This review highlights the need for a classification system that will make it possible to fully describe the sexual strategies employed by self-fertile fungi. It also provides a number of molecular and genetic determinants that can aid in the delineation of homothallism into its discrete subcategories. Clearly, homothallism is far more complex than was originally believed, and much work still needs to be done to fully understand the various mechanisms underlying its behaviour. The availability of genome sequences for non-model species is already aiding in this research, allowing for the characterization of the *MAT1* locus in addition to classic mycological techniques used to identify the sexual status of a fungus. Unisexual reproduction has emerged as an intriguingly unique reproductive strategy in fungi. Its unexpected discovery in *H. moniliformis* leads us to question whether it might not be much more common in fungi than has previously been imagined. That fungi with already well-described homothallic or heterothallic sexual cycles have now also been described as unisexual suggests that many other species could also exhibit this dual mating behaviour. For example, the tree pathogen *Cryphonectria parasitica* is described as homothallic but it preferentially outcrosses in nature, a situation relatively common in species of *Cryphonectriaceae* and *Dothideomycetes* ([@R32]). Could it be that these fungi also have the capacity to undergo unisexual mating? The numerous and varied mechanisms *via* which self-fertility has been maintained in fungi suggests that homothallism represents an evolutionarily significant mating strategy. Self-fertility provides these species with the numerous benefits associated with sexual reproduction while minimizing its costs, particularly the costs associated with locating a mating partner. The ability of many species to reproduce sexually both *via* a heterothallic and homothallic cycle illustrates a fascinating level of reproductive plasticity in fungal mating systems. It allows species to outcross or inbreed depending on the particular environment which they face ([@R47]). That most fungal species do not sporulate actively in culture has posed a major challenge relating to the understanding of sexual reproduction in fungi. It is for this reason that most genetic studies have been undertaken in so-called model species, which are typically easy to maintain and mate in culture. However, the increasing availability of fungal genome sequences will allow a much more comprehensive understanding of the reproductive strategies in the fungi. This will be for both model and non-model species and makes for exciting prospects in the near future. ACKNOWLEDGEMENTS {#s6} ================ This project was financed by the University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the Genomics Research Institute (University of Pretoria Institutional Research Theme). This work is based on research supported in part by a number of grants from the National Research Foundation of South Africa (including Grant specific unique reference number (UID) 83924). The Grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by NRF supported research are that of the author(s), and that the NRF accepts no liability whatsoever in this regard. We would also like to acknowledge Glenda Brits of Education Innovation at the University of Pretoria for producing the illustrations used in this review. {#F1}

State of the Art and Perspectives of *Vibrio* Research in Europe ================================================================ According to the European Environment Agency the rise of global sea surface temperature (SST) is one of the major physical impacts of climate change. However, SST in coastal European seas has increased 4--7 times faster over the past few decades than in the global oceans ([@B59]). This local increase in SST has been linked to outbreaks of *Vibrio*-associated human illness caused by *Vibrio cholerae* non O1-non-O139, *V. parahaemolyticus*, and *V. vulnificus* in several European countries (Table [1](#T1){ref-type="table"}). However, the lack of mandatory notification systems for *Vibrio*-associated illnesses prevents accurate estimates of the number of *Vibrio* infections occurring in Europe. Also mass mortalities of marine animals increase in frequency (Table [1](#T1){ref-type="table"}), particularly in heavily polluted coastal areas, suggesting human activities as a factor favoring disease epidemics. Prominent examples include several *Vibrio* species associated with the recent great devastation of oyster beds in France. The salmonid farming industry is constantly threatened by *V. salmonicida* and *V. anguillarum*. Moreover, different subspecies of *Photobacterium damselae* are associated with diseases in cultured fish species like sole, sea bass, sea bream and turbot, while *V. vulnificus* causes hemorrhagic septicaemia in eel, derbio, tilapia, trout and shrimps but can also cause septicemia in humans. Finally, evidence has accumulated linking *Vibrio* infections (e.g., *V. coralliilyticus*) to increasing mass mortalities of benthic corals (e.g., *Paramuricea clavata*) in the NW Mediterranean Sea. ###### **Recent Vibrio-associated diseases caused by Vibrio in Europe**. **Agent** **Pathogenic to** **Country** **References** ---------------------------------------------------------------------- ------------------------------------------------ ----------------------------- ---------------- *V. parahaemolyticus, V. vulnificus and non-O1/non-O139 V. cholerae* Human Germany [@B25] *V. parahaemolyticus* Human France [@B56] *V. parahaemolyticus* Human Spain [@B41] *V. parahaemolyticus* Human Italy [@B49] *V. cholerae* Human Sweden [@B3] *V. cholerae non-O1-non-O139* Human Italy [@B48] *V. cholerae non-O1-non-O139* Human Finland [@B39] *V. cholerae non-O1-non-O139* Human Poland [@B76] *V. cholerae non-O1-non-O139* Human Austria [@B26] *V. cholerae non-O1-non-O139* Human Austria [@B28] *V. vulnificus* Human Denmark [@B10] *V. vulnificus* Human Israel [@B5] *V. vulnificus* Human Spain [@B78] *V. vulnificus* Human Turkey [@B52] *V. vulnificus* Human, finfish, crustacean USA, Europe, Asia [@B2]; [@B47] *V. alginolyticus* Human Guernsey [@B58] *Vibrio* spp. Human North and Baltic Seas [@B68] *V. coralliilyticus* Coral Italy [@B84] *V. crassostreae* Oyster France [@B32] *V. aestuarianus* Oyster France [@B24] *Harveyi clade* Finfish, crustaceans, mollusks Mediterranean countries [@B55] *V. anguillarum* Finfish Northern European countries [@B20] *Photobacterium damselae* subsp. *Damselae* Fish, humans, crustaceans, mollusks, cetaceans Mediterranean countries [@B64] To cover the large diversity of infectious vibrios, the development of operational tools to identify and detect emergent pathogens is essential to zoosanitary monitoring of cultivated species as well as on wild animal populations. Yet, compared to human pandemic strains, little is known about the virulence mechanisms of emergent environmental vibrios. This lack of knowledge may be attributed to the high genetic diversity of *Vibrio* isolates and the diversity/plurality of virulence mechanisms. To date pathogenic capacity cannot be inferred by taxonomic affiliation, because virulence factors (e.g., secretion systems, toxins) are rarely species-specific and are often shared between *Vibrio* species by lateral gene transfer. On top of that there are very few animal models to distinguish pathogenic strains and extend our understanding of the mechanisms involved in host-microbe interactions. Hence the elucidation of virulence for agent and target is a prerequisite to develop prophylactic methods to fight infectious diseases. Due to the extent of the environmental, economical, and public health consequences resulting from *Vibrio* infections, a large scientific community is working on these bacteria in Europe. In order to join fundamental and applied research teams and to investigate the emergence of pathogens in natural *Vibrio* populations, we organized the first European workshop dedicated to the research on vibrios in Paris (11--12th March 2015), that provided a forum for experts in *Vibrio* ecology, evolution and pathogenesis to address societal issues involving ocean health and food security. *Vibrio* Spread in Europe linked with climate change ---------------------------------------------------- Vibrios preferentially grow in warm (\>15°C) saline aquatic environments. Warming of marine and saline inland waters is likely to support larger numbers of *Vibrio* populations and consequently an increased risk of *Vibrio* infections. An increase in the prevalence of human infections caused by *V. parahaemolyticus*, *V. cholerae* non-O1-non-O139 and *V. vulnificus* has been recorded in Europe even at high latitudes ([@B4]). In northern Europe, the increase in reported infections corresponds both in time and space with spikes in domestically-acquired *Vibrio* cases in "heatwave" years. Similarly, samples collected in the last 60 years by the continuous plankton recorder (CPR) survey ([@B82]) showed that the genus *Vibrio*, including the human pathogen *V. cholerae*, has increased in prevalence in the last 44 years in the coastal North Sea, and that this increase is correlated with warming SST. Elevated water temperatures might also facilitate the successful invasion of pathogenic variants via food trade ([@B44]), ballast water ([@B16]), travelers ([@B19]) or natural animals. For example, migrating birds may act as vectors of intercontinental transport of *V. cholerae* ([@B83]). The direct comparison of the population structure of *V. cholerae* from a major bird sanctuary (Lake Neusiedl, Austria), with strains collected from six other European countries revealed that several strains in the lake shared the same alleles with other European strains, consistent with pan-European transport between distant ecosystems via birds (A. Kirschner, unpublished data). As a future challenge, macro-ecological studies on the impact of climate change on *Vibrio* persistence and spread in the aquatic environment combined with studies investigating climate change effects on epidemiologically relevant variables, such as host susceptibility and exposure are needed to significantly improve prediction and mitigation strategies against the future occurrence of *Vibrio* disease outbreaks. Virulence as a Function of Biotic Interactions With Host and Microbiome ----------------------------------------------------------------------- Virulence is a widespread phenomenon across the *Vibrio* phylogeny ([@B85]). Its expression critically depends on biotic interactions with the host but also with other resident microbiota. On the host side, spatially-structured cross-infection experiments indicated that virulence of only distantly related *Vibrio* strains was lower when infecting oysters from the same geographic location. This suggests that oyster hosts are locally adapted and have evolved resistance to genetic factors shared within *Vibrio* populations ([@B86]). When considering interactions of *Vibrio* with the resident microbiome, the hemolymph microbiome modulates infections but is vulnerable to environmental disturbance ([@B38]). Accordingly, *Vibrio* disease cannot be seen as an isolated event but needs to be considered in the context of the microbiome, which includes other non-virulent *Vibrio.* Indeed, the successive replacement of non-virulent with virulent strains during oyster infections occurs in the natural environment ([@B32]) and the amplification of virulence in the presence of non-virulent strains suggests that also non-virulent strains contribute directly or indirectly to the development of disease. Future research on *Vibrio* disease should therefore focus on the higher order biotic interactions between the environment, the host and the pathogenic as well as the non-pathogenic fractions of microbial communities. A key feature of the interaction between microbes within a community is the production of molecules that determine behavior like antagonism, competition or cooperation. Cell-to-cell communication in vibrios coordinates virulence gene expression based on the biotic and abiotic environment ([@B11]). For example, the three-channel quorum sensing (QS) system of *V. harveyi* controls the pathogenicity of the bacterium toward different aquatic hosts ([@B13]; [@B51]), and our most recent research revealed that another signaling molecule, indole, controls the virulence of *V. anguillarum* toward sea bass larvae ([@B35]). Another potential signaling mechanism has been described based on the production and release of high concentrations of D-amino acids into the extracellular milieu ([@B30]). First discovered in *V. cholerae*, these D-amino acids are different from those known to be part of the cell wall in bacteria (D-Ala and D-Glu) and were therefore called non-canonical D-amino acids (NCDAAs; [@B8]). NCDAAs released into the media by producer strains can affect non-producer organisms beneficially or detrimentally in a particular niche ([@B9]; [@B1]). The possible implications of NCDAAs in the biological processes of co-inhabitants still remains to be investigated but the enormous energy demand suggests that these molecules should have a great impact in poly-microbial communities. Finally several strains of *Vibrio* have been demonstrated to produce potent antibacterial agents (andrimid and holomycin) or agents that block QS regulated genes (solonamides, ngercheumicin) in human pathogens ([@B87]; [@B40]; [@B29]; [@B46]). Comparative and functional genomics using software like antiSMASH ([@B42]) could identify the genetic determinants of these secondary metabolites (polyketide synthases and non-ribosomal peptide synthetases). Hence further elucidation of virulence regulatory mechanisms will enable us to better understand *Vibrio*-host interactions and ecology, and to identify targets for the design of novel agents to control disease caused by vibrios. Horizontal Gene Transfer, Genome Plasticity, and Chromosome Partitioning ------------------------------------------------------------------------ Evolution of *Vibrio* species is often driven by mobile genetic elements via horizontal gene transfer (HGT). However, very little is known about HGT in environmental *Vibrio* isolates infecting marine organisms. In 75 marine *Vibrio* spp. isolated from the broad-nosed pipefish, *Syngnathus typhle*, associated prophages were characterized and the virulence of strains carrying different prophages was then assessed by comparing the relative expression of 44 immune genes during controlled infection experiments on juvenile pipefish. Preliminary results suggest that virulence is significantly influenced by the associated prophages, further supporting a role for bacteriophages in manipulating the virulence of environmental *Vibrio* isolates (C. Wendling unpublished data). Virulence of *V. vulnificus* and *P. damselae* subsp. *damselae* in fish is determined by transferable plasmids (pVvbt2 in *V. vulnificus* and pPHDD1 in *P. damselae*). pVvbt2 contains two highly conserved virulence genes involved in serum resistance (*vep07*) and the ability to grow from eel transferrin (*vep20*) ([@B50]). Interestingly, pPHDD1 also contains *vep07* and *vep20* homologs suggesting that both genes are involved in resistance to fish innate immunity. pVvbt2 also encodes RtxA1~3~, a toxin belonging to MARTX (multifunctional, autoprocessive, repeat in toxin) family. RtxA1~3~ is considered a host-non-specific virulence factor because it is involved in resistance to phagocytosis by murine and human phagocytes as well as in eel death ([@B31]). The other virulence plasmid, pPHDD1, encodes phospholipase-D damselysin (Dly) and the pore-forming toxin HlyA~*pl*~ ([@B62]). A second HlyA (HlyA~*ch*~) is encoded in chromosome I ([@B63], [@B65]) and the three toxins contribute to hemolysis and virulence, and are secreted by a type-two secretion system ([@B66]). While the two HlyA hemolysis produce an additive effect, Dly and any of the two HlyA interact in a synergistic manner, being responsible for maximal virulence for fish and for mice ([@B63]). Due to their host range and their duality as pathogens for both poikilotherm and homeotherm animals, *P. damselae* and *V. vulnificus* constitute valuable biological models to study the role of mobile genetic elements in the rise of novel pathogenic strategies. Vibrios contain large chromosomal integrons ([@B7]) and belong to the group of naturally competent bacteria, which allows them to absorb free DNA from their surrounding environment and recombine it into their genome ([@B69]). For *V. cholerae*, entry into competence is tightly regulated and requires growth to high cell densities on chitinous surfaces ([@B43]; [@B36], [@B37]). Uptake of external DNA is accomplished by a sophisticated DNA-uptake machinery ([@B70], [@B71]; [@B72]). As the competence regulon also encompasses the type VI secretion system-encoding gene clusters, HGT is enhanced through deliberate killing of neighboring non-sibling cells followed by the transfer of their DNA ([@B6]). The presence of two chromosomes is another characteristic feature of vibrios. While distinctive localization patterns have been described for the two chromosomes, the selective advantages brought by this bipartite architecture are still under debate ([@B79], [@B80]). Replication of both chromosomes is tightly coupled so that replication termination is synchronized ([@B57]). Moreover, the chromosomal position of genes determines the relative copy number during growth thereby impacting the bacteriums physiology ([@B75]). Notably, mechanistic aspects of chromosome organization, architecture, and cell cycle-dependent dynamics are only starting to be deciphered ([@B88]; [@B14]). The elucidation of the mechanisms that coordinate the interplay between chromosomes, accessory replicons, mobile DNA and HGT mechanisms is essential to better apprehend the evolution and niche adaptation of *Vibrio* species. Adaptation of Pathogenic Vibrios to Intracellular Life ------------------------------------------------------ The pathogenic *V. tasmaniensis* strain LGP32, a member of the *V. splendidus* clade ([@B22]) was found to be a facultative intracellular pathogen of oyster immune cells called hemocytes ([@B18]). This is a rare example of *Vibrio* adapted to intracellular life. The virulence of LGP32 in oysters correlated with the ability to enter hemocytes ([@B17], [@B18]). Both cellular invasion and pathogenicity depend on the major outer membrane protein OmpU, which serves as an adhesin to invade host cells. Once inside the phagosome, LGP32 releases outer membrane vesicles (OMVs) that protect the organism against antimicrobial peptides and act as vehicles for the delivery of virulence factors ([@B15]; [@B81]). Moreover, entry into hemocytes and intracellular survival of LGP32 are required for expression of LGP32 cytotoxicity toward hemocytes. This capacity to survive intracellularly relies on potent antioxidant and copper tolerance responses, both of which are highly induced in the hostile environment of the phagosome. Small regulatory RNAs have been shown to play important roles in regulating virulence gene expression in response to conditions encountered in the host. sRNAs present in multicopies such as Qrrs and CsrBs were found in several *Vibrio* spp. to mediate QS regulation of virulence gene expression ([@B45]). One peculiarity of the *Splendidus* clade seems to be the presence of four highly expressed copies of the CsrB sRNAs in their genome, instead of 2--3 found in other vibrios ([@B33]; [@B77]). CsrB sRNAs are highly transcribed inside oyster hemocytes suggesting a role in adaptation to the intracellular environment (Vanhove et al., submitted). The landscape and phylogeny of putative sRNAs encoded by LGP32 demonstrate rapid vertical evolution, with a vast majority of sRNAs being species/strain specific, and only a small number (28/250) conserved in all *Vibrio* sequenced so far ([@B77]). Thus, sRNAs contribute to a high diversity between species and provide opportunities for adaptation/colonization of new hosts and virulence emergence, a question that will be tackled by comparative functional studies of conserved *Vibrio* sRNAs. Model Systems to Study Pathogenicity Mechanisms and *Vibrio*-host Interactions ------------------------------------------------------------------------------ Microbiologists are increasingly aware that how organisms behave *in situ* in the "real world" might be distinct from those that occur in laboratory monocultures grown under tightly controlled conditions ([@B74]). Thus, model systems, which replicate at least part of the natural processes of infection, are needed in order to examine the relevance and biological impact of *in vitro* findings. *In vivo* models that reproduce the main clinical and pathological signs of disease seen following the consumption of contaminated food or water, are available for toxigenic and non-toxigenic *V. cholerae*, and for *V. parahaemolyticus* ([@B60], [@B61]; [@B73]). In these studies, a combination of microbiological, histological and genetic analysis was used to identify key virulence factors and the pathologic mechanisms associated with the respective strains (e.g., see [@B89], [@B90]). However, a growing number of *Vibrio*-associated illnesses are associated with a diverse group of strains, some of which lack known virulence factors ([@B21]; [@B27]; [@B49]). A future challenge will be to examine the pathogenesis of these strains and identify additional virulence markers, which should be used to improve risk assessment tools targeted to the different pathogens. Furthermore, a growing number of human *Vibrio* infections in Europe were not food-borne, but instead associated with the ability of non-O1-non-O139 *V. cholerae*, *V. parahaemolyticus*, or *V. vulnificus* to cause septicemia via wound infections (Table [1](#T1){ref-type="table"}). Models to examine this aspect of their pathogenicity are currently lacking and should become a high priority given the poor prognosis of individuals acquiring this type of infection. Next to models for human pathogens there is also an increasing need for aquatic animal models. However, studies aimed at investigating the pathogenicity mechanisms in aquatic hosts are often confounded by the presence of the natural microbiota (which usually contains *Vibrio* spp.). Gnotobiotic animals provide researchers with a means to examine host-microbe interactions without interference or influence from unknown microbiota ([@B23]). A model based on the use of gnotobiotic 1-day old larvae of brine shrimp (*Artemia franciscana*) has been recently developed to study *V. campbellii*, *V. harveyi*, or *V. anguillarum* pathogenesis ([@B12]). An alternative model system for *V. anguillarum* involves the use of gnotobiotic European sea bass (*Dicentrarchus labrax*) larvae, where survival is monitored over 1 week ([@B34]). Finally, specific-pathogen-free (SPF) juveniles of *C. gigas* ([@B54], [@B53]) have been developed to investigate the diversity and dynamics of microbial populations in an oceanic environment during disease. When combined with methods to monitor gene expression and activity of vibrios during infection (e.g., [@B67]; [@B13]), a better understanding of the infection process(es) will emerge. Conclusion ========== This workshop clearly demonstrated the importance of vibrios to our understanding of emergent diseases in marine and inland aquatic ecosystems as well as their potential impact on society. The rising frequency of disease events not only affects humans directly but also indirectly by reducing food security and ecosystem health. The synergistic investigation of mechanistic and ecological processes contributing to disease is therefore paramount for our understanding of the larger scale consequences of changing *Vibrio* populations. A better understanding of *Vibrio* ecology is pivotal for the development of prevention and mitigation strategies. In addition, the mechanistic knowledge of virulence regulatory mechanisms could ultimately be used to inhibit disease. However, these tasks are complicated by the high diversity present within *Vibrio* populations, and the fact that biotic interactions within and between microbial communities, modify disease expression on different levels. Therefore, we have to consider *Vibrio* disease as an emergent, multi-faceted phenomenon that will require experimental model systems covering molecules to whole organisms. Expertise for most of these crucial challenges already exists and became united at the workshop under the European umbrella of *Vibrio* research thereby fostering a more productive combination of basic and applied research in the future (Figure [1](#F1){ref-type="fig"}). {#F1} Conflict of Interest Statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We warmly thank EUROMARINE, EMBRC-France, the Agence Nationale de la Recherche (ANR) for their financial support and EFOR organization to this first workshop. [^1]: Edited by: *Maurizio Labbate, University of Technology, Sydney, Australia* [^2]: Reviewed by: *Daniela Ceccarelli, University of Maryland, USA; Yan Boucher, University of Alberta, Canada; Fabiano Thompson, Federal University of Rio de Janeiro, Brazil* [^3]: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology.

Main Text {#sec1} ========= Introduction {#sec1.1} ------------ The hippocampus is placed firmly at the centre of a network supporting memory function [@bib1], [@bib2], [@bib3], [@bib4], [@bib5]. In humans, hippocampal damage is associated with wide-ranging impairments in autobiographical memory [@bib1], [@bib6] as well as profound deficits in spatial memory, manifesting as a loss in the ability to navigate flexibly through the world [@bib7], although some spatial knowledge can be retained [@bib3]. In rodents, lesions made to the hippocampus and associated structures have generated complementary results, including deficits in spatial working memory [@bib8], [@bib9], impairments in navigation to hidden spatial goals [@bib10], [@bib11], and a more general failure to recognise familiar environments [@bib12], [@bib13]. However, the hippocampus is far from being a simple static repository of past experiences. In patients, memory for distant events can be preserved even when that for more recent events is disrupted by hippocampal damage [@bib1], [@bib14], [@bib15]. This temporally graded retrograde amnesia has been taken as evidence that, with time, some memories become less dependent on the hippocampus and increasingly dependent on the cortex: a process known as systems consolidation [@bib16], [@bib17], [@bib18], [@bib19]. Whether all initially hippocampal-dependent memories are subject to consolidation is a point of some controversy [@bib20], [@bib21]. Nevertheless, careful lesion studies in animals provide support for this hypothesis, suggesting that under some circumstances an offline process governs the modification of hippocampal memories, rendering them less susceptible to hippocampal damage [@bib15], [@bib22], [@bib23], [@bib24]. Equally, a separate body of work points to a role for the hippocampus in planning and future thinking, that is, constructing potential scenarios. For example, patients with hippocampal damage have difficulty imagining the future [@bib25] and are unable to describe rich fictitious scenes [@bib26]. Moreover, functional magnetic resonance imaging (fMRI) indicates a distinct overlap between a network of brain areas, including the hippocampus, that are engaged during remembering as well as imagining events [@bib27], [@bib28]. Electrophysiological investigations of the hippocampus and associated regions in rodents and other animals, including humans, have identified some of the key neural elements supporting memory and spatial cognition. Place cells, typically pyramidal neurons from areas CA1 and CA3 of the hippocampus, exhibit stable, spatially constrained firing fields, known as place fields [@bib2], [@bib29], [@bib30], [@bib31] ([Figure 1](#fig1){ref-type="fig"}). When an animal is in motion, the activity of a population of such place cells provides an accurate representation of its self-location [@bib32], [@bib33]. Moreover, the fidelity of the place cell representation covaries with navigational accuracy, strongly implying that these cells are instrumental in guiding spatial decisions [@bib34], [@bib35]. Subsequently, several complementary classes of neurons have been identified, representing other aspects of an animal's position within the world: head direction cells, found throughout the limbic system, signal direction of facing [@bib36], [@bib37], [@bib38]; grid cells in the medial entorhinal cortex and para-subiculum represent self-location with an efficient periodic code [@bib39], [@bib40]; and border cells as well as boundary vector cells signal proximity to elongated features of the environment [@bib41], [@bib42], [@bib43]. Clearly the representation of self-location provided by these neurons is likely to play a role in spatial memory [@bib44]. It is also evident, however, that information about the animal's current position alone is insufficient to account for either consolidation or the apparent role of the hippocampus in future thinking and navigational planning.Figure 1Place cells are characterised by their stable spatial firing fields.(A) Standard configuration for place cell recording. A rodent with chronically implanted extracellular electrodes forages in an open enclosure. Upper right: the animal's path over the course of a 10-minute trial is indicated by the black line, action potentials from a single place cell are superimposed in red. Lower right: firing rate-map of the raw data indicating the mean firing rate of the cell per spatial bin. 'Hotter' colours indicate higher rates, peaking at 8.3 Hz (shown above the map); dark blue indicates low rates (0--20% of the peak rate); white bins are unvisited locations. (B) On exposure to an unfamiliar enclosure place cells 'remap', rapidly generating a novel representation; individual cells change their firing rate and field locations relative to each other and the environment [@bib32], [@bib64], [@bib158]. Recordings of four CA1 place cells (columns) made in similarly sized (70 cm square) enclosures located in different rooms (rows). Remapping is evident as a change in firing correlates and rates. Interestingly, in the last twenty years, it has become clear that the activity of place cells do not simply represent an animal's self-location but, under certain circumstances, also 'replay' past experiences [@bib45], [@bib46], [@bib47] and potentially construct new spatial trajectories [@bib48], [@bib49], [@bib50]. Here we present a review of the replay literature and critically assess evidence supporting its hypothetical role in memory consolidation and planning. Further, we describe key questions for future studies to address, emphasising the need for novel behavioural tasks and the development of new technologies that can more precisely identify and perturb hippocampal activity patterns in order to provide firm evidence for a functional role of replay in mnemonic function and goal-directed behaviour. Reactivation of Past Place Cell Sequences {#sec1.2} ----------------------------------------- It has been known for some time that hippocampal neurons transiently increase their firing rate during sleep [@bib51], [@bib52]. Indeed, in 1978, O'Keefe and Nadel [@bib2] noted that, during quiescence and quiet wakefulness, the local field potential (LFP) of CA1 was interrupted by short bouts (40--120 ms) of high frequency (140--250 Hz) oscillations superimposed on lower frequency deflections; the high frequency components were termed 'ripples' and the composite signal is known as a sharp wave ripple [@bib45], [@bib53]. It was during these sharp wave ripples that CA1 pyramidal cell activity was observed to dramatically increase [@bib2]. A decade passed, however, before the link between this phenomenon and prior experience was identified, with the first demonstration that place cells which have been active during recent exploration are most likely to be reactivated during a subsequent sleep session [@bib54]. Subsequent studies of hippocampal reactivations were aided greatly by the development of tools enabling concurrent recordings of large (\>50) numbers of place cells. In this context, Wilson and McNaughton [@bib45] recorded from 50--100 place cells per session while rats foraged on an elevated circular platform, as well as during prior and subsequent sleep sessions. They observed that place cell pairs which were temporally correlated during exploration also tended to exhibit correlated activity during the following period of sleep; in other words, cells with overlapping place fields fired together during rest. This relationship was not present in sleep recordings made before foraging. As such, they proposed that the preserved correlation between place cells represented a 'reactivation' of previous wakeful experience [@bib45]. Importantly, correlated activity was most pronounced during sharp wave ripples. Subsequently, the same group [@bib46] extended their work, showing that reactivations during sharp wave ripples explicitly recapitulated the relative timing of activity between cell pairs, strongly suggesting that sequences of spiking seen during awake behaviour were being reinstated; this effect was labelled 'replay'. Contemporary investigations of replay use similar methods, comparing the dynamic activity of cell pairs recorded during exploration with those observed during rest. Moreover, many recent studies explicitly compare sequences of place cells corresponding to entire trajectories, rather than the coactivation of cell pairs, effectively decoding the content of the replay event ([Box 1](#tbox1){ref-type="boxed-text"} and [Figure 2](#fig2){ref-type="fig"}). To facilitate sequence matching, the experimental environment typically consists of a track on which animals can follow only relatively simple routes, reducing the awake experience to a limited number of place cell sequences that can be identified during replay [@bib47], [@bib55], [@bib56]. Lee and Wilson [@bib47] were the first to apply this approach, recording place cells while rats ran back and forth on an elevated track. They showed that, during slow wave sleep, particularly in close temporal proximity to sharp wave ripples, replayed sequences of place cells corresponded to temporally compressed versions of trajectories that the animals had made in the preceding session. Indeed, the typical duration of a replay event varies between 100 and 300 ms [@bib47], [@bib56], [@bib57], a ∼20 times increase in speed over the actual experience [@bib58] --- though replayed trajectories occurring during REM sleep progress at a more natural speed [@bib59]. As with earlier work, replay of trajectories along the track were not detected in recordings made before the animal was placed into that environment.Figure 2Typical methodology for detecting and analysing replay.(A) Linearised ratemaps are generated based on recordings made while rodents traverse a track. (B) In a subsequent rest period or during pauses in a task, hippocampal replay is marked by a high frequency 'ripple' oscillation in the LFP (top trace), which is associated with elevated multi-unit place cell activity, lasting ∼100 ms (middle, top). (C) A Bayesian approach is used to calculate, for each temporal bin (x-axis, typically 10 ms bins), the probability of the animal's location on the track given the observed action potentials. Hot colours designate higher probability. A fitting procedure, typically enforcing a fixed velocity, is used to find the most likely trajectory encoded by the posterior probability matrix (shown in white, goodness-of-fit value indicated above the line). (D) To determine statistical significance a shuffling procedure is conducted. The cell ID of each place cell is randomly permuted such that the spike trains observed during the putative replay events are associated with different place fields. This process is repeated at least 100 times; on each iteration, the posterior probability matrix is recalculated and a goodness-of-fit for the best trajectory determined. (E) The goodness-of-fit obtained for the original event is ranked against the shuffled distribution, determining the probability of obtaining the goodness-of-fit value by chance.Box 1Analysing replay.Replay analyses typically consist of three distinct phases: first, detection of putative replay events; second, decoding the spike activity recorded during the putative events; and third, identification of *bona fide* replay events representing trajectories through the animal's environment.The most common strategies for detecting putative events are either to identify the ripple component (140--250 Hz) of sharp wave ripples in the LFP [@bib48], [@bib64], [@bib82], [@bib156] or to directly detect brief periods (40--500 ms) when multi-unit firing rates are markedly elevated (more than three standard deviations above the mean, for example) [@bib55], [@bib56], [@bib71], [@bib95].The content of putative replay events is then analysed by comparing the sequence of spikes emitted during the event with the sequence expected to arise as an animal traverses the environment ([Figure 2](#fig2){ref-type="fig"}, left). Because the vast majority of studies are conducted on tracks and runways, which constrain the animal's movements, this is a tractable problem (though see [@bib71]). Comparisons of the observed and expected spike sequences can be made using rank-order correlations (for example [@bib50], [@bib55], [@bib56]) but probability-based methods are more powerful. Commonly, the observed spike sequence is divided into short time windows (∼10 ms); Bayes formula with a uniform prior is then used to calculate the probability of the animal being at each position on the track given the observed spikes [@bib33], [@bib57], [@bib64], [@bib66], [@bib95], [@bib142], [@bib157]. Ratemaps, which define the expected firing rate of cells across the track, are generated during track running and firing rate probability distributions are assumed to be Poisson. Thus, each putative replay event results in a posterior probability matrix which expresses the probability of the animal's location at different points in the environment as a function of time ([Figure 2](#fig2){ref-type="fig"}, centre).Finally, replay events corresponding to trajectories through the recording environment are detected by assessing the extent to which the posterior probability matrix depicts incremental movement across some portion of the environment. Practically, this is often achieved by assuming that the replayed trajectory has a constant velocity, in which case a linear fit can be applied to the posterior probability matrix [@bib57], [@bib95]; the goodness-of-fit then provides a measure of the extent to which a contiguous path is represented.To determine significance and to control for differences in the number and distribution of spikes, this linear fit is compared against fits generated from shuffled data. A common strategy is to either randomise the identity of the cells from which the observed spikes originated or to randomise the location of the place fields on the track [@bib57]. Data from each putative event are shuffled \>100 times, each time calculating a posterior probability matrix, and establishing the best linear fit. The rank of the fit obtained for the experimental data relative to the shuffled population establishes the significance level ([Figure 2](#fig2){ref-type="fig"}, right). Different shuffling procedures effectively make different assumptions about what constitutes a valid null hypothesis and some shuffles are likely to be more conservative than others [@bib95], [@bib113]. For example, permuting spike assignments such that spikes from the same cell are allocated to different cells assumes a degree of independence that is not present in biological data, and is likely to result in an overly liberal criterion for the identification of replay. One possible solution is to conduct several different types of shuffle, accepting only replay events that are identified reliably by all methods [@bib57]. Subsequent work found that replay was not limited to 'offline' periods --- during sleep or quiet rest --- but could also be observed while animals were awake and engaging in simple tasks, such as eating, grooming, or while paused at decision points [@bib55], [@bib56], [@bib60] ([Figure 3](#fig3){ref-type="fig"}A). For example, Foster and Wilson [@bib55] found that replay trajectories were emitted when animals paused to consume a food reward at the ends of a track [@bib55]. Although 'online', such replay shares many of the characteristics observed during offline periods: both occur while the animal is stationary and theta-band oscillations (6--12 Hz) are absent from the LFP (though see [@bib61], [@bib62] for examples of replay, or forward 'sweeps', during theta states while animals were located at decision points during a spatial task). Indeed, while sensory features of the current environment can shape the content of online replay --- in larger environments, for example, the replayed trajectories tend to be longer [@bib57] --- that does not mean that it is a simple recapitulation of recent sensory experience. Like offline replay, it can encode paths remote to the animal's current position [@bib48], [@bib57] ([Figure 3](#fig3){ref-type="fig"}C) or through enclosures entirely separate from the current one [@bib63], [@bib64]. Finally, during both online and offline replay, the relative proximity of cells in the sequence is preserved [@bib55], [@bib56], [@bib65], [@bib66], but the absolute order can be reversed [@bib55] ([Figure 3](#fig3){ref-type="fig"}B), inverting the sequence experienced during awake behaviour. Indeed, because place cells are typically directional on a linear track and therefore disambiguate travel in either direction [@bib67], [@bib68], the place cell sequences generated during 'reverse' replay are never observed during normal exploration.Figure 3Replay types.(A) Replay occurs during rest/slow wave sleep ('offline' replay, left) [@bib45], [@bib47], [@bib85] as well as during brief pauses in awake behaviours ('online' replay, right), such as when stopping at a decision point during navigation [@bib48], [@bib55], [@bib56], [@bib117]. (B) Top: As an animal runs down a track, place cells are sequentially activated. Bottom: during replay, place cells can be reactivated in the same sequence as was experienced during running ('forward' replay, left) [@bib46], [@bib47] or in the opposite sequence ('reverse', replay, right) [@bib55], [@bib56], [@bib66]. (C) Online replay can depict locations proximal to the animal's current location ('local' replay, left) [@bib71], [@bib85], [@bib122] or be distant to the animal ('remote' replay, right) [@bib63], [@bib64], [@bib122]. Thus, a considerable canon of research describing the characteristic features of replay now exists. Although first identified during sleep, it is now evident that replay is not limited to offline states. The content of replay and conditions that modulate its occurrence seem to be equally diverse, and accordingly a number of distinct hypotheses describing a putative role for replay in mnemonic function and spatial cognition have been proposed. The two most prominent theories are that replay represents the mechanism underlying memory consolidation [@bib45], [@bib69] and that it may support planning during spatial decision making and goal-directed navigation [@bib56], [@bib70], [@bib71], [@bib72]. Replay as Systems Consolidation {#sec1.3} ------------------------------- The role of the hippocampus in episodic memory indicates that it is particularly important for the rapid --- 'one-shot' --- learning of events and places [@bib19]. However, theoretical studies indicate that this renders the hippocampal memory system prone to catastrophic interference, whereby new learning can rapidly disrupt or erase previously encoded memory traces [@bib73]. Hence, a process of consolidation after the initial encoding of a new memory trace is essential to transform that trace from a temporary, labile state to a more stable and permanent form (see Squire *et al.* [@bib74] for a recent review). Interestingly, early neuropsychological data also indicated that the hippocampus plays a time-limited role in memory storage, best characterised by a temporal gradient of retrograde amnesia [@bib1], [@bib3]. These findings led several authors [@bib16], [@bib17], [@bib18], [@bib19] to propose a theory of systems consolidation, whereby the 'offline' reactivation of hippocampal memory traces allows the gradual strengthening of complementary memory traces in the slower learning neocortex. Subsequent research, however, has both complicated the standard model of systems consolidation and indicated that memory consolidation processes extend far beyond simple hippocampus--cortex interactions. First, evidence suggests that intracellular and intra-regional processes aimed at stabilising changes in synaptic strength also contribute to the consolidation of recently acquired or reactivated memory traces. In addition, there is some debate over whether memory traces ever become fully independent of the hippocampus, given that the hippocampus can be involved in the retrieval of both recent and remote memory traces [@bib20], [@bib75] and that memory consolidation appears to operate on a timescale of decades in humans [@bib1], [@bib3], [@bib76]. In either case, the selective strengthening of specific intra-hippocampal cell assemblies seems likely to play an equally important role in memory consolidation. Second, the rate of consolidation has been shown to depend on the amount of related prior knowledge that is available --- the existence of appropriate schema [@bib23], [@bib77] --- and it has been suggested that the process of consolidation acts to compress mnemonic information by extracting the principal statistical features from multiple unique experiences [@bib24], [@bib73]. Hence, consolidation may be an active process by which new memory traces are selected and incorporated into the existing corpus of knowledge at variable rates and with differential success according to their content. Whatever the specific nature and function of the memory consolidation processes may be, it seems clear that the reactivation of neural activity patterns at the population level following encoding is likely to contribute to the permanence of cell assemblies and therefore behaviourally relevant memory traces. Moreover, this reactivation is likely to occur during sleep or other quiescent states which provide relief from potentially interfering sensory activity, and involve neural activity on a timescale that is well-suited to the induction of synaptic plasticity and therefore the stabilisation of cell assemblies [@bib78], [@bib79], [@bib80], [@bib81]. Given these requirements, Wilson and McNaughton [@bib45] hypothesised that hippocampal replay might represent the neural mechanism supporting systems consolidation; and subsequent research has generated considerable, though not unanimous, support for this proposition. If, as proposed, replay is important for stabilising behaviourally relevant memories, it might be expected that novel, recent, and salient experiences are preferentially reactivated: this appears to be the case. Replay, particularly reverse replay, more frequently represents novel as oppose to familiar environments [@bib55]. This effect, measured by cell-pair co-activations, was found to be most pronounced on the first day of exposure to a novel environment and diminished on subsequent days [@bib82]. Equally, during acquisition of a spatial alternation task, sharp wave ripples and related activity occurred more commonly after rats made a correct turn and were rewarded, as opposed to after unrewarded incorrect turns [@bib83]. In a similar vein, Csicsvari and colleagues [@bib84], [@bib85] demonstrated an explicit link between sharp wave ripple associated hippocampal reactivations and animals' subsequent performance on a spatial memory task. Specifically, rats were required to remember the location of multiple unmarked food wells and navigate quickly between them. The number of goal location reactivations during both rest and task engagement predicted performance; though a causal relationship was not established. Ensuing studies went further, however, and showed that the electrical interruption of sharp wave ripples, and presumably replay, reduced the rate at which animals acquired a spatial memory task [@bib69], [@bib86] ([Figure 4](#fig4){ref-type="fig"}A). Hence there is a compelling, albeit incomplete link between the occurrence of replay and successful retention of information. Nonetheless, the location and extent of plasticity being instantiated by replay remain unclear.Figure 4Replay as consolidation.(A) Sharp wave ripples in CA1 were detected and disrupted (via electrical stimulation) while animals slept (left) following training on a spatial reference memory task (learning the location of food on an eight-arm radial maze, middle). Animals acquired the task more slowly and consistently performed worse than control animals receiving stimulations outside sharp wave ripples (right) [@bib69]. (B) Grid cells from the deeper layers of the medial entorhinal cortex and CA1 place cells were co-recorded while animals ran on linear runways (top) and during a subsequent rest session. Grid cell activity was significantly coordinated with place cell activity during hippocampal replay events recorded during rest, such that grid cells expressed similar spatial positions to place cells during replay (bottom) [@bib95]. (C) Rats encoded the location of two objects in a rectangular arena (left), sharp wave ripples (from CA1) and delta-spindle sequences (from medial prefrontal cortex) were recorded during subsequent sleep (middle). The co-occurrence of hippocampal and cortical rhythms was associated with memory of the object locations, indexed by preferential exploration of a displaced object in the post sleep session (right). If the duration of encoding was shortened to impair learning, consolidation could be rescued by stimulating the cortex, when sharp wave ripples were detected, thereby inducing delta-spindle sequences [@bib103]. The standard theoretical view of systems consolidation has focused on the diminishing requirement for hippocampal involvement in memory retrieval that occurs with time [@bib16] whereas alternative explanations assume a sustained role for the hippocampus in conjunction with intra-hippocampal reorganisation of memories [@bib21]. While it is too early to settle this long-standing debate, it is clear that replay is well placed to generate offline changes in both hippocampal and cortical memory networks. In the Csicsvari group studies [@bib84], [@bib85] described above goal location reactivations were observed to co-occur with a clustering of CA1 place fields around the goal, though curiously not those in CA3, suggesting that offline replay shapes hippocampal memories. This link was made explicit by recent work [@bib87] in which the activity of a sub-population of CA1 place cells was disrupted during sharp wave ripples and the stability of the manipulated place fields alone was reduced, although in this case the manipulation was made while animals remained in the test environment. It is also clear that sharp wave ripple associated activity propagates from the hippocampus to neighbouring structures such as the pre-subiculum and para-subiculum, as well as medial entorhinal cortex [@bib88], [@bib89]. In fact, a number of authors [@bib90], [@bib91], [@bib92] have investigated similarities between activity patterns during wakefulness and quiescence in various extra-hippocampal regions. These studies have frequently used an 'explained variance' [@bib92] approach to identify reactivations, that is, a measure of the degree to which activity patterns observed during post task rest can be accounted for by wakeful activity patterns. Recording from the ventral striatum while rats performed T-maze spatial alternation, Pennartz *et al.* [@bib92] found significant reactivation of wakeful activity patterns during post task sleep. Many ventral striatal cells were also modulated by hippocampal sharp wave ripples, and those that were showed stronger reactivations. Similarly, recordings made in macaques during and after a sequential reaching task found significant reactivations in posterior parietal, motor, somatosensory and dorsal prefrontal cortex [@bib90]. The authors also found evidence for reactivations of wakeful inter-regional activity patterns. A smaller number of studies [@bib93], [@bib94], [@bib95], [@bib96] have explicitly shown that coordinated cortical-hippocampal activity associated with awake behaviours is present during replay events. Ji and Wilson [@bib93] were the first to demonstrate this using simultaneous recordings from visual cortex (V1) and hippocampus made while rats ran on a track. They observed many V1 cells that had spatial firing fields on the track, resulting in distinct sequences of activity which were replayed during a subsequent sleep session. Moreover, replay of similar trajectories in hippocampus and V1 tended to co-occur [@bib93]. To be consistent with the simple consolidation hypothesis, however, one would expect hippocampal replay to invariably precede cortical replay; in fact, in this study it was not possible to establish if replay was initiated in the hippocampus and, although replay in the two regions did coincide, in some cases it appeared that V1 trajectories were generated with no hippocampal contribution. This result may simply reflect the difficulty of making population level inferences from a relatively small number of recorded single units. Alternatively, it might point to some form of bidirectional control over replay or suggest that, even during rest, the role of replay extends beyond simple consolidation. Subsequently, other authors have applied similar methods to identify coordination between the hippocampus and prefrontal cortex [@bib94], auditory cortex [@bib96], and grid cells in the deep layers (V and VI) of the medial entorhinal cortex [@bib95] ([Figure 4](#fig4){ref-type="fig"}B) --- the principal cortical projection target of the hippocampus [@bib97], [@bib98]. In the case of the study by Olafsdottir *et al.* [@bib95], during rest, replay in the medial entorhinal cortex was seen to lag CA1 by around 10 ms; consistent with a hippocampal initiation of replay. Interestingly, a similar study examining activity during sharp wave ripples in superficial layers of the medial entorhinal cortex found quite different results: entorhinal replay frequently occurred without coordinated activity in the hippocampus [@bib99]. Further, recent work from Yamamoto and Tonegawa [@bib100] found that input from layer III of the medial entorhinal cortex was required for extended awake CA1 replay. In part, these findings may be accounted for by anatomical differences as superficial entorhinal layers are primarily an input to the hippocampus [@bib97], [@bib98]. Equally, the latter two analyses were predominantly based on recordings made while animals were engaged in spatial tasks, and the observed replay may therefore be more strongly related to spatial planning and less to consolidation processes, in turn suggesting a differential involvement of superficial and deep medial entorhinal cortex layers in planning and consolidation, respectively. Clearly then, there is ample evidence for coordinated cortical-hippocampal activity during sharp wave ripples, albeit with more complex dynamics than might be expected from the simplest model of systems consolidation. Still, if hippocampal replay activity is involved in systems consolidation processes, then one might expect replay to be associated not only with increased activity in cortex, but also with increased plasticity. Several authors (for example [@bib81]) have noted that the rapid sequences of place cell activity observed during replay are optimal for inducing plasticity in post-synaptic targets. Although direct evidence for this is currently lacking, cortical oscillations that strongly modulate the depolarisation of principal neurons have also been associated with hippocampal sharp wave ripples during sleep. Two of the main oscillatory events are delta waves [@bib101], slow 0.1--4 Hz oscillations observed in a variety of cortical regions; and thalamo-cortical spindles [@bib102], [@bib103], 10--20 Hz oscillations originating in the thalamus. These two oscillatory patterns are often observed in close temporal proximity to each other and, importantly, to sharp wave ripples in the hippocampus [@bib104], [@bib105], [@bib106], [@bib107]. Indeed, Maingret and colleagues [@bib103] found that the successful recall of object locations following sleep was associated with enhanced coupling between hippocampal sharp wave ripples, cortical delta-waves, and spindle events ([Figure 4](#fig4){ref-type="fig"}C). Furthermore, when the duration of encoding was shortened to impair learning, the authors saw that performance could be rescued by stimulating the prefrontal cortex when hippocampal sharp wave ripples were detected, thereby generating delta waves and spindles. Similarly, it is clear that plasticity related to the spatial content of replayed trajectories can be induced during rest. This was beautifully demonstrated by de Lavilleon *et al.* [@bib108]: During sleep, though not just during periods of sharp wave ripples, reward in the form of medial forebrain bundle stimulation was triggered whenever a specific CA1 place cell was active; subsequently, during exploration, animals spent more time in the place field of the corresponding cell. While the foregoing discussion makes a convincing case for a role of hippocampal replay in memory consolidation, several important caveats must be considered. First, the majority of studies examining the modulation of replay by novelty, recency, and saliency have done so while animals are awake and engaging in tasks (for example [@bib55], [@bib82], [@bib83]). In contrast, the reduced sensory interference present during offline states are believed to render them favourable for consolidation. Nevertheless, this does not preclude the possibility that replay occurring during brief pauses in active behaviour also contributes to consolidation. Second, the disruption of sharp wave ripples has been shown to impair spatial learning [@bib69], [@bib86], but it remains to be seen whether this effect is due to the disruption of replay specifically or simply due to disruption of the sharp wave ripple state. For example, stimulation during sharp wave ripples --- when neurons in CA1 are highly active [@bib2], [@bib81] --- may simply disrupt existing spatial memory traces in the hippocampus (for example [@bib87]) rather than interfere with consolidation. Third, although replay may be important for learning, whether it genuinely represents the mechanism supporting systems consolidation, or simply reflects an intra-hippocampal process stabilising newly formed memory traces, remains to be established. A convincing demonstration of the former would be to identify the cortical correlates of a hippocampal memory and show how these develop or mature with the occurrence of replay. This might be achieved, for example, using a similar method to Kitamura *et al.* [@bib109] in which *c-Fos* expression was used to identify a cortical engram, hence determining its involvement in memory retrieval at different time points following learning. Alternatively, confirmation that memories do not become hippocampus-independent if replay is prevented from propagating to cortex would be compelling evidence. To address this latter point, it seems plausible that the interruption of sharp wave ripples could be used in conjunction with an approach similar to that used by Tanaka *et al.* [@bib110]. Namely, an experimenter would probe the point at which reinstatement of a cortical representation occurred independently of the hippocampus, that is, after CA1 silencing. Fourth, recent studies by several groups [@bib50], [@bib111], [@bib112] have shown that replay can be observed even before animals have any experience of an environment. In other words, place cell sequences recorded during rest are subsequently found to correspond to paths through a novel environment. This 'preplay' is a controversial topic, and there is some disagreement regarding its existence [@bib113]. Potentially, the effect might arise due to a failure in the statistical assumptions used to determine the significance of the observed sequences [@bib113] ([Figure 2](#fig2){ref-type="fig"}). For example, if the place cell representation of the novel environment is not orthogonal to familiar environments, then replay of those familiar places might be construed as preplay. Equally, though, preplay may result from activity in preconfigured cell assemblies, possibly supported by attractor networks, that are primed for inclusion into future spatial representations [@bib111], [@bib112], [@bib114], [@bib115]. Either way, preplay seems to be a weaker phenomenon than replay [@bib112]; indeed, the level of correlation in place cell activity that existed prior to exploration of a novel environment was used as a control condition in early replay studies [@bib45], [@bib47]. Hence, replay can still be understood as a process that predominantly reflects prior experience, though the extent to which it is influenced by the pre-existing configuration of hippocampal networks remains to be seen. Replay as Planning {#sec1.4} ------------------ The fact that replay occurs during awake behaviour suggests that, beyond its potential role in learning, it might also provide a mechanism for guiding navigational decisions, planning goal-directed trajectories or simulating the outcome of a given choice (for example [@bib56], [@bib70], [@bib71], [@bib72], [@bib116], [@bib117]). This view was established by Diba and Buzsaki's [@bib56] observation that, during track running, replay occurring as rats stopped for reward at the track's ends tended to be in the reverse direction, while movement initiation was mainly preceded by forward replay. The authors posited that reverse replay was important for learning from recent experiences, while forward replay was required for planning forthcoming actions. Although the functional distinction between forward and reverse replay is no longer clear cut (for example [@bib65]), broadly similar results have been noted elsewhere. For example, as animals pause at a junction on a working memory task, replay events tend to depict routes ahead of the animal [@bib117]. Similar forward 'sweeps' have been observed by others (for example [@bib61], [@bib62], [@bib118]), yet in these instances the sweeps seem to occur during theta states rather than sharp wave ripples. Although theta sweeps have been proposed to support decision making about immediate actions [@bib61], it remains to be seen whether and how they differ functionally from forward replay observed during awake behaviour. Finally, various theoretical propositions suggest replay as a candidate mechanism for exploring potential routes or extracting goal-directed heading vectors [@bib72], [@bib116], [@bib119]. If replay does represent a mechanism for planning spatial trajectories, then its occurrence should, in general, predict accurate behaviour. Indeed, one might specifically expect replayed trajectories to prefigure the path an animal is about to take. To a certain extent this appears to be true. For example, an increase in the probability of cell-pair co-activation during sharp wave ripples has been seen to precede correct turns on a W-maze and, in the same experiment, a non-significant trend was noted for preferential replay of the 'correct' arm [@bib117]. Similarly, when animals were engaged in open field navigation to a hidden goal, Pfeiffer and Foster [@bib71] found a tendency for replay trajectories to propagate in the direction animals were about to travel --- an effect that was not present when the rats were foraging randomly. The analysis did not, however, establish whether the replayed trajectory predicted the exact path the animal was about to take. Indeed, in the absence of a clear mechanism linking replay and navigational guidance it is not obvious if such a relationship really should be expected. Several groups [@bib72], [@bib116], [@bib119] have proposed, based on theoretical analyses, that replay in entorhinal grid cells provides the means to calculate goal-directed vectors. Current models, though, are agnostic as to how place cells should respond during bouts of grid cell replay; indeed, recent experimental work suggests that grid cells and other spatial cell types in the superficial layers of the medial entorhinal cortex exhibit replay independently of CA1 [@bib99]. More broadly, if online replay does provide a mechanism for planning trajectories, then one might occasionally expect to observe novel sequences of activity; for example, corresponding to a new combination of turns through an environment. This does appear to occur: in fact, replayed sequences have been reported to combine elements of an environment that were experienced separately and even to represent paths through a section of the environment the animal had seen but not experienced [@bib48], [@bib49]. Indeed, in the latter case, despite the fact that replay was recorded during rest, activity was seen to preferentially represent a section of the environment leading to reward, implying a link with planning [@bib49]. More generally, normal hippocampal sharp wave ripple-related activity is necessary to support spatial decision making under some circumstances. Clear evidence for this was provided by Jadhav and colleagues [@bib69] using closed-loop electrical stimulation of the ventral hippocampal commissure to truncate sharp wave ripples while animals performed a W-maze alternation task. Interruption of the sharp wave ripples decreased performance and the rate at which the task was acquired [@bib120] ([Figure 5](#fig5){ref-type="fig"}A). Despite causally connecting sharp wave ripple-related activity and spatial decision-making, the specific mechanism linking replay with behavioural output is still unclear, and so it is hard to discern the functional contribution of replay in this scenario.Figure 5Replay as planning.(A) Disrupting sharp wave ripples at decision points in a spatial alternation task ('W maze') was associated with impaired performance compared to control animals (left). When sharp wave ripples were disrupted at non-decision points performance was preserved (right) [@bib120]. (B) Replay was recorded at the corners of a Z-shaped track preceding correct and incorrect turns. When replay depicted positions consistent with the animals' current positions (for example, proximal locations, left) the rats were more likely to make the correct turn (right). Whereas if replay depicted positions not immediately relevant to current behaviour (middle), animals were less likely to make the correct turn (right) [@bib122]. (C) Following training on an inhibitory avoidance task (learning to associate the end of a linear runway with a foot shock), replay during pauses preceding entry to a shock zone preferentially depicted paths towards the feared zone (top) and was associated with the animals turning away from the shock zone and running in the opposite direction (bottom) [@bib121]. As intimated above, one of the central difficulties in ascribing a specific function to 'online' replay has been the fact that the trajectories depicted do not obviously relate to ongoing behaviour, often representing non-local positions [@bib48], [@bib57], [@bib63], [@bib64] de-coupled from the animal's future path [@bib121]. For example, when animals were required to run laps on one section of a track, Gupta *et al.* [@bib48] observed that replay trajectories preferentially represented portions of the maze that were not currently in use and which had not been recently visited. Similarly, recent work [@bib121] has demonstrated preferential replay of paths that animals were incentivised to avoid, and these trajectories were most often replayed prior to animals turning away from the aversive region and hence were anti-correlated with subsequent behaviour ([Figure 5](#fig5){ref-type="fig"}C). In part, some of these apparent discrepancies can be accounted for by immediate behavioural relevance to the animal, with replay providing a mechanism by which prior associations or memories are recalled and used to direct behaviour. But online replay is also known to depict trajectories through environments distinct from that in which the animal is currently located, which were experienced earlier in the same recording session [@bib64] or even on previous days [@bib63]. It is more difficult to envisage the immediate behavioural relevance of a distant environment or indeed even for replay in the absence of an ongoing task. What could account for these divergent findings? A viable hypothesis seems to be that, in part, this variability arises because ongoing task demands influence the nature and content of replay. Namely, during periods characterised by a low cognitive load when animals are not engaged in a demanding behavioural task --- such as sitting still or shuttling back and forth on a linear track --- replay occurs in order to support the consolidation of existing memories, as it does during slow wave sleep. Conversely, when animals actively engage in a task, such as at a decision point, replay becomes task focused. Consistent with this view, O'Neill *et al.* [@bib84] observed that shortly after animals arrived at a reward site replay was task-focused, preferentially expressing the goal location; whereas, when animals lingered at the site --- disengaging from the task --- replay was more likely to express places remote from the current position. Similarly, recent work from our own laboratory [@bib122] examined the dynamics governing this switch. We saw task-related replay immediately as animals arrived or departed from a decision point, depicting forward trajectories focused on the current location. Conversely, when animals remained at the decision point, replay changed to preferentially encode trajectories in both forward and reverse directions that were distributed across the apparatus, rather than concentrated on the current location; suggestive of consolidation processes. In addition, we found that accurate spatial choices tended to be preceded by forward replay focused on the animal's current position, as one would expect if these dynamics contributed to spatial decision making ([Figure 5](#fig5){ref-type="fig"}B). Furthermore, even when animals are actively engaged in a task, the observed replay is expected to be variable. Although replay under these circumstances might be task related, this does not mean that replay should exclusively depict, and consequently predict, future paths. If adaptive behaviour requires the retrieval of past actions or experiences then replay might equally, or indeed preferentially, express prior behaviour. This proposition could account for the varied findings relating to online replay (for example [@bib48], [@bib71], [@bib84], [@bib117], [@bib121]). In this context, replay may be considered to mediate the retrieval of information needed for accurate behaviour, rather than planning of a specific trajectory; and any distinction between online and offline replay is not particularly useful. 'Offline replay', typically recorded while an animal is either sleeping or resting in a holding environment, is likely to consist almost entirely of consolidative replay. 'Online replay', on the other hand, will be mixed; varying between task-relevant and consolidative replay according to the animal's current behavioural state and motivation. To summarise, the content of online replay has been found to be surprisingly variable. Although it may support spatial planning under some conditions, equally often it seems to serve other functions. We suggest that an animal's engagement with ongoing task demands may account for some of these differences. When engaged in a task, replay contributes to spatial decision making and navigation, and hence is largely focused on the animal's current milieu. In the absence of specific task demands, replay subserves consolidation, meaning the content of replay will likely be more varied, potentially representing the full range of the animal's recent experiences. Replay in Human and Non-human Primates {#sec1.5} -------------------------------------- Because of the relative paucity of single unit electrophysiology studies outside of the rodent, there is limited evidence for the existence of hippocampal replay in humans and non-human primates, though a number of studies have reported spatial and non-spatial firing correlates of individual neurons in hippocampal and parahippocampal regions that would permit such analyses in future [@bib30], [@bib123]. Nonetheless, several intracranial recording studies have characterised sharp wave ripple activity in both the human [@bib124], [@bib125], [@bib126] and primate [@bib127], [@bib128] hippocampal LFP. These ripples appear at a frequency of approximately 100 Hz (lower than that typically observed in the rodent), have a duration of around 50 ms, are observed during both quiet rest and non-rapid eye movement (NREM) sleep and tend to co-occur with, and be phase locked to, cortical slow waves [@bib126], [@bib128], [@bib129], [@bib130]. Only a few studies have co-recorded single unit activity, however, showing that burst firing activity is increased in the human hippocampus during slow wave sleep [@bib131], and in primate CA1 during sharp wave ripples [@bib127], but not examining the finer temporal structure of that activity. Alternative approaches have provided inferential evidence for the replay of previous experience in the human brain during quiet rest and sleep. The best example comes from applying multivariate pattern classification techniques to magnetoencephalography (MEG) recordings, allowing the offline reoccurrence of neural activity patterns associated with different visual stimuli to be identified [@bib132]. Using this technique, it has been demonstrated that sequences of stimuli initially encoded during a non-spatial navigation task were reactivated in reverse order on a timescale consistent with hippocampal replay during a subsequent 30 second planning period, although the signal appeared to originate from occipital/posterior temporal sources. Similar methods, applied to fMRI data, have shown that, during rest, the frequency with which stimulus representations are reactivated correlates with subsequent memory performance [@bib130], [@bib133]. Furthermore, other neuroimaging studies have shown that manipulations which bias replay in rodent models --- i.e. the presentation of sound or odour cues during sleep --- generate an increase in the hippocampal BOLD signal [@bib134], [@bib135], [@bib136]. Moreover, these interventions can enhance subsequent memory performance (but see van Dongen *et al.* [@bib137]), and the memory benefit correlates with hippocampal volume [@bib138]. Interestingly, one study [@bib126] showed that the number of sharp wave ripples in the human medial temporal lobe during a short rest period after learning correlated with mnemonic performance. Importantly, however, none of these studies provide direct evidence for hippocampal replay. Moreover, similar performance benefits are seen on procedural tasks that are not hippocampal dependent, suggesting that these interventions may access a more general mechanism of memory enhancement [@bib139]. Hopefully, future studies, making use of single-unit data derived from patients with intra-cranial depth electrodes, will be able to investigate the existence of replay in humans; providing an important translational connection between rodent and human hippocampal research. Future Directions {#sec1.6} ----------------- Replay is known to occur during sleep, rest and active navigation, and has been ascribed a number of functional roles including the stabilisation of newly formed memories, planning and decision-making during spatial tasks. In general, there is strong support for each of these propositions, although the relationship is more complex than was originally envisaged and specific mechanisms are still lacking. Indeed, a number of clear caveats exist. First, to assess whether replay supports systems consolidation, it is not sufficient to simply manipulate hippocampal replay (or indeed sharp wave ripples) and assess the effects on behaviour. Rather, it is also necessary to demonstrate that this manipulation either enhances or delays the maturation of memory traces, be they in the hippocampus or cortex. Second, the relationship between awake replay and ongoing behaviour is complex. In part this likely reflects the fact that, during awake periods, replay might support consolidation as well as task-related processes. Further, even when replay appears to be related to an ongoing task, the specific link between replayed trajectories and subsequent behaviour is not trivial. A fuller understanding of the network mechanisms that control the apparent 'switch' between replay for consolidation and replay for planning will resolve some of this variability. Beyond this, untangling the relationship between replay and behaviour will also require carefully constructed behavioural tasks with clearly delineated demands in which performance depends on defined and spatially localised sources of information (see for example [@bib66]). Third, the role played by extra-hippocampal regions in the generation and control of replay has received relatively little attention. Both the medial septum and mesopontine median raphe region have been shown to modulate the occurrence of sharp wave ripples [@bib140], [@bib141], but the extent to which these regions govern the occurrence of replay during the course of normal behaviour is unknown. Similarly, it appears that cortical regions have a greater autonomy to initiate and potentially guide replay than was previously imagined (for example [@bib99]), but again, the behavioural relevance of cortical replay and how it relates to hippocampal replay requires further study. More specifically, the mechanism controlling which precise sequence of place cells is replayed is also unknown. One possibility described above is that cortical activity, possibly itself resembling replay, might prompt the reactivation of hippocampal sequences (for example [@bib93], [@bib99]). Consistent with this view, it is known that presentation of familiar auditory stimuli during sleep biases hippocampal replay to represent the locations in which those stimuli were encountered during wakefulness [@bib142]; presumably auditory cue presentation causes the reinstatement of stimuli-specific activity patterns in the auditory cortex, ultimately promoting activity in place cells associated with that cue during training. An alternative mechanism is provided by dopamine signalling from the ventral tegmental area (VTA) [@bib143] and locus coeruleus [@bib144]. Dopamine signalling in the hippocampus is known to be important for the stabilisation of place cell activity [@bib145] and the retention, but not initial encoding, of spatial memories [@bib146]. Indeed, optogenetically triggering dopamine release in mice during exposure to a novel environment results in stronger CA1 reactivations of that environment during rest and a concomitant increase in memory retention [@bib147]. However, it is not known whether dopamine directly modulates the occurrence of replay or whether the apparent increase in reactivation results from other mechanisms that stabilise CA1 activity. Interestingly, VTA neurons were found to have elevated firing rates during sharp wave ripples emitted while animals conducted a spatial task, but not during sleep [@bib148]. This is particularly relevant because VTA neurons are known to represent reward prediction error [@bib149] and are central for reinforcement learning [@bib150]. In turn, reverse replay during behaviour has been implicated as a possible solution to the temporal credit assignment problem in reinforcement learning [@bib55], [@bib70]. The juxtaposition of VTA activity and replay supports this general view but, again, does not necessarily implicate dopamine as a modulator of replay itself. Lastly, evidence for the existence of replay outside of the rodent is at best indirect. It will be exciting to see if emerging technologies in human intracranial recordings are able to resolve large populations of single neurons --- and thus, replay --- in the human brain. Such studies would provide essential insight into potential similarities between rodent and human hippocampal function and clarify the hypothesised role of hippocampal replay in mnemonic functions and goal-directed behaviour. In general, looking to the future, it appears that several technical developments are of particular relevance to this field. Historically one of the problems in studying the long-term effects of replay on hippocampal and cortical networks has been the difficulty in stably recording large populations of neurons for significant periods of time. The increasing availability of two-photon calcium imaging coupled with rodent virtual reality now means that activity of hundreds of cells can be monitored across days, while animals perform spatial tasks [@bib151]. Indeed, the temporal resolution of the most recent generations of calcium indicators is sufficient to allow replayed sequences to be detected optically [@bib152], and so it is already possible to monitor the incremental changes induced in a hippocampal network as a result of replay. Going further, the combination of optogenetics and optical imaging, allowing for the rapid manipulation of visually identified cells, provides a powerful means of perturbing, or even artificially generating, specific replay sequences [@bib153]. Such an approach would provide the means to explicitly link replay of a sequence with specific mnemonic or behavioural outcomes. To be viable though, this would likely require a control system triggered by the rapid decoding of replay trajectories, and some progress towards this goal has already been made [@bib154], [@bib155]. Conclusions {#sec1.7} ----------- To conclude, research over the past twenty-five years has contributed to understanding the role hippocampal replay plays in cognition. Current evidence suggests that replay is used to selectively strengthen newly acquired memories for retention and guide adaptive decisions during active behaviour. Yet, numerous questions regarding the underlying mechanisms remain. Does replay mediate the maturation of cortical memories, stabilise newly formed hippocampal cell assemblies, or instigate changes in both regions? Is the content of replay prescribed or can it adapt to changing demands, dictated by current tasks and behavioural state? Newly available techniques are well placed to address these questions, and it seems likely that the next decade will reveal a clearer understanding of the functional roles replay performs, the mechanisms by which it acts, and the systems by which it is controlled. This work was funded by a Sir Henry Dale Fellowship to C.B. jointly funded by the Wellcome Trust and Royal Society.

Abbreviations {#nomen0010} ============= ATT : Antithrombotic Therapy CRT : Cardiac Resynchronization Therapy ICD : Implantable Cardiac Defibrillator INR : international normalized ratio UGAVP : Ultrasound Guided Axillary Vein Puncture US : Ultrasounds VKA : Vitamin K Antagonist 1. Introduction {#sec1} =============== Several anatomical access points and methods to gain central venous access have been described. The axillary, cephalic, and subclavian veins, as well as the internal and external jugular veins, have all been used to insert pacemaker or defibrillator leads. The axillary vein has become an emerging technique for the placement of pacing and defibrillation leads for several reasons. Unlike the cephalic vein, the main advantage of the axillary vein is that it is almost always large enough to accommodate multiple pacing leads. When compared to the subclavian vein, the properly-accessed axillary vein affords a less angulated course ([Fig. 1](#fig1){ref-type="fig"}). This potentially decreases mechanical stress (subclavian crushing syndrome) on the implanted leads or catheters, hence resulting in a lower incidence of mechanical lead failure or vein occlusion \[[@bib1],[@bib2]\]. Compelling evidence has implicated the infraclavicular musculotendinous complex in mechanical lead failure and occlusion of subclavian catheters \[[@bib3],[@bib4]\].Fig. 1Fluoroscopic comparison of the course of the leads with ultrasound-guided axillary vein puncture (left image), and subclavian puncture (right image). The angulation and potential mechanical stress on the leads are significantly marked with the subclavian access (white arrow).Fig. 1 Additionally, subclavian access portends the risk of inadvertently accessing the non-compressible subclavian artery and the potential for increased mechanical stress on the lead or indwelling catheter from crossing the subclavius muscle and the clavipectoral fascia. Finally, unlike the jugular system, the use of the axillary system does not require tunneling of the leads over or under the clavicle. Techniques for accessing the axillary system with the use of fluoroscopic (either with or without venography) or ultrasounds (US) imaging have also been used \[[@bib5],[@bib6]\]. The landmark (fluoroscopy) approach is associated with a significant risk of arterial puncture, pneumothorax or failed access \[[@bib7]\]. In addition, the current trend is to implant under antithrombotic therapy (ATT), because the perioperative bridging of anticoagulation is associated with a higher risk of thromboembolic events \[[@bib9]\]. A previous study reported a greater use of pressure dressings with ultrasound-guided axillary vein puncture (UGAVP) \[[@bib10]\]. This may suggest a higher risk of bleeding in comparison with the cephalic approach. It is to note that cardiac resynchronization therapy (CRT) with triple leads placement by UGAVP, and upgrade procedures (i.e. in the presence of preexisting leads) have not been described.^[4,11]{.smallcaps}^ We aimed to assess the incidence of complications using UGAVP in patients under ATT, including CRT and upgrade procedures. The learning curve of UGAVP use for routine practice will also be assessed in this study. 2. Methods {#sec2} ========== 2.1. Patients selection {#sec2.1} ----------------------- Prospectively, all consecutive patients eligible for cardiac devices implantation (i.e. pacemaker, defibrillator, CRT or upgrading) in whom an UGAVP was performed were included in this study, at two centers: Princess Grace Hospital in Monaco between September 2014 and September 2015, and Mohammed VI university hospital in Marrakech Morocco between October 2016 and April 2018. All the patients gave their written consent for the procedure. 2.2. Ultrasound-guided venous puncture {#sec2.2} -------------------------------------- To access the vein with sonography, the patient was placed in the supine position, without Trendelenburg, and the patient was prepared in the usual sterile manner ([Fig. 2](#fig2){ref-type="fig"}).Fig. 2Patient installation in the catheter laboratory with position of the probe during puncture.Fig. 2 A surface vascular US probe was inserted into a sterile plastic sleeve and used to image the axillary vasculature. Real-time US imaging of the spatial relationship of the artery and vein, and of the course of the access needle visually guided the venous puncture. A local anesthesia by lidocaine hydrochloride 2%, under US visualization was made along the course of the puncture needle. Using an out-of-plane technique, the vein was centered in the middle of the screen with the probe held with the left hand perpendicular to the skin ([Fig. 2](#fig2){ref-type="fig"}, [Fig. 3](#fig3){ref-type="fig"}). An 18-gauge, 7-cm length Cook bevel-tipped needle was introduced and advanced with the right hand below the US probe towards its center while watching for tissue movement on the US screen and maintaining negative pressure on the plunger. Once the needle is seen to enter the vein and blood flashes into the syringe, the syringe was removed and a guidewire was placed into the lumen. From this point, a sheath and dilator may be placed in the usual fashion.Fig. 3Ultrasound image of a left axillary vasculature.Fig. 3 No time limit was set. No internal jugular access was used. Puncture time was defined as time between US visualization of the axillary vein to the insertion of the guidewire in the superior vena cava. All procedures were performed by a single operator, experienced with UGVP for femoral access, and fluoroscopy-guided axillary vein puncture \[[@bib12]\]. A learning curve defined as UGAVP time evolution was established. Procedure time, but also complications were systematically studied: hematoma, pneumothorax, hemothorax. 2.3. Management of antithrombotic therapy {#sec2.3} ----------------------------------------- ATT (Aspirin, Clopidogrel, Ticagrelor, Dabigatran, Rivaroxaban, Apixaban, low weight molecular heparin and Vitamin K Antagonists \[VKA\]) were continued until and after the procedure. In VKA patients, International normalized ratio (INR) target was 2--3 the day of the procedure. The implantation was postponed if the INR was greater than 4. 2.4. Follow-up {#sec2.4} -------------- All patients were monitored in the hospital at least two nights after the implantation. After hospital discharge, patients were followed in our outpatient clinic at 1, 6, and 12 months. Axillary access points checks were performed at the end of the procedure, the following day after dressing removal and before discharge. Vascular access complications, including hematomas, were categorized as major if they resulted in prolongation of hospitalization, repeat hospitalization, blood transfusion, or surgical intervention; or minor (hematoma without hospital stay lengthening). 2.5. Statistical analysis {#sec2.5} ------------------------- The statistical analysis was made with GraphPad Prism 5 (San Diego, CA, USA). Numerical variables are expressed as mean ± SD. 3. Results {#sec3} ========== 3.1. Patients population {#sec3.1} ------------------------ Patients characteristics are summarized in [Table 1](#tbl1){ref-type="table"}. 200/457 (43.76%) patients were included: 164 (82%) patients received a pacemaker and 36 patients (18%) received an ICD. The study population was composed by 54 (27%) patients with CRT. Among them, 26 patients (13%) underwent a triple insertion of new leads implanted in the axillary vein. 180 (90%) patients were under ATT ([Table 1](#tbl1){ref-type="table"}).Table 1Baseline characteristics and procedural data.Table 1Number of patients200Age (y)77.8 ± 10 \[44--94\]Male, n (%)116 (58)Body Mass Index (kg/m^2^), n (%)\<25: 58 (29)\ \>25: 148 (74)Type of procedure (devices), n (%)VVI: 44 (22) PM 36 (18) ICD 8 (4)CRT/CRT-D: 54 (27) CRT-D: 24 (12) Upgrade:6 (3) 3C: 10 (5) BiV: 8 (4) CRT-P: 30 (15) Upgrade: 8 (4) 3C: 16 (8) BiV: 6 (3)DDD: 100 (50) PM: 98 (49) ICD: 2 (1)VDD: 2 (1) ICD: 1 (1)ICD/PM: PM: 164 (82) ICD: 36 (18)Side of implantation, n (%)Left: 174 (87)\ Right: 26 (13)Major vascular complications, n (%)0 (0)Minor vascular complications, n (%)1 (0.5)Procedure time (min)75.13 ± 44.3 \[25--205\]Fluoroscopy time (min)8.46 ± 10.71 \[0.5--50\]Antithrombotic therapy, n (%)No ATT 20 (10)/ATT 180 (90)Anticoagulation 104 (52) VKA 48 (24) DOAC 46 (23) Apixaban 14 (7) Dabigatran 4 (2) Rivaroxaban 28 (14) LWMH 10 (5)Antiplatelet therapy 82 (41) Single APT 74 (37) Aspirin 62 (31) Clopidogrel 4 (2) Prasugrel 2 (1) Ticagrelor 2 (1) Dual APT 8 (4)Anticoagulant + APT 10 (5)Dual ATT 18 (9)[^1] 3.2. Ultrasound-guided venous puncture performance {#sec3.2} -------------------------------------------------- UGAVP was successfully achieved in 182 patients (91%), this rate increased to 95.78% after excluding anatomic variations: non-visualized vein or very small caliber (\<2 mm maximal diameter). Axillary vein visualization was obtained in 95% of the cases ([Table 2](#tbl2){ref-type="table"}). The vein presented with a very small caliber in 4% of the cases confirmed by angiography during subclavian approach.Table 2Ultrasound-guided axillary vein puncture performance.Table 2Global puncture time (min)4.68 ± 3.64 \[0.5--15\]First puncture time (min)3.03 ± 2.9 \[0.5--15\]Puncture time after 15 first patients4.46 ± 3.38 \[0.5--15\]Puncture time per guidewire (min)2.52 \[0.5--15\]Mean number of guidewires inserted per patient1.8 ± 0.6 \[1--3\]Success rate, n (%) Global success182/200 (91) After excluding anatomic variations (Non-visualized veins or very small caliber)182/190 (95.7)Failure rate, n (%)8/190 (4.2)Table 3Comparison to prior experience with ultrasound-guidance for axillary vein.Table 3Nash A \[[@bib5]\] (1998)Orihashi K \[[@bib17]\] (2005)Jones DG \[[@bib8]\] (2006)Franco E \[[@bib22]\] (2016)Liccardo M \[[@bib23]\] (2018)Our serie (2019)Number of patients, n\ Number of leads, n70\ 9518\ 3260\ 8350\ 86116\ 304200\ 360Visibility of axillary vein (favorable anatomy for puncture)N/A100%N/A100%98.2%95%Success of axillary puncture, n (%)56 (80)2753 (88)49 (98)106/116\ 91%182/190 (95.7)Time considerations31 s time for vein cannulation82.1 s time for entry in vein8 min time for lead placement56 s time for entry in vein5 min as time limit (mean time N/A)4.68 min visualization of axillary vein - all GW in SVCVascular complications, n (%)NoneNonePocket hematomas 2 (3.3) Pressure dressings 26 (43)Minor pocket hematoma 1 (2)NoneNonePneumothorax001 (1.6)000Devices implanted, n (%)PM38 (76)N/A164 (82)VVI45 (64.3)4 (22.2)37 (62)16 (32)44 (22)DDD25 (35.7)14 (77.8)23 (38)31 (62)100 (50)ICD0N/A010 (22)36 (18)CRT-P, CRT-D0004 (6)54 (27)Learning curve, number of patientsafter 35N/Aafter 15After 5-7Training phase:23after 15[^2] Mean puncture time was 4.68 ± 3.64 (0.5--15) minutes. Mean puncture time per guidewire was 2.52 min. Mean puncture duration evolution over the time is illustrated in [Fig. 4](#fig4){ref-type="fig"}. The learning curve associated with this technique was estimated to 15 patients, corresponding to the beginning of puncture time plateau ([Fig. 4](#fig4){ref-type="fig"}).Fig. 4Ultrasound-guided axillary vein puncture learning curve for devices.Fig. 4 3.3. Complications {#sec3.3} ------------------ There was only one minor complication (hematoma: it was the 5th case with failure of UGAVP and conversion to a blind subclavian vein puncture), after a mean follow-up of 45 ± 10 months. This patient (with prosthetic mitral valve) was excluded from analysis because the complication occurred with a subclavian puncture. 4. Discussion {#sec4} ============= The present study supports a wide and safe use of UGAVP for cardiac devices implantation (pacemakers, ICDs and CRT), especially in patients under ATT. UGAVP resulted in low incidence of complications. This is a fast and short-learning curve technique. The "blind" or fluoroscopy-guided axillary vein puncture often implies a collapse of the vein in patients in a fasting state, while US allow direct visualization and can be of a precious help by predicting inter-individual anatomical variations. 4.1. Prior experience with ultrasound guidance ([Table 3](#tbl3){ref-type="table"}) {#sec4.1} ----------------------------------------------------------------------------------- Nash et *al* first described the use of two-dimensional US for pacemaker lead implantation in 70 patients in 1998 \[[@bib8]\]. The authors found that the use of US for placement of pacemaker leads was a safe technique but needed a significant "learning curve" in that nearly all of the unsuccessful cases were in the first half of the series. No major complications were reported. Orihashi et *al* described their experience in 18 patients and found a 90% success rate within two attempts using longitudinal imaging within the pacer pocket and a freehand technique \[[@bib13]\]. The authors observed the ease of compressibility of the vein by the needle, and the utility of short jabbing motions to image the needle tip and facilitate venipuncture. Finally, Jones et *al* demonstrated in 60 patients that the learning curve for US access was short, and that US guidance led to a reduction in lead placement time (8 min versus 12 min) and fluoroscopy time compared with the cephalic approach even after inclusion of training. Nevertheless, there was a significant greater use of pressure dressings in comparison with the cephalic approach \[[@bib7]\]. In comparison to the subclavian puncture, the absence of pneumothorax can be explained by the extra-thoracic course of the puncture ([Fig. 1](#fig1){ref-type="fig"}). In the present study, no complications were observed with UGAVP. Our series also reported a higher number of leads implanted in comparison with previous studies (139 leads in total), confirming the possibility to implant multiple leads with this technique (including ICD leads), but also the potential benefit in case of upgrade procedures. 4.2. Anatomic variations and role of ultrasounds {#sec4.2} ------------------------------------------------ In 2003, Galloway and Bodenham published their experience in using US guidance to define the axillary system \[[@bib14]\]. The authors examined 50 patients with US. Their data showed that the Trendelenburg position only afforded a 1 mm (12--13 mm) increase in the diameter of the axillary vein and that the arm position did not cause significant differences in the vessel size or US visibility. In this study, it was observed that as the axillary vein coursed laterally, its diameter decreased (from 12.2 mm to 8.5 mm), its depth increased (from 19.5 mm to 32.2 mm), and its proximity to the axillary artery decreased (from 3.4 mm to 8.9 mm). Anatomic variations of the axillary vein and its tributaries were noted in 27.5% with duplicated axillary vein in 5% \[[@bib15]\]. Additionally, the variations in branching pattern of the axillary artery were found in 62.5% \[[@bib16]\]. Furthermore, the rib cage to vein distance is variable (0.2--2.2 cm) \[[@bib11]\]. In cases the BMI \> 25 kg/m^2^, there was a significant difference in depth, and a trend to significant differences in diameter. However, age-specific differences in depth and diameter were not observed \[[@bib17]\]. These anatomical variations are clinically significant and can increase the risk of vascular complications and pneumothorax with a blind technique. The use of US allows the operator to appreciate anatomic variations in arterial, venous and rib cage spatial relationships, as well as of the vessels themselves. Such imaging provides visualization of the access needle tip course and trajectory. It has been well recognized that the use of real-time US guidance during central line insertion is one of the patient's safety practices with the greatest strength of supporting evidence \[[@bib18],[@bib19]\]. A randomized controlled trial reported a higher first-attempt success rate and fewer needle passes with real-time US guided puncture compared with the anatomic landmark approach \[[@bib20]\]. 4.3. No perioperative bridging for anticoagulation {#sec4.3} -------------------------------------------------- **In our department which is a reference center for atrial fibrillation ablation, ATT is routinely maintained, especially in patients with atrial fibrillation. This may explain that 90% of patients were on anti-thrombotic therapy which is unusually high compared to routine practice.** This practice is supported by data from large series, demonstrating that periprocedural continuation of anticoagulation not only confers protection against thromboembolic events but is also safe, as evidenced by the overall low rates of bleeding complications \[[@bib5],[@bib6]\]. In contrast with previous studies, despite the fact that implantations were performed with uninterrupted ATT, no major bleeding complications were observed, as opposed to previous studies \[[@bib7]\]. 4.4. UGAVP for ICD, CRT and upgrade procedures {#sec4.4} ---------------------------------------------- In previous studies concerning UGVAP, ICD, CRT and upgrade procedures were excluded \[[@bib4]\]. In contrast, UGVAP was successfully performed for ICD (18 patients), CRT implantation (24 patients) and upgrade procedures (8 patients) in our study. Recent studies using the fluoroscopy-guided axillary vein puncture included some CRT devices, but this technique was not extended to the three leads, while it was possible in our series (11 patients) \[[@bib21]\]. 4.5. Limitations {#sec4.5} ---------------- This study is bicentric and not randomized. The lack of a control group is a significant limitation of the study but this does not seem to diminish the quality of this study whose main objective was to verify the feasibility and safety of UGVAP in patients under ATT. No vascular complication was reported in the present study with UGAVP, but the analysis involved a limited number of patients (n = 200). There have been no randomized trials between the US-guided technique and either the cephalic approach, traditional landmark axillary technique, or fluoroscopic and venogram-guided techniques for pacemaker or ICD placement. However, the limited published literature concerning axillary access with US, including lead placement, has demonstrated a consistent reduction in time to access, number of attempts, and complications. US guidance has plausible benefits in reducing the risk of lead crush, pneumothorax, and hematoma, and may have particular utility in patients with preexisting leads. With advances in US imaging technology, increasing emphasis on patient safety, and trainees who are more familiar with US-guided access, the use of US in device implantation is likely to expand. The additional cost associated with this technique has been approximated to 18.85€/procedure (cost of the sterile plastic sleeve). This cost may be added to the initial cost of a dedicated vascular probe, if not present in the catheter laboratory/operating room. 5. Conclusion {#sec5} ============= The present study, as well as the recent literature, support wide use of UGAVP in patients under antithrombotic therapy undergoing cardiac devices implantation including ICD and CRT. The short learning curve should encourage every cardiologist to adopt this technique. Author's contribution section {#sec6} ============================= Dr Mohammed El Jamili and Dr Sok-Sithikun Bun have equally participated in the preparation of this manuscript. Mohammed El Jamili[:]{.ul} Concept/Design; Data analysis/interpretation; Drafting article; Critical revision of article; Approval of article; Data collection. Sok-Sithikun Bun[:]{.ul} Concept/design; Data analysis/interpretation; Drafting article; Critical revision of article; Approval of article; Data collection. Decebal Gabriel Latcu[:]{.ul} Critical revision of article; Approval of article. Tahar Delassi[:]{.ul} Data collection. Mustapha Elhattaoui[:]{.ul} Critical revision of article; Approval of article. Nadir Saoudi[:]{.ul} Critical revision of article; Approval of article. Declaration of competing interest ================================= None. Peer review under responsibility of Indian Heart Rhythm Society. [^1]: APT: antiplatelet therapy; ATT: antithrombotic therapy; BiV: biventricular; DOAC: direct oral anticoagulant; LWMH: low weight molecular heparin; PM: pacemaker; VKA: vitamin K antagonist; 3C: three chambers. [^2]: GW: guidewires; N/A: non-available; PM: pacemakers; SVC: superior vena cava.

All relevant data are within the manuscript and its Supporting Information files. Introduction {#sec001} ============ Cytotoxic T Lymphocytes (CTLs) are key effectors in the adaptive immune response, therefore CTL function---or lack thereof---is relevant in many pathologies. A greater quantitative understanding of CTL effector function will aid in interpretation of prior experiments and should yield useful insights for the treatment of diseases in the future. However, the rate at which CTLs kill infected or malignant cells remains poorly characterised. Estimates of CTL killing based on *in vitro* and *in vivo* CTL killing assays vary, with some variation explained by e.g. different susceptibility of target cells to CTL killing or the type of antigen expressed by the targets \[[@pcbi.1007972.ref001],[@pcbi.1007972.ref002]\]. Moreover, especially *in vivo* the presence of stimulatory or suppressive factors and difficulty in controlling or estimating the ratio of CTLs to target cells at the site of killing might confound CTL killing estimates \[[@pcbi.1007972.ref001],[@pcbi.1007972.ref002]\]. As a frequently discussed example, consider the *in vivo* CTL killing assay of Barber *et*. *al*. \[[@pcbi.1007972.ref003]\], in which CTLs demonstrated rapid killing against Lymphocytic Choriomeningitic Virus (LCMV). Although Barber *et*. *al*. initially estimated that CTLs took 15 minutes to kill targets, subsequent modelling studies based on the same data have estimated much faster killing rates \[[@pcbi.1007972.ref004],[@pcbi.1007972.ref005],[@pcbi.1007972.ref006]\], with one study implying an expected target survival time of 16 seconds after contact from a CTL \[[@pcbi.1007972.ref004]\] (see also \[[@pcbi.1007972.ref001]\] for a detailed summary of these estimates). Given that killing in those experiments was perforin-dependent\[[@pcbi.1007972.ref003]\], these fast estimates seem to contradict recent *in-vivo* imaging showing that the perforin-dependent killing process requires a minimal contact time. For example, long-lasting (median: 80s) calcium fluxes linked with CTL killing of virally infected cells occurred, on average, 480s (median) after CTLs established contact with virally infected targets\[[@pcbi.1007972.ref007]\]. Such killing times of around 10 minutes are consistent with the duration of killing events that can be observed in various supplemental videos elsewhere \[[@pcbi.1007972.ref008],[@pcbi.1007972.ref009]\]. Given this lower bound it is difficult to see how solely granule-mediated killing could plausibly lead to killing rates in excess of \~6 hour^-1^, even in optimal situations where CTLs are not limited in their supply of targets and do not require time to search for new targets between killing events. A major limitation of many prior estimates of CTL killing is that analysis is performed on population level data in *in vivo* settings, with no direct measurements of the killing process. This approach has a number of drawbacks: First, it can be challenging to accurately assess the frequency of CTLs and target cells. Second, other immune cells may contribute to the killing process, confounding estimates of the true CTL killing rate. Third, the processes underlying CTL killing are complex and it may be insufficient to describe them with a single, time invariant rate constant. Indeed, recent observations have indicated that target cells may require multiple hits before death either *in vitro* \[[@pcbi.1007972.ref010]\], or *in vivo* \[[@pcbi.1007972.ref007]\]. We have previously shown that such multiple-hitting can lead to a time-increasing killing kinetic when CTLs are exposed to fresh targets \[[@pcbi.1007972.ref011],[@pcbi.1007972.ref012]\], further complicating the killing rate estimation procedure. Besides analysing CTL killing performance at the population level, a potentially useful approach is to analyse CTL killing at the single cell level. Such analysis can yield greater insights into the dynamics of the killing process. This was exemplified in studies undertaken in the 1970's in which the killing kinetics of CTLs conjugated with 1--4 EL4 tumour cell targets were examined under the microscope for a period of 3 hours \[[@pcbi.1007972.ref013],[@pcbi.1007972.ref014]\]. Subsequent mathematical analysis of these studies indicated that the CTL killing process was well described as a Poisson process \[[@pcbi.1007972.ref015]\], indicating that CTLs kill targets sequentially rather than simultaneously. This analysis allowed the authors to conclude that CTL killing was mediated by secretory lysosomes, several years before this was demonstrated conclusively \[[@pcbi.1007972.ref016]\]. The aforementioned studies also revealed that the rate of CTL killing was not diminished after target lysis, an observation which led the authors to deduce that CTLs were able to discriminate between viable and killed targets. More recently, *in vitro* studies of individual natural killer cells have shown that killing occurs via both granzyme and death receptor mediated pathways, each having different kinetics \[[@pcbi.1007972.ref017],[@pcbi.1007972.ref018]\]. Despite the utility of studying CTL killing at the single-cell level, there remains a shortage of *in vitro* CTL killing studies with statistical power sufficient to check the validity of the Poisson model first proposed in the 1980's by Perelson et. al. \[[@pcbi.1007972.ref015]\]. Recently one such a study was performed: Over a 12 hour period, image-based killing measurements were taken from human-derived CTL clones, each CTL being separately confined within small micro-wells that contained an excess of JY target cells \[[@pcbi.1007972.ref019]\]. During the studied time period, the killing rate of CTLs was dynamic, exhibiting a marked increase in the final hours of the experiment. The total number of targets killed per CTL was overdispersed compared to the Poisson distribution, implying greater heterogeneity between individual CTL killing performance than anticipated. Vasconcelos et. al. [(Vasconcelos et al. 2015)](https://paperpile.com/c/oMBHuP/ZTAi) found the data was well described by a Poisson mixture model, and they postulated the existence of a subset of "high rate killers" comprising 30% of the population that emerged 8--10 hours after first exposure to target cells. However, no mechanistic explanation could be found to explain this result, despite a search for membrane markers that might identify and/or explain the variability of CTL killing characteristics. We hypothesised that a requirement for "multiple hits" to kill targets before apoptosis induction might explain heterogeneous killing amongst clonal CTLs *in vitro*. Perelson et. al. \[[@pcbi.1007972.ref020]\] previously considered the possibility of multiple-hitting, noting however that such a model was excessively complex to describe the limited experimental data available at that time. Recent evidence has directly shown that multiple-hitting does occur at least in some settings \[[@pcbi.1007972.ref007],[@pcbi.1007972.ref010],[@pcbi.1007972.ref021]\], and our previous modelling work has demonstrated that multiple-hitting can indeed lead to population-level killing kinetics increasing over time when CTLs are exposed to fresh targets \[[@pcbi.1007972.ref011]\]. Therefore, we here used stochastic simulations to investigate the compatibility of the multiple-hitting hypothesis with the findings of Vasconcelos et. al. \[[@pcbi.1007972.ref019]\]. We found that multiple-hitting was indeed able to explain the late onset, high-rate bursting kinetic of individual CTLs, with physiologically plausible parameters. We also highlight that multiple-hitting is expected to lead to a complex dependence of realised killing rate upon the number of available targets and on the ability of individual CTLs to form and abort conjugates with target cells. We subsequently developed spatially explicit, agent based simulations of CTLs killing targets in micro-wells as a means of generating realistic yet noisy artificial data and assessing methods of recovering CTL hitting parameters from future microscopy data. Using these spatial simulations, we demonstrate how parameter estimation is substantially improved if contacts of individual targets with CTLs can be tracked throughout the duration of the experiments. Results {#sec002} ======= Multiple-hitting CTLs exhibit heterogeneous late onset killing {#sec003} -------------------------------------------------------------- We first sought to establish whether the multiple-hitting hypothesis was a feasible explanation for the heterogeneous, delayed onset, "burst" killing kinetics observed and defined by Vasconcelos *et*. *al*. \[[@pcbi.1007972.ref019]\]. In brief, these high rate killer CTLs were a subset among a clonal population whose killing suddenly accelerated after 8--10 hours of experimentation, with no explanation readily apparent (Methods). In the current study, we used Monte Carlo simulations of individual CTLs killing targets to identify conditions under which multiple-hitting might lead to heterogeneous, "burst" killing. In these Monte Carlo simulations, CTLs hit targets at a constant rate *λ*, then targets died after receiving *η* hits. We simulated single- and multiple-hitting scenarios on the basis that the expected (mean) time for one target in contact with a CTL to be killed was 1 hour, i.e., we set *λ*/*η* = 1 (valid for entire [Fig. 1](#pcbi.1007972.g001){ref-type="fig"}). {#pcbi.1007972.g001} Firstly we simulated CTLs with *η* = 1,2, or 10, with each simulation containing one CTL interacting with a single target. For such a strictly 1:1 CTL:target ratio, the waiting times for target death were gamma distributed with rate parameter *λ* and shape parameter *η* ([Fig 1A](#pcbi.1007972.g001){ref-type="fig"}). The gamma distributions ([Fig 1B](#pcbi.1007972.g001){ref-type="fig"}, top panel), together with their accompanying survival probability functions ([Fig 1B](#pcbi.1007972.g001){ref-type="fig"}, middle panel), define the hazard function ([Fig 1B](#pcbi.1007972.g001){ref-type="fig"}, bottom panel), which is the momentary rate of death experienced by a target, given that the target has already survived an interaction for some time, *t*. When *η* = 1, the hazard experienced by contacted targets does not change with time. In contrast, when *η*\>1 the hazard experienced by contacted targets increases over time, as contacted targets become increasingly likely to have received (*η*−1) hits and thus be killed by the next hit. For the case where CTLs interact with targets in a strictly 1:1 ratio, the gamma(η, λ) distribution parameters could be estimated from the mean and variance of the samples of the waiting time (y~o~): $\overline{y_{o}} = \frac{\eta}{\lambda}$ and $\left. {Var\left( y \right.}_{o} \right) = \frac{\eta}{\lambda^{2}}$ ([Fig 1C](#pcbi.1007972.g001){ref-type="fig"}). We next extended our Monte Carlo simulations to allow CTL:target interactions in a 1:n ratio, for variable numbers of targets, *n*. CTLs were individually assigned their initial number of targets by drawing *n* from a Poisson distribution, with mean $\overline{n} = 16$ ([Fig 1D](#pcbi.1007972.g001){ref-type="fig"}, blue bars). The total number of targets killed by a CTL during one simulation, *x*, should also follow the Poisson distribution, if the killing rate of each simulated CTL would be the same. Moreover, the mean and variance should be approximately equal for any set of Poisson distributed samples. Therefore, observation of a ratio $\frac{var\left( x \right)}{\overline{x}} > 1$ for a set of killed targets would imply that the killing was more heterogeneous than expected under Poisson assumptions. For single-hit killing (*η* = 1), the variance of the 12 hour killing samples was in fact slightly below the mean ([Fig 1D](#pcbi.1007972.g001){ref-type="fig"}, upper row), resulting from some simulations where CTLs killed all their targets before the simulation had finished. However, for *η* = 2 ([Fig 1D](#pcbi.1007972.g001){ref-type="fig"}, central row), the variance approached the mean and for *η* = 10 far exceeded the mean ([Fig 1D](#pcbi.1007972.g001){ref-type="fig"}, bottom row). In the latter case, a bimodal distribution occurred, which could be interpreted as a subpopulation of high-rate killers, yet importantly such a population did not exist in our simulations. In our simulations, the additional variability in killing performance of multiple-hitting CTLs was due to the allocation of subsequent hits amongst several different targets. When a group of targets share hits evenly, the time for a specified target to be hit is proportional to the number of other targets sharing. This has no effect on the killing rate observed if *η* = 1, so the mean killing rate for our simulated single-hitting CTLs initially remained constant over time ([Fig 1E and 1F](#pcbi.1007972.g001){ref-type="fig"}, top row; time\<8h), gradually decreasing as some CTLs eliminated all their targets ([Fig 1E and 1F](#pcbi.1007972.g001){ref-type="fig"}, top row; time\>8h). In contrast, hit sharing in the case of multiple-hitting CTLs led to a delayed onset of killing ([Fig 1E and 1F](#pcbi.1007972.g001){ref-type="fig"}), with the length of the delay dependent on the number of targets sharing hits (compare [Fig 1A and 1B](#pcbi.1007972.g001){ref-type="fig"} with single targets to [Fig 1F](#pcbi.1007972.g001){ref-type="fig"} with multiple targets, for identical *η*). The interaction between *η* and the number of initial targets can also be understood from heatmaps of targets killed ([Fig 1G](#pcbi.1007972.g001){ref-type="fig"}). The expected cumulative number of kills increases over time for *η*,*λ* = 1, but this increase is independent of the initial number of targets except for the censorship implying a maximum target number that can be killed. For *η*,*λ* = 10, the dependency of the observed kills on the initial number of targets is very clear, with killing happening earlier in those wells with initially fewer targets. Moreover, these effects did not only depend on the initial number of targets. When we performed simulations with the hitting rate *λ* a random variable, this in turn increased the variability of killing amongst multiple-hitting CTLs to a greater extent than was the case for single-hitting CTLs ([S1 Fig](#pcbi.1007972.s003){ref-type="supplementary-material"}), implying that the killing of multiple-hitting CTLs is more sensitive to environmental variables than the single-hitting CTLs. Taken together, these results imply that multiple-hitting CTLs could explain both heterogeneous and delayed onset killing among clonal CTL populations. Multiple-hitting is not identifiable based on population killing statistics only {#sec004} -------------------------------------------------------------------------------- We asked if population-level killing statistics (as e.g. examined in \[[@pcbi.1007972.ref019]\])) could be used to identify the hitting parameters (*λ* and *η*) of CTLs. In our previous simulations ([Fig 1](#pcbi.1007972.g001){ref-type="fig"}) we studied a scenario of simultaneous risk for target cells, yet this may be an oversimplification. For example, due to physical constraints the number of targets CTLs can simultaneously contact and thus hit must be limited. Therefore, we extended our *1*:*n* Monte Carlo simulations to allow dynamic contacts between CTLs and targets, in order to check how the parameter estimates (*λ* and *η*) would be impacted. To achieve this, we included an additional state for target cells, now distinguishing between targets that are contacting the CTL, versus those not in-contact ([Fig 2A](#pcbi.1007972.g002){ref-type="fig"}; Methods). The killing kinetics realized by CTLs in these dynamic simulations indeed differed from those in our previous simulations ([Fig 1](#pcbi.1007972.g001){ref-type="fig"}), where all the targets shared risk and so the killing rate of each CTL was dictated by the total number of yet-living targets. In contrast, for our Monte Carlo simulations allowing dynamic conjugate formation, only the targets presently being contacted were relevant. Here, small bursting events occurred throughout the simulations, which was the result of accumulating hits followed by rapid sequential killing among a subset of contacted targets ([Fig 2B](#pcbi.1007972.g002){ref-type="fig"}). ![Burst killing and non-identifiability of dynamically interacting, multiple hitting CTLs.\ A) Schematic of the dynamic model (example with *η* = 3). Target cells are represented by circles containing fractions (numerator: hits received; denominator: *η*). The observable state \[*C U*\] consists of the total number of contacting, *C*, and non-contacting targets, *U*. The complete state of the system is represented by a matrix, with *η* rows indicating the number of hits received (subscript) and with columns indicating whether the target is contacting (*c*~*i*~) or non-contacting (*u*~*i*~). B) Measured killing events (red dots, filled white) during Monte Carlo simulations with *N*~*w*~ *= 10* CTLs and the number of targets drawn from a Poisson distribution with mean $\overline{n} = 16$ (top panels; *λ*,*η* = 10,*k*~*off*~ = 0,*k*~*on*~→∞), or a subset of targets at risk (bottom panels; *λ*,*η* = 10,*k*~*on*~ = 1*hr*^−1^,*k*~*off*~ = 0.3*hr*^−1^). Each horizontal strip is one single simulation, the right panel strips are colored according to the total number of alive targets and the left panel strips are colored according to the number of targets that are in contact with a CTL. C) Each line (coloured according to *η*) is the mean cumulative killing over time (CTL^-1^) from N~w~ = 10^4^ CTLs, simulated using parameters estimated by fitting the case with $\overline{n} = 16$ targets (central column). Straight lines show target values for fitting. All parameters except $\overline{n}$ are constant across columns. D-F) Measured statistics within simulations with $\overline{n}$ = 16 shown in the central column of C. Shown are the distribution of killed targets after 12 hours, with the mean and variance as indicated for each *η* (D), and the mean killing rate over time for CTLs grouped by *η* and shown either separately (E, rows), or together (F, colors), calculated as (*kills*⋅(6 *min*⋅*N*~*w*~)^−1^).](pcbi.1007972.g002){#pcbi.1007972.g002} Using our simulations including dynamic conjugate formation we searched for parameters consistent with the statistics reported previously \[[@pcbi.1007972.ref019]\]concerning high rate "burst killing" CTLs. For fitting we used the reported group mean (4) and variance (6.9) of the number of killed targets per CTL over 12 hours. Additionally, we aimed for a breakpoint in the mean killing rate such that half (2) of the observed kills occurred in the interval 0--9 hours and the other half in the interval 9--12 hours (see [Methods](#sec009){ref-type="sec"}). We performed this fit using different values for *η* (ranging from 1--5, or 10), and a Poisson variable with $\overline{n} = 16$ for the initial number of targets ([S2 Fig](#pcbi.1007972.s004){ref-type="supplementary-material"}). We obtained good fits for different values of *η*; in particular for all *η*\>2 the cumulative killing was very closely matched ([Fig 2C](#pcbi.1007972.g002){ref-type="fig"}, intersecting lines for $\overline{n} = 16$). Moreover, for values *η*\>3 the fits to the mean and variance for cumulative targets killed at 12 hours were all similarly close to their target values of 4 and 6.9, respectively ([Fig 2D](#pcbi.1007972.g002){ref-type="fig"}). Some differences for different *η* were apparent, for example as the number of hits increased towards *η* = 10 the breakpoint marking transition from low to high rate was more distinct ([Fig 2E](#pcbi.1007972.g002){ref-type="fig"}). However, overall differences between *η* were quite small ([Fig 2F](#pcbi.1007972.g002){ref-type="fig"}), and many simulated CTLs were required for these differences to emerge consistently (at least N~w~ = 10^3^ CTLs). Our results were also sensitive to the distribution for the initial number of targets per CTL: for simulations with $\overline{n} = 12$ or 20, substantial differences in the cumulative kills over time occurred ([Fig 2C](#pcbi.1007972.g002){ref-type="fig"}). Thus, we conclude that multiple-hitting is not only qualitatively, but also quantitatively consistent with the experimental results reported previously [(Vasconcelos et al. 2015)](https://paperpile.com/c/oMBHuP/ZTAi). However, our analysis shows that the mean and variance of the killing process measured for a group of CTLs are insufficient statistics to determine the number of hits CTLs require to kill targets, so CTL:target interactions should be explicitly accounted for if killing due to multiple-hitting is to be modelled accurately. An Agent Based Model of Multiple-hitting CTLs to test methods for estimation of killing parameters {#sec005} -------------------------------------------------------------------------------------------------- Since we found that in many situations the true hitting parameters for CTLs could not be determined based on group level killing mean and variance, we sought methods to compare the likelihood of different hitting models. We did not wish to consider a particular model for the process of CTLs finding targets, preferring a method that could be applied to determine the CTL hitting behaviour in general situations (i.e., in the absence of knowledge on contact dynamics). As a framework for testing we employed an agent based cellular Potts model (CPM) to generate 2D simulations of CTLs interacting with and killing targets ([Fig 3A](#pcbi.1007972.g003){ref-type="fig"}). The resulting datasets were visually similar to realistic microscopy data and could be used to investigate methods for recovering the hitting parameters (*η* and *λ*) of CTLs from experimental data under various conditions. For all CPM simulations we maintained the same underlying gamma model of CTL hit generation as was used for our Monte-Carlo simulations (Figs [1](#pcbi.1007972.g001){ref-type="fig"} and [2](#pcbi.1007972.g002){ref-type="fig"}), however we made several modifications that would lead to different (yet not [predictable]{.ul} *a priori*) distributions of hits amongst targets. Specifically, instead of allocating hits to all contacted targets with equal probability, target risk of receiving a hit was proportional to the length of the interface between CTL and target at the moment of hit generation ([Fig 3A](#pcbi.1007972.g003){ref-type="fig"}, target coloring on left images indicates interface length). We also considered the effect of a lower bound on the time required for a CTL to complete a hit, by introducing a delay condition that prohibited targets from being hit within an initial time window after contacting a CTL, which was reset every time the target broke contact with the CTL ([Fig 3A](#pcbi.1007972.g003){ref-type="fig"}, target coloring on right images). Note that the delay condition was applied per target and therefore does not preclude the possibility of CTLs hitting other contacted targets simultaneously. ![Characterisation of high- and low-motility *in silico* CTLs within CPM simulations.\ A) Still images of a high motility CTL with 15 minute minimal hitting time, interacting with targets. Left color scheme: CTLs are red, uncontacted targets are grey, and contacted targets have various shades of blue based on their share of total CTL:target interface, which determines their probability of receiving a hit. Targets are overlaid with the number of hits they have received. Right color scheme: Lattice sites inhabited by the CTL are colored according to actin activity \[[@pcbi.1007972.ref022]\]. Targets are black, turning yellow after 15 minutes of continuous contact with the CTL. Elapsed simulation time is displayed in the upper left corner of the stills, presented in minutes since the first frame shown. B-C) Track plots showing movement of 3 randomly sampled CTLs of high (B) and low (C) motility throughout a simulation, for simulated η as shown. D-E) Frequency at which CTLs form new conjugates (D) and mean number of simultaneously contacted targets per CTL (E) for low- and high-motility CTLs. Plots are based on 100 simulations per condition, with each dot representing one CTL, and circles and error bars indicating mean +/- SD.](pcbi.1007972.g003){#pcbi.1007972.g003} Finally we varied CTL migration to create two groups of CTLs which we termed "high-motility" ([Fig 3B](#pcbi.1007972.g003){ref-type="fig"}, [S1 Video](#pcbi.1007972.s009){ref-type="supplementary-material"}) or "low-motility" ([Fig 3C](#pcbi.1007972.g003){ref-type="fig"}, [S2 Video](#pcbi.1007972.s010){ref-type="supplementary-material"}) CTLs. For both motility conditions the migration of the CTLs was influenced by the presence of the targets, as CTLs became corralled by surrounding targets. The difference between these models was that high-motility CTLs exhibited an increased propensity to break free from confinement and roam the well. This roaming ensured that over the course of 12 hours the high-motility CTL made new contacts with far greater frequency than low-motility CTLs ([Fig 3D](#pcbi.1007972.g003){ref-type="fig"}), although the average number of simultaneously contacted targets at any time was similar ([Fig 3E](#pcbi.1007972.g003){ref-type="fig"}). Thus, the high-motility CTL is expected to approach the previously modeled 'all targets at risk' scenario more closely than the low-motility CTLs. We used the CPM model to simulate CTLs (with *η*,*λ* = 1,2, *or* 10), in either high- or low-motility scenarios. The total amount of targets killed by each CTL depended on the interaction between the parameters *λ* and *η*, the CTL motility, and the presence or absence of the delay condition. In particular, the combination of high motility plus 15 minute delay resulted in a substantial decrease in killing in comparison to the other simulation groups, for all values of *η* ([Fig 4A](#pcbi.1007972.g004){ref-type="fig"}). Together with the high rate of contact formation in that group ([Fig 3D](#pcbi.1007972.g003){ref-type="fig"}), this is consistent with targets spending significant time in transient contacts with the CTL, too short to result in successful hit delivery. The killing rate of the low-motility CTLs was initially greater than of high-motility CTLs, in particular for large *η* ([Fig 4B](#pcbi.1007972.g004){ref-type="fig"}), due to the more stable nature of the contacts leading to greater accumulation of hits among the contacted targets ([Fig 4C](#pcbi.1007972.g004){ref-type="fig"}). High-motility CTLs reduced this deficit over the course of the simulations due to an accumulation of latent hits among uncontacted targets ([Fig 4D](#pcbi.1007972.g004){ref-type="fig"}). These spatial simulations therefore illustrate how CTL:target contact dynamics can play a role in determining killing performance. Moreover, since in these models CTLs with the same killing parameters---but different motility parameters---generated different killing kinetics, they are useful to test how underlying killing parameters might be recovered from microscopy data that are similar to data emanating from our realistic simulations. {#pcbi.1007972.g004} Estimating CTL hitting parameters through analysis of contact time and target survival {#sec006} -------------------------------------------------------------------------------------- Since we found that hitting parameters *η* and *λ* could not be recovered via analysis of population averages only, we employed a parametric survival analysis to study the hazard experienced by individual targets contacting CTLs. Our analysis considers the different hazard functions identified earlier ([Fig 1B](#pcbi.1007972.g001){ref-type="fig"}), which distinguish CTLs on the basis of their intrinsic hitting rate *λ* and the number of hits required for killing targets, *η*. Specifically, we analyse the cumulative duration of CTL:target contact events from the perspective of the target cells (example in [Fig 5A](#pcbi.1007972.g005){ref-type="fig"}). To take into account shared hazard amongst a set of co-contacting targets, we recorded for each sampled frame the statistic *θ* = (*c*)^−1^ (per-target), representing the probability that each separate target out of the subset of *c* targets co-contacting the CTL is presently being hit ([Fig 5B](#pcbi.1007972.g005){ref-type="fig"}). Note that targets not in contact with the CTL were assigned *θ* = 0. Subsequently, we integrated the *θ* values over time to arrive at a set of 'adjusted' contact times, *τ*, for each target ([Fig 5C](#pcbi.1007972.g005){ref-type="fig"}), which takes into account uncertainty with respect to hitting of multiple co-contacted targets ([Fig 5C](#pcbi.1007972.g005){ref-type="fig"}). This approach has the advantage that no explicit account needs to be taken of the CTL-target interaction dynamics. Moreover, estimation of cellular contact times occurs already frequently in time-lapse imaging data \[[@pcbi.1007972.ref007],[@pcbi.1007972.ref023]\], hence is feasible. {#pcbi.1007972.g005} Applying the concept of adjusted contact times, *τ*, on all our CPM simulations, we established maximum likelihood estimates for the hitting parameters within the simulations ([S1 Text](#pcbi.1007972.s002){ref-type="supplementary-material"}; [S3 Fig](#pcbi.1007972.s005){ref-type="supplementary-material"}). This yielded excellent estimates for the parameters in simulations without delay ([Fig 5D](#pcbi.1007972.g005){ref-type="fig"}; *η*~*CPM*~,*λ*~*CPM*~ indicate input CPM parameter values, and $\hat{\lambda},\ \hat{\eta}$ indicate estimated values). We also tested our method of parameter recovery by fitting our model to small subsets taken from the CPM simulations each containing only N~w~ = 10 CTLs ([Fig 5E](#pcbi.1007972.g005){ref-type="fig"}), which led to good estimates. Additionally, we tested our model on sample data generated from a mixed dataset with two subpopulations of single-hitting CTLs, each with a different killing rate ([S1 Text](#pcbi.1007972.s002){ref-type="supplementary-material"}), in order to examine the high-rate-killer hypothesis put forth by Vasconcelos *et*. *al*. \[[@pcbi.1007972.ref019]\]. We found that the multiple-hitting model would not predict multiple-hitting unless multiple-hitting was indeed underlying the data, instead predicting a single-hitting population whose killing rate was the mean of the individual subpopulations ([S1 Text](#pcbi.1007972.s002){ref-type="supplementary-material"}; [S4 Fig](#pcbi.1007972.s006){ref-type="supplementary-material"}, [S5 Fig](#pcbi.1007972.s007){ref-type="supplementary-material"}). Thus, our maximum likelihood approach based on contact time monitoring can distinguish multiple-hitting from alternative hypotheses and is expected to work for a relatively small number of samples. Impact of a hitting threshold on killing parameter estimation {#sec007} ------------------------------------------------------------- Given the time needed for formation of a cytotoxic synapse that is required for hit delivery, brief interactions between CTLs and targets may not contribute to killing. Taking such brief interactions into account in our parameter estimation may thus interfere with correct estimation. Therefore, we tested our parameter recovery on those CPM simulations wherein a 15 minute minimal bound (+15m) was set for the time CTLs required to successfully execute each hit upon a target. In these CPM simulations, we generally obtained robust estimates for the number of hits needed to kill targets, $\hat{\eta}$ ([S6A Fig](#pcbi.1007972.s008){ref-type="supplementary-material"}). However, after rounding to the nearest integer value for *η*, the estimated hitting rate parameter, $\hat{\lambda}$, was underestimated compared to the generating value (*λ*~*CPM*~) in simulations with the 15m minimal hitting time. Since the realised killing was reduced in the 15m-delay simulations, particularly for high-motility CTLS ([Fig 4](#pcbi.1007972.g004){ref-type="fig"}), the estimated $\hat{\lambda}$ could be considered more appropriate than the generating value *λ*~*CPM*~. Nevertheless, to investigate further we performed additional simulations, using high motility CTLs, with variable hitting delays in the interval between 0--15 mins (Figs [6A](#pcbi.1007972.g006){ref-type="fig"} and [S6B](#pcbi.1007972.s008){ref-type="supplementary-material"}). We found that for the important boundary between single-hitting (*η* = 1) or multiple-hitting (*η* = 2), the estimated number of hits parameter $\hat{\eta}$ was accurately classified for limited delays of less than 15 minutes ([Fig 6A](#pcbi.1007972.g006){ref-type="fig"}, top row). {#pcbi.1007972.g006} A particular advantage of a parametric survivorship analysis such as that we employ here is that, having estimated the hitting parameters (*η*,*λ*), we can revisit the sample data and ask whether different subsets of targets were killed according to our expectation. We selected the high-motility +15m simulations with *η*~*CPM*~ = 1 for further study, since for this simulation group there was an ambiguous estimate of $\hat{\eta}$. For comparison, we also analysed the data from CPM simulations with multiple-hitting CTLs (*η*~*CPM*~ = 2). First, we inspected the Kaplan-Meier estimates of the survival functions ([Fig 6B](#pcbi.1007972.g006){ref-type="fig"}, black lines) marking close agreement when the correct parameter estimate ($\hat{\eta} = 2$) was applied to CPM data generated by multiple-hitting CTLs ([Fig 6B](#pcbi.1007972.g006){ref-type="fig"} bottom, blue line, *η*~*CPM*~ = 2), but not when the incorrect $\hat{\eta} = 1$ was applied ([Fig 6B](#pcbi.1007972.g006){ref-type="fig"} bottom, red line, *η*~*CPM*~ = 2). For data generated by single-hitting CTLs, the Kaplan-Meier estimate lay exactly between the estimates using $\hat{\eta} = 1$ or 2, yet the shape of the survival function over the entire length better matched that for the single-hitting estimate $\hat{\eta} = 1$ ([Fig 6B](#pcbi.1007972.g006){ref-type="fig"} top, red line, *η*~*CPM*~ = 1) than for the estimate $\hat{\eta} = 2$ ([Fig 6B](#pcbi.1007972.g006){ref-type="fig"} top, blue line, *η*~*CPM*~ = 1). Second, visual inspections of the hazard experienced by individual targets throughout the simulations ([Fig 6C](#pcbi.1007972.g006){ref-type="fig"}), revealed that in many CPM simulations with *η*~*CPM*~ = 1 and a +15m hitting delay there was substantial killing of targets that had not yet undergone long interactions with the CTL ([Fig 6C](#pcbi.1007972.g006){ref-type="fig"}, first two grey bars), as would not be expected for multiple-hitting. Thus, both results ([Fig 6B and 6C](#pcbi.1007972.g006){ref-type="fig"}) supported $\hat{\eta} = 1$ as the most likely candidate for the data derived from simulations with *η*~*CPM*~ = 1. However, the most conclusive result was obtained by evaluating the mean hazard experienced by contacted targets according to either of the two candidate estimates for the number of hits ($\hat{\eta}$ = 1 or 2). Integrating this value over the duration of the experiments ([Fig 6D](#pcbi.1007972.g006){ref-type="fig"}, black lines) led to predictions for the killing rate over time which closely followed the data whenever a correct estimate for $\hat{\eta}$ was applied ([Fig 6D](#pcbi.1007972.g006){ref-type="fig"}, comparing black and red lines in the upper panel, or black and blue lines below). In contrast, killing predictions from incorrect estimates of $\hat{\eta}$ were extremely poor, thus allowing for correct identification of the underlying *η*. Thus, our analysis shows that monitoring of cumulative interaction times between targets and single CTLs allows for proper estimates of the number of hits required for target cell death even when brief contacts between CTLs and targets cannot lead to hits, although the hitting rate may be underestimated in that case. Discussion {#sec008} ========== Here we have used stochastic simulations to show that 'multiple-hitting' is a plausible explanation for the heterogeneous and time-inhomogeneous killing activity recently observed for CTLs *in vitro* \[[@pcbi.1007972.ref019]\]. We showed that multiple-hitting leads to an increase in realised killing rate over time. Moreover, the extent of this late onset killing increases when more hits are required to kill targets, or when a greater number of antigen-presenting targets are simultaneously contacted. Furthermore, identical CTLs displayed varying killing performance depending on the number of targets available. Simulating CTLs with variable hitting rates, we also found that the killing performance of multiple-hitting CTLs is more heterogeneous than killing of single-hitting CTLs, given similar variation in underlying hitting rate. Overall, we conclude that multiple-hitting is sufficient to explain heterogeneous killing amongst clonal CTLs and there is no need to invoke an unobserved subpopulation of high-rate killers. Given the dependence of the killing performance of multiple-hitting CTLs on several parameters that we describe here, we developed spatially explicit CPM simulations to assess methods for investigating whether multiple hitting occurs *in vitro* or *in vivo*. Our specific goal was retrieval of the hitting rate and number of hits required for CTLs to kill targets. Our model of dynamic conjugate formation can be conceptualised using Kendall's notation as an M/E~r~/1 queue \[[@pcbi.1007972.ref024]\]. Within this framework there is 1 "server" (in our case the CTL), with markovian arrival times (M: in our case conjugate formation events), and Erlang distributed (E~r~) "service times" which represent the killing process. It is known that for such models, the mean and variance (or any similar measure of variability) are insufficient for estimating the true parameters, and can only be used to approximate the distribution \[[@pcbi.1007972.ref024]\]. Instead of using population-level killing statistics, we were able to accurately recover model parameters from the CPM data by analysing the 'adjusted' cumulative contact durations between CTLs and individual targets, i.e. the total length of the interaction until either the target was killed or the experiment ended. Importantly, we found that measurements for both killed and surviving cells are required for this approach to be successful. This is because the limited time window of observation renders data that are in part censored, yet elapsed contacts that have not yet resulted in killed targets also contain information on underlying killing parameters. In a similar fashion, we previously developed a method to estimate absolute (i.e., not cumulative) cellular interaction times based on time lapse imaging data \[[@pcbi.1007972.ref023]\]. Although CTL cooperativity and multiple-hitting have now been described in a number of settings \[[@pcbi.1007972.ref007],[@pcbi.1007972.ref010]--[@pcbi.1007972.ref012]\], a detailed quantitative description of the sequence of intracellular events which might underlie multiple-hitting does not yet exist. Several mechanisms can be envisaged which separately or collectively might result in target cells enduring sustained attacks from CTLs before death. A first factor which may explain the ability of target cells to endure sustained attacks is death occurring via the 'extrinsic apoptosis pathway', i.e., via tumour necrosis factor (TNF) or FAS-L. In the study of Vasconcelos et. al. \[[@pcbi.1007972.ref019]\], blockade of FAS-L did not diminish overall killing, suggesting that FAS-L was not involved in CTL killing. Moreover, separation of CTLs and targets in a transwell assay showed that contact was required before target cell apoptosis could occur. Although this result suggests that diffusible TNF did not contribute to target cell apoptosis, TNF is also expressed in transmembrane form \[[@pcbi.1007972.ref025]\] and may have contributed to contact-dependent killing, or could have synergised with other effector pathways. Furthermore, TNF or interferon-γ---another hallmark cytokine produced by activated CTLs---have been linked to an upregulation of FAS-L receptors in different cell types \[[@pcbi.1007972.ref026],[@pcbi.1007972.ref027]\], or might otherwise synergise with FAS-L to induce target cell apoptosis \[[@pcbi.1007972.ref028]\]. The possibility of synergistic activators of the extrinsic apoptosis pathway is intriguing since activation of such mechanisms might explain delayed onset of burst-killing. This was observed in recent studies in which natural killer cells initially controlled tumour cell targets with a fast-acting, perforin-dependent mechanism, before switching to a mechanism primarily depending on engagement of death receptors \[[@pcbi.1007972.ref017],[@pcbi.1007972.ref018]\]. It would be useful to investigate whether CTLs also utilise this mechanism. A second factor which may account for multiple-hitting is heterogeneity in delivery of perforin and granzymes. Perforin alone induces rapid pore formation in target cell membranes, with such membrane disruption expected to increase the metabolic burden on target cells. Even if insufficient to directly induce apoptosis, one would expect such depletion to divert resources from adaptive cellular stress responses, thereby sensitising cells to death from other mechanisms. Granzymes are a diverse set of cytotoxic proteases with a broad array of intracellular targets \[[@pcbi.1007972.ref029]\]. A recent review highlights that perforin-mediated pore formation may or may not be accompanied by delivery of granzyme molecules into the cytosol \[[@pcbi.1007972.ref030]\]; a requirement for granzyme delivery appears to be the establishment of a sufficiently large pore at the point of contact between CTL and target. Examination of recent 4D images of CTL--target engagement highlight potential for heterogeneous delivery of cytotoxic molecules \[[@pcbi.1007972.ref008]\]. That study showed the capability of a single CTL to rapidly organise lytic molecules around the centrosome upon initial target recognition and to subsequently polarise the centrosome towards the target. This sequence of events results in a strong and stable cytotoxic synapse with a high local density of perforin and granzymes. Anecdotal evidence from this same work indicates that there can also be an alternative outcome: In one observation a CTL attempted to form two immunological synapses with one target, with the result that effective centrosome polarisation towards either synapse did not occur and ultimately both synapses were aborted without target cell death (see S8 Video in reference \[[@pcbi.1007972.ref008]\]). Other observations of CTLs simultaneously polarising granules towards multiple targets \[[@pcbi.1007972.ref009]\] demonstrate that the formation of multiple immunological synapses does not necessarily preclude CTLs from killing. Taken together, these observations suggest that due to the diversity of possible damage pathways activated by CTLs as well as the potential for heterogeneity in delivery of granules, several mechanistic explanations for multiple-hit induced killing remain open. Given the breadth of cytotoxic weaponry available to a single CTL, it is apparent that experimental interference with one or more CTL effector functions is insufficient to conclude that one or another pathway is primarily involved in target cell death in a given experiment. We suggest high resolution, *in vitro* imaging as an effective means of achieving insight into the CTL killing process. Such high-resolution imaging would have several benefits: clear visualization of the polarisation of the lytic granules towards target cells would allow acquisition of statistics regarding the lethality of hits. Moreover, monitoring of individual target cells over time would provide statistics regarding the formation and abortion rates of immunological synapses and regarding the probability of target cell death after multiple hits. In addition, such approaches would allow investigation of the possibility of target cell recovery between successive hits, along with assessing the timescale over which such recovery might occur. Although such spatio-temporal resolution might be challenging to achieve experimentally, recent approaches using structured environments \[[@pcbi.1007972.ref031],[@pcbi.1007972.ref032]\] provide a possible means of achieving more refined control of CTL-target interactions. In conclusion, in addition to recent efforts to further characterise heterogeneity amongst CTLs, greater attention is needed to simultaneous monitoring of mechanisms activated in target cells after the target has been contacted by a CTL, assisted by statistical analyses and computational methods such as those presented here. Experimental research particularly involving use of e.g. caspase-8 reporters or reporters of granzyme activity to compare the relative importance of different killing mechanisms, as recently done in NK cells \[[@pcbi.1007972.ref017],[@pcbi.1007972.ref018]\] is crucial. Computational models can then be used to compare results between different experimental assays, thereby quantitatively assessing the contribution of identified CTL effector functions in different contexts. Methods {#sec009} ======= Monte Carlo simulations {#sec010} ----------------------- We devised stochastic simulations representing different "wells" in which individual CTLs killed targets. The setup of the simulations was based on published data by Vasconcelos *et al*. \[[@pcbi.1007972.ref019]\]. In brief, Vasconcelos *et al*. incubated pre-activated human-derived CTL clones with Epstein-Barr virus transformed B cell targets for 12 hours in microwells (*N*~*w*~ = 259). Each microwell contained a single CTL confined with an indeterminate (approximately 10--20, see Fig 4A in reference \[[@pcbi.1007972.ref019]\]) number of targets. Microwells were approximately cylindrical and had a cross-section diameter of approximately 100μm. A caspase reporter was used to determine the killing rate of individual CTLs over time. Similarly, our simulations featured *N*~*w*~ independent simulations, each containing *n* initially unhit targets and lasting for a simulated time period of 12 hours, or until all targets had been killed. The simulations proceed as follows: 1. A random variable *x*~*wait*~, representing the waiting time until the next CTL hit, is drawn from the exponential distribution with rate parameter equal to the CTL hitting rate *λ*. The current simulation time is increased by *x*~*wait*~. 2. A random target is selected and its number of hits is increased by one. 3. If a target has received sufficient hits for death (i.e., *η* hits), it is immediately removed from the simulation. In some simulations, we extended the rules in order to reflect typical *in vitro* assays more accurately: 1. ***Variable target numbers*.** Each simulation contained a single CTL and a variable number of targets *n*. For each well the number of targets was drawn from a Poisson distribution with mean $\overline{n}$. 2. ***Variable hitting rate*.** For each simulation the hitting rate *λ* of each CTL was a normally distributed random variable. The standard deviation of this distribution was used as a model parameter, with larger standard deviation reflecting CTL populations with greater intrinsic heterogeneity in killing performance between individuals. 3. ***Dynamic conjugate formation*.** We considered that hit delivery had to be preceded by conjugate formation and that at *t* = 0 *hrs* the CTL has not yet encountered any targets, and that CTLs form new conjugates with targets at constant rate *k*~*on*~ and abort conjugates with constant rate *k*~*off*~. Thus these simulations consider 4 distinct types of event: in addition to hitting and dying, we now have conjugate formation and conjugate abortation. The Gillespie algorithm was used to determine the type of event and waiting time between subsequent events \[[@pcbi.1007972.ref033]\], except for target cell death which occurs immediately after the lethal hit just as in our "all at risk" simulations. Parameter estimation for the dynamic conjugate formation model was based on four reported values from Vasconcelos *et*. *al*. \[[@pcbi.1007972.ref019]\]: the population killing averages for the high rate killers (6.4 targets killed per 12 hours) or low rate killers (2.8 targets killed per 12 hours), the fraction of the population reported to be high rate killers (⅓), and the breakpoint after which the high rate phenotype appeared (8--10 hours; we took 9 hours for this value). From these 4 reported values we derived three statistics for fitting our Monte Carlo simulations with dynamic conjugate formation: the mean (a~1~) and variance (a~2~) of the number of killed targets per CTL after 12 hours, and the expected number of killed targets per CTL at the breakpoint of 9 hours (a~3~). We estimated the killing at 9 hours by noting that the high rate group had not yet emerged at 9 hours, before which all cells killed at an approximately constant rate. Thus extrapolating from the low rate killing average at 12 hours (2.8 x 9/12) gives approximately 2 targets killed at the 9-hour breakpoint (note that this is also consistent with Fig 4B of Vasconcelos et. al \[[@pcbi.1007972.ref019]\]). Thus, the experimental estimates were: a~1~ = 4, a~2~ = 6.9 and a~3~ = 2. To fit to these estimates, we measured the same statistics (b~1,2,3~) from our simulations and then minimised the root mean squared error: $$RMSE = \sqrt{}\left( 1/3 \cdot {\sum_{i:1,2,3}\left( {a_{i} - b_{i}} \right)^{2}} \right),$$ for different values of the parameters *η*, *λ*, *k*~*on*~, and *k*~*off*~. For the stochastic optimisation we performed 10 repeats for all combinations of selected discrete values of *η*, *k*~*on*~, and *k*~*off*~ ([S2 Fig](#pcbi.1007972.s004){ref-type="supplementary-material"}), and then for each combination we estimated *λ* based on *N*~*w*~ = 1000 repeats and the optimise function in R. Dynamic conjugate simulations were written in C++ using the Rcpp package. Biological interpretation of parameters for the stochastic simulations are summarised in [Table 1](#pcbi.1007972.t001){ref-type="table"} and the parameter values used throughout the manuscript are provided in [S1 Table](#pcbi.1007972.s001){ref-type="supplementary-material"}. 10.1371/journal.pcbi.1007972.t001 ###### Stochastic simulation parameters. {#pcbi.1007972.t001g} parameter biological interpretation --------------------- ------------------------------------------ *η* number of hits required for target death $\overline{n}$ mean number of targets in a well *λ* (hr^-1^) hitting rate *k*~*on*~ (hr^-1^) conjugate formation rate *k*~*off*~ (hr^-1^) conjugate dissociation rate Spatial simulations {#sec011} ------------------- We developed spatial simulations of CTLs killing in microwells, with the aim of generating noisy and undersampled artificial data representative of data generated by microscopy, data which can be used to test methods for recovery of parameters governing CTL hitting. To this end we employed the cellular Potts model (CPM) framework \[[@pcbi.1007972.ref034]\], a formalism we used previously to simulate T cell-target cell interactions \[[@pcbi.1007972.ref011],[@pcbi.1007972.ref035],[@pcbi.1007972.ref036]\]. The CPM is a lattice based model, with entities such as cells represented by assigning individual lattice sites a 'spin' value, to identify them as belonging to a specific entity. The model evolves via minimisation of an energy function, the Hamiltonian: $$H = H_{sort} + H_{l} + H_{act}.$$ Here, *H*~*sort*~ represents interactions between cell surfaces and deviations from a target cell area; *H*~*sort*~ is defined as \[[@pcbi.1007972.ref034]\]: $$H_{sort} = {\sum_{{(a(\sigma) - A_{q(\sigma)})}^{2}}{J\left( {q\left( {\sigma\left( {i,j} \right)} \right),q\left( {\sigma\left( {i^{\prime},j^{\prime}} \right)} \right)} \right)\left( {1 - \delta_{\sigma({i,j}),\sigma({i^{\prime},j^{\prime}})}} \right) + \zeta_{a}{\sum_{spin\ types\ \sigma}\left( a(\sigma) - A_{q(\sigma)} \right)^{2}}},}$$ where *σ*(*i*,*j*) is the spin of an individual cell of type *q* at grid point with *x* coordinate *i* and *y* coordinate *j*; *J*(*q*,*q*′) is the surface energy between cells of type *q* and *q*′; *δ*~*σ*,*σ*′~ represents the Kronecker delta; *a*(*σ*) represents the actual area of a cell and *A*~*q*(*σ*)~ the target area for a cell of type *q* (we refer to this as area rather than volume because we employ 2D simulations); *ς*~*a*~ is a weighting term for the area constraint; Note that the sum of the surface energies are calculated over each third order neighbour of a 2D grid site. Our model also includes a term for surface area conservation of individual cells \[[@pcbi.1007972.ref037]\]: $$H_{l} = \zeta_{l}{\sum_{\sigma}\left( l(\sigma) - L_{q(\sigma)} \right)^{2},}$$ where *L*~*q*(*σ*)~ is the target perimeter for cells of type *q*, *l*(*σ*) is the current perimeter of a cell with type *σ* (determined as the total length of the boundary interfaces with grid sites of differing spin), and *ζ*~*l*~ is the weight of the perimeter constraint. We set $L_{q} = 2\pi\sqrt{A_{q}}$, i.e., the ratio of a circle's perimeter to its area, so that the term *H*~*l*~ is minimised when cells become perfectly circular. We set *ζ*~*l*~ lower for the CTLs than for the target cells, implying that the targets retained a spherical shape whereas CTLs were much more deformable in our simulations. Finally, the Hamiltonian includes a term *H*~*Act*~ to drive the motility of CTLs \[[@pcbi.1007972.ref022]\]: $$H_{Act} = \frac{\varsigma_{Act}}{{Max}_{Act}}\left( {GM}_{Act}\left( u \right) - {GM}_{Act}\left( v \right) \right).$$ This follows an actin-driven cell motility model with protrusions driving the migration of cells. In this model actin is modelled explicitly and when a cell occupies a new site on the lattice, the site is given an actin value *Max*~*Act*~. The actin activity *Act* in that site then decreases by one at every Monte carlo step until it reaches 0. The function: $${GM}_{Act}\left( u \right) = \left( {\prod_{y \in V(u)}{Act\left( y \right)}} \right)^{1/|{V(u)}|}$$ calculates the geometric mean actin activity around site *u*, where \|*V*(*u*)\| are the second order Moore neighbours of site *u* (see Fig 1 of reference \[[@pcbi.1007972.ref022]\]). The model favours updates from sites *u* with high actin activity into neighbouring sites *v* with low actin activity, resulting in local positive feedback. The CPM parameter *ς*~*Act*~ is a weighting term the strength of which we varied to control the motility of the CTLs. The *H*~*act*~ term was not applied to target cells, which are moved only passively via interactions with the CTL and other targets. In our spatial simulations we also implemented a contact-limited hitting behaviour for the CTL. We take CTL killing of targets to occur primarily via the perforin/granzyme pathway so we consider only contacted targets to be at risk, although our model should also apply to FAS-Ligand mediated killing, which is also contact-limited. When multiple targets are contacted by a CTL, it seems likely that the risk of getting hit is not equal for all targets, as polarisation of the lysosome towards specific targets should occur in order to permit delivery of lytic molecules to the target \[[@pcbi.1007972.ref008],[@pcbi.1007972.ref009]\]. Although we did not model the polarisation of the lysosome explicitly, we do take into account a tendency for CTLs to unequally distribute hits towards contacted targets. To achieve this, we implement the same baseline hitting probability as in the Gillespie simulations, and multiply this by *θ*~*i*~(*t*), the proportional fraction of CTL: target membrane interface occupied by the target at time point *t*: $$\theta_{i}\left( t \right) = \frac{l_{i}\left( t \right)}{L_{i}\left( t \right)},$$ where *l*~*i*~(*t*) is the length of the interaction interface between target *i* and the CTL inhabiting the same well, *L*~*i*~(*t*) the total interaction interface length of the CTL that contacts target *i*, including any other co-contacting targets. Because CTLs are considered to hit targets at a constant rate *λ*, for simulations without delayed hitting each target's risk of being hit during a brief time interval *Δt* equals *λθ*(*t*)⋅*Δt*. For some simulations we introduced a rule preventing CTLs from hitting targets for a specified delay period each time a CTL contacted or recontacted a target. This was implemented by means of a counting variable inside each target, such that hits would not register until the target had been in continuous contact with the CTL for the specified interval. Simulations had a spatial scale of 1 μm pixel^-1^ and were 100 μm^2^ in area. The simulation space consisted of a circular area representing a microwell within which one CTL and usually between 10--20 targets were constrained to move. Simulations had a temporal scale of 1 second per Monte Carlo step. Parameters employed in the CPM simulations are given in [Table 2](#pcbi.1007972.t002){ref-type="table"}. Simulation output was produced every 120 Monte Carlo steps (2 minute intervals), corresponding to a typical sampling frequency in time-lapse imaging data with multiple wells \[[@pcbi.1007972.ref019]\]. CPM simulations were developed within the morpheus framework \[[@pcbi.1007972.ref038]\]. 10.1371/journal.pcbi.1007972.t002 ###### Cellular Potts simulation parameters. {#pcbi.1007972.t002g} --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- parameter value Description --------------- ----------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------- *J*~*σ*,*σ*′~ *J~tar,tar~* = 0.7; *J*~*ctl*,*tar*~ = −3; *J*~*tar*,*well*~ = 0; *J*~*ctl*,*well*~ = 0 surface energies between cell types *A*~*q*~ *A*~*ctl*~ = 140 *μm*^2^\ the target area for a cell of type *q* *A*~*tar*~ = 340 *μm*^2^\ *L*~*q*~ $2\sqrt{\pi A_{q}}$ the target perimeter for a cell of type *q* *ς*~*l*~ *ς*~*l*,*ctl*~ = 0.1\ strength of cell perimeter constraint *ς*~*l*,*tar*~ = 0.25 *ς*~*a*~ *ς*~*a*,*ctl*~ = 1\ strength of cell area constraint *ς*~*a*,*tar*~ = 1 *ς*~*Act*~ *ς*~*Act*,*low*~ = 2\ strength of actin protrusion dynamics: *ς*~*Act*,*low*~ for low-motility and *ς*~*Act*,*high*~ for high motility CTLs *ς*~*Act*,*high*~ = 10 *Max*~*Act*~ 50 Actin activity value when CTLs occupy a new lattice site --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Supporting information {#sec012} ====================== ###### Summary of parameters used in stochastic simulations. Data and code used in this project are available (<http://doi.org/10.17605/OSF.IO/6GQYP>). (PDF) ###### Click here for additional data file. ###### Fitting procedure and hypothesis comparison for multiple-hitting model and subpopulation model. (PDF) ###### Click here for additional data file. ###### Multiple hitting increases inherent variability in killing performance between individual CTLs. A-B) Distribution of killed target numbers after 12 hours (A) when intrinsic hitting rates *λ* (B) are drawn from a normal distribution with mean $\overline{\lambda}$ and standard deviation $\sigma_{\lambda}\left( \lambda \sim Normal\left( \overline{\lambda},\sigma_{\lambda} \right) \right.$). C) Overdispersion for the variance in killed targets in A relative to the variance expected for a Poisson distribution, i.e., the ratio of the variance (*var*(*x*)) to the mean ($\overline{x}$) number of targets killed after 12 hours (vertical axis). The horizontal axis is the ratio of the standard deviation to the mean value of the intrinsic hitting rate. (TIFF) ###### Click here for additional data file. ###### Parameter estimation for Monte Carlo simulations with dynamic contacts. A\) Estimated hitting rates (*λ*, represented by colour) for various combinations of the number of hits (*η*, rows), contact formation rates (k~on~, vertical axes in sub-panels), or contact escape rates (k~off~, horizontal axes in sub-panels). Ten repeats (across columns) were performed for the optimisation step, using N~w~ = 10^3^ CTLs per tested value of *λ*. After fitting we validated our results by performing N~w~ = 10^4^ simulations with each best fitting parameter combination, which is shown here. B) Root mean square residual errors for the best fitting parameter estimates (panel arrangement is as described in S2A Fig legend). Results are from validation simulations, using N~w~ = 10^4^ simulations per parameter combination. (TIFF) ###### Click here for additional data file. ###### Monte Carlo simulated CTL:target interaction durations amongst surviving and killed targets. A\) Sample density of killed targets in Monte Carlo simulations lasting until all targets were killed, with different numbers of hits (*η*, on different rows). B) Sample density of killed targets in Monte Carlo simulations stopped after 12 hours. C) Sample density of surviving targets, corresponding to the 'absent' portion of the distribution for killed targets in B. The red line in A and B is the function f~k~, which describes how the relative probability until targets receive η hits arriving at a constant rate λ depends on the cumulative interaction time τ, for a gamma distributed waiting time. For all S3 Fig: N~w~ = 100, n = 12 targets per well, all targets equally at risk. Parameter combinations used were: (*η* = 1,*λ* = 0.34; *η* = 2,*λ* = 1.17; *η* = 3,λ = 2.12; *η* = 4,*λ* = 3.14; *η* = 5,*λ* = 4.22). (TIFF) ###### Click here for additional data file. ###### Maximum likelihood estimation for the killing rate of single-hitting CTLs. A\) Poisson distributions for the number of targets used to start simulations in S4 Fig, with mean $\overline{n} = 8\ or\ 16$ as shown. B) Number of killed targets after 12 hours for N~w~ = 2 x 3 x 1000 simulations, each group of N~w~ = 1000 started with one of the 2 distributions in A, and with one of the 3 indicated parameter settings. C) Density of killed targets after 12 hours from 'Mixed' distributions resulting from *η* = 1,*λ*~***LR***~ = **0**.2,*λ*~*HR*~ = 0.7 and either $\overline{n}$ = 8 *and m* = 0 (left panel), or $\overline{n}$ = 16 *and m* = 0.67 (right panel). Note that for $\overline{n}$ = 8 the killing of multiple-hitting CTLs became greater than the high rate subpopulation of single-hitting CTLs; $\overline{n}$ = 8 was only used for testing robustness of the estimators on heavily censored data. D) Relative likelihood of candidate hitting rate estimates, $\hat{\lambda}$, compared to the maximum likelihood estimate, ${\hat{\lambda}}_{ML}$, resulting from application of the Poisson estimator separately to each of the single-hitting (*η* = 1) datasets shown in B. Relative likelihood are shown either for the dataset in its entirety (dashed lines), or for a randomly selected sample of N~W~ = 10 (solid lines). E) Examples of testing datasets derived from the multiple-hitting population (B, $\overline{n}$ = 16, *η* = 10) or from a mixture of single-hitting CTLs (B, $\overline{n}$ = 16, *η* = 1, where the true density of killed targets in the mixture distribution is in C). F) Relative likelihood of candidate hitting rate estimates, $\hat{\lambda}$, compared to the maximum likelihood estimate, ${\hat{\lambda}}_{ML}$, for constrained fits constructed from either the subpopulation datasets, or from multiple-hitting datasets, for three samples with either N~w~ = 30,100, or 1000 (note the multiple-hitting-generated data (*η* = 10) is therefore fully represented by the N~w~ = 1000 case). (TIFF) ###### Click here for additional data file. ###### Testing for multiple-hitting CTLs versus subpopulations of single-hitting CTLs. A\) Maximum likelihood estimates for the hitting rate, ${\hat{\lambda}}_{ML}$, with either the gamma or Poisson estimators, both constrained to a uniform single-hitting population (i.e. by forcing *η* = 1 for the gamma estimator and by forcing *m* = 1 for the Poisson estimator). Each of the 2x4x10 = 80 points represents one of the 4x10 testing populations from [S4E Fig](#pcbi.1007972.s006){ref-type="supplementary-material"} (here indicated by facet labels), fit with both of our estimators (x-axis). B) Difference between the log likelihood function evaluated with the constrained versus unconstrained Gamma estimator $log\ L\left( {\hat{\lambda}}_{ML},{\hat{\eta}}_{ML} \right)$ (dark bars); or with the constrained versus unconstrained Poisson estimator $log\ L\left( {\hat{\lambda}}_{HR,ML},{\hat{\lambda}}_{LR,ML},{\hat{m}}_{ML} \right)$ (light bars). Each of the 40 testing populations occupies one horizontal bar, with the details of the testing populations as indicated in facet labels. For the x-axis scaling (negative values are not possible), the relative size of the dark v.s. light bars is proportional to the strength of the evidence for the multiple-hitting hypothesis (dark bars) versus the subpopulation hypothesis (light bars). C) The constrained estimates for the hitting rate parameters, ${\hat{\lambda}}_{ML}$, (circles; also shown in A) or their unconstrained counterparts (red asterisks) for each testing population (points on x-axis). For the Gamma estimator (top row) the estimated ${\hat{\eta}}_{ML}$ is shown only where ${\hat{\eta}}_{ML} > 1$. For the Poisson estimator (bottom row), the unconstrained estimates for ${\hat{\lambda}}_{HR,ML},{\hat{\lambda}}_{LR,ML}$ are above and below their counterpart constrained estimates, and the Gamma and Poisson estimators can be compared per population. D) Distribution of all cumulative interaction times, *τ* (killed and surviving targets shown separately in columns), for all N~w~ = 1000 members of each of the 3 generating populations (as shown in [S4B Fig](#pcbi.1007972.s006){ref-type="supplementary-material"}). Although the 2 single-hitting populations were combined (upper row), the separate contribution of the *λ*~*HR*~ (red) or *λ*~*LR*~ (blue) populations is indicated by color. Multiple-hitting CTLs (green) are shown separately (bottom row). (TIFF) ###### Click here for additional data file. ###### Maximum likelihood estimation for CPM simulations with a hitting threshold time. A-B) Heatmaps of the likelihood function around the maximum likelihood estimates for the killing parameters *η* and *λ*, in CPM simulations under various conditions. In A, results are shown for various *η* values (rows) and for both high-motility (left colum) and low-motility (right column) conditions for simulations with 15 minute hitting delay. In B, results are shown for high motility CTLs at all tested values of the delay (in range 0--15 minutes, across columns). The horizontal and vertical lines in A-B mark the values of the CPM parameters used to generate the data for each group and the boundary enclosing the 95% confidence region is marked with a thin blue line. (TIFF) ###### Click here for additional data file. ###### Simulation of high-motility CTL, requiring 5 hits to kill targets. (AVI) ###### Click here for additional data file. ###### Simulation of low-motility CTL, requiring 5 hits to kill targets. In all videos, CTLs are shown in red whilst uncontacted targets are in grey. Contacted targets are shaded blue based on their share of total CTL:target interface, i.e. the probability that they will receive the next hit generated by the CTL. Targets are overlaid with the number of hits they have received. Elapsed simulation time (hours:minutes) is displayed in the upper right corner of the videos. (AVI) ###### Click here for additional data file. We would like to thank Delphine Guipouy and Loïc Dupré for useful discussions on the set-up of previously published microwell experiments. 10.1371/journal.pcbi.1007972.r001 Decision Letter 0 De Boer Rob J. Deputy Editor Yates Andrew J. Associate Editor © 2020 De Boer, Yates 2020 De Boer, Yates This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 12 Feb 2020 Dear Dr. Beltman, Thank you very much for submitting your manuscript \"Heterogeneous, Delayed-Onset Killing by Multiple-Hitting T Cells: Stochastic Simulations to Assess Methods for Analysis of Imaging data\" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers\' comments. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers\' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation. When you are ready to resubmit, please upload the following: \[1\] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. \[2\] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments. Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts. Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don\'t hesitate to contact us if you have any questions or comments. Sincerely, Andrew J. Yates Associate Editor PLOS Computational Biology Rob De Boer Deputy Editor PLOS Computational Biology \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\* Reviewer\'s Responses to Questions **Comments to the Authors:** **Please note here if the review is uploaded as an attachment.** Reviewer \#1: Comments: Beck et al simulate CTL-mediated immune responses in different settings to better understand how CTL-mediated killing of target cells is regulated in vitro. They use different simulation approaches to address the very important open question of how CTL-mediated killing of target cells can be better understood. This remains a very important question, given the high clinical importance of CTL-mediated immune responses against infectious diseases and cancer in the current age of immune-cell based therapeutic approaches. Reviewer comments regarding the text: Regarding the Monte Carlo Simulations in Figure 1, it remains a little unclear how a small increase in simulated target cell numbers (plus 1) would change the outcome of the simulation in the way presented in Figure 1B (for the 100 hit case). Obviously, the "100 hit case" is a very extreme situation -- but maybe this outcome of the simulation could be discussed and explained in more detail. Maybe a plotting of the effector-to-target cell ratio of these simulations would help to better understand this result? Regarding the "over-dispersion" -- maybe a short explanation of how such a conclusion was reached might be helpful (how was dispersion measured, and how can the cut-off that determines over-dispersion be justified). Regarding the spatial model, it remains unclear how the "two CTL motility patterns" are used in the simulations: how are the two states set for individual cells? Does target cell contact decrease motility? Based on what experimental data are these motility features defined? Finally, to better understand how "cumulative contact time" in the simulations can be interpreted a short description of how the simulated CTL deliver the "lethal hits" in relation to contact duration would be helpful. How is "hit delivery" defined in the spatial model? Could one contact event lead to multiple "hits"? Reviewer comments on Material and Methods section: 1.) Monte Carlo simulations: How were these simulations performed? How was the data from Vasconcelos et al used exactly and where can the used data be found? How many simulations were performed? 2.) Spatial simulations How was target cell motility defined? Why are the targets motile -- how much does that affect the different conclusions (Maybe check results with non-motile and motile targets). Reviewer comments on Figures: Figure 1: Fig 1B: what does the Y-axis label really mean ("frequency")? Maybe this can be described in the figure legend. Fig 1D: The Y axis is cut at 10 -- is this really necessary? Fig 1E: what does the Y-axis label mean -- is it different to Fig 1B? Figure 2: Usually, target cell binding is expected to alter CTL migration -- so how does target binding affect the CTL motility here? How are the high vs low motility states "regulated" in these simulations? Reviewer \#2: In this study, the authors use stochastic simulations to examine if heterogeneity in CTL killing dynamics can be explained by the requirement of target cells to receive multiple lytic hits. Using explicit spatial simulations based on the CPM-framework, they further investigate how this hitting rate and the number of required hits per target cell can be inferred from imaging data. Based on their analyses they show that measuring the cumulative duration of CTL-target contacts would improve the accuracy of estimating CTL killing kinetics. The topic is timely and relevant as understanding CTL efficacy is an important aspect for designing optimal therapeutic vaccination and treatment strategies, as e.g. against cancer. The study is well written and thoroughly structured. With this theoretical analysis the authors provide a reasonable explanation for previous observations from experimental studies and propose required measurements for further experiments. However, the study remains a bit limited by focusing on only one explanation for CTL killing dynamics. \# Major points: (1.) The authors state that with the multiple-hitting-scenario, there is no need to invoke an unobserved subpopulation of high-rate killers (p. 22) as suggested by other studies. While this is true and nicely shown, I think the study would substantially benefit if the authors also show how the proposed information from imaging studies should differ in case of the latter scenario or others. It could be quite interesting to know on what type of measurements one has to focus in order to differ between different hypotheses, such as having cell populations that are heterogeneous (either in their killing efficacy (CTL) or susceptibility to killing (target cell)), having serial or multiple-killing, or assuming single- or multiple hit killing. (2.) The ability to infer the hitting parameters in case of multiple-hitting CTL from CTL-target (cumulative) contact times in case of multiple-hitting is quite robust and interesting. However, measuring the cumulative contact times might be not sufficient in case that active conjugate formation would require the stopping of CTLs to allow them to deliver their lethal hits (Wiedemann et al., PNAS 2006), i.e., requiring a minimal contact time. This seems not to be directly implemented within the CPM as the hitting probability only considers the contact length for distributing the hit across multiple target cells. (3.) Another question concerns the parameterizations of the CPM. Do "high-" and "low-motility" CTL correspond in some way to the dynamics observed within the experiments or were they arbitrarily chosen? (4.) Why is the average number of target cells killed after 12 hours for a density of 15 target cells higher when several hits are required compared to only one? This seems to be counterintuitive unless the hitting rates differ between the scenarios, maybe due to the adjustment done before and mentioned in the text? However, Figure 1E actually shows that for a given target cell concentration, the distribution gets shifted to the left the more hits are required. The values of the rates used for simulation should be mentioned within the text or the figure legends. The corresponding Table (Table 1) seems to be empty. \# Minor points: \- Figure 4A and Figure 5F are very difficult to read. Maybe there are better ways of representation, e.g. using different colors for each given (correct) parameter combination and similar colors with point sizes corresponding to frequencies for the estimates? \- Figure 1B: Does the red dotted line always refer to the 16 targets case for each row separately, i.e. for each number of hits (1,10,100)? \*\*\*\*\*\*\*\*\*\* **Have all data underlying the figures and results presented in the manuscript been provided?** Large-scale datasets should be made available via a public repository as described in the *PLOS Computational Biology* [data availability policy](http://journals.plos.org/ploscompbiol/s/data-availability), and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information. Reviewer \#1: None Reviewer \#2: No: The parameters used for simulating the results shown in Figure 1 seem to be missing. \*\*\*\*\*\*\*\*\*\* PLOS authors have the option to publish the peer review history of their article ([what does this mean?](https://journals.plos.org/ploscompbiol/s/editorial-and-peer-review-process#loc-peer-review-history)). If published, this will include your full peer review and any attached files. If you choose "no", your identity will remain anonymous but your review may still be made public. **Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our [Privacy Policy](https://www.plos.org/privacy-policy). 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This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: <http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5>. [Reproducibility:]{.ul} To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions, please see [[http://journals.plos.org/compbiol/s/submission-guidelines\#loc-materials-and-methods](http://journals.plos.org/plospathogens/s/submission-guidelines)]{.ul} 10.1371/journal.pcbi.1007972.r002 Author response to Decision Letter 0 13 May 2020 ###### Submitted filename: review response.pdf ###### Click here for additional data file. 10.1371/journal.pcbi.1007972.r003 Decision Letter 1 De Boer Rob J. Deputy Editor Yates Andrew J. Associate Editor © 2020 De Boer, Yates 2020 De Boer, Yates This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 21 May 2020 Dear Dr. Beltman, We are pleased to inform you that your manuscript \'Heterogeneous, Delayed-Onset Killing by Multiple-Hitting T Cells: Stochastic Simulations to Assess Methods for Analysis of Imaging data\' has been provisionally accepted for publication in PLOS Computational Biology. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated. IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript. Should you, your institution\'s press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS. Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology.  Best regards, Andrew J. Yates Associate Editor PLOS Computational Biology Rob De Boer Deputy Editor PLOS Computational Biology \*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\* 10.1371/journal.pcbi.1007972.r004 Acceptance letter De Boer Rob J. Deputy Editor Yates Andrew J. Associate Editor © 2020 De Boer, Yates 2020 De Boer, Yates This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 22 Jun 2020 PCOMPBIOL-D-19-02233R1 Heterogeneous, Delayed-Onset Killing by Multiple-Hitting T Cells: Stochastic Simulations to Assess Methods for Analysis of Imaging data Dear Dr Beltman, I am pleased to inform you that your manuscript has been formally accepted for publication in PLOS Computational Biology. 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1. Introduction {#sec1-sensors-15-27142} =============== Accurate exhaust gas recirculation (EGR) rate control and air-fuel ratio (AFR) control are important technologies to satisfy the increasingly stringent emission regulations, which are dependent on the precise calculation of the EGR rate and AFR \[[@B1-sensors-15-27142],[@B2-sensors-15-27142]\]. The accuracy of the EGR rate and AFR can be improved by a mass air flow (MAF) sensor, in which a sensor element is heated to a fixed temperature, and the difference in temperature attributed to heat transfer from the heating element to the air flow is a measure of the air mass flow \[[@B3-sensors-15-27142],[@B4-sensors-15-27142],[@B5-sensors-15-27142]\]. However, there are many different local flow fields within the inlet piping due to the three-dimensional turbulence flow, leading to measurement biases in the MAF sensor installed between the air filter and the intake manifold. In addition, the MAF sensor is also subjected to aging phenomena owing to the accumulation of dust on the sensing element, which causes the deterioration of the measurement accuracy \[[@B6-sensors-15-27142],[@B7-sensors-15-27142]\]. These errors will bring about an inaccurate EGR rate and AFR and have adverse impacts on the emission performance of diesel engine. It is difficult to accurately establish an analytical model for the MAF sensor error. In view of the relatively low computational load, maps (or lookup tables) have been widely used to characterize systems where the functional relationship is unavailable or too complex to represent analytically \[[@B8-sensors-15-27142]\]. Therefore, the relative error of the MAF sensor is described as a one-dimensional (1D) map taking compressor mass air flow as input \[[@B2-sensors-15-27142]\]. In order to track MAF sensor aging, the extended Kalman filter (EKF) for updating maps is presented in \[[@B9-sensors-15-27142],[@B10-sensors-15-27142],[@B11-sensors-15-27142]\], in which the 1D map is represented as a piecewise linear interpolation model and the map parameters are considered as parameter states. Due to the piecewise linear interpolation model having the characteristic of partition calculation and due to the the map input being able to enter only one input interval of the 1D map at any time, then only two parameter states participating in linear interpolation are observable and the other not. Therefore, the error covariance matrix elements of EKF corresponding to the locally unobservable parameter states will increase linearly. Although the solution is to limit this growth in \[[@B9-sensors-15-27142],[@B10-sensors-15-27142],[@B11-sensors-15-27142]\], the convergence of EKF with a confined covariance matrix cannot be guaranteed. In addition, the measurement error of the MAF sensor depends on the engine operating point, which is usually defined as fuel mass injection quantity and engine speed. The 1D map representing MAF sensor error ignores the engine speed, reducing the accuracy when the diesel engine is run over a wide speed range. The adaptive observer with the advantage of simple convergence conditions is an alternative method for updating maps. Recursive algorithms designed for joint estimation of states and parameters in state space systems are usually known as adaptive observers, and some early works with adaptive observers to jointly estimate states and parameters in multi-input-multi-output linear time varying systems can be found in \[[@B12-sensors-15-27142],[@B13-sensors-15-27142]\]. In order to estimate sensor faults, adaptive observers for linear time varying systems with unknown parameters in output equations have been studied \[[@B14-sensors-15-27142],[@B15-sensors-15-27142]\]. However, the existing adaptive observers cannot directly update maps. In this paper, an adaptive observer is developed to update the map, in which the MAF sensor error is described as a two-dimensional (2D) map taking the operating point as the input to improve the model accuracy comparing the 1D map. Then, two problems are studied. First, in order to expediently analyze and design the parameter estimation method, the input-output relationship of the MAF sensor error 2D map is expressed as a dot product between the regression vector and the unknown parameter vector. Second, based on the linear parameter varying (LPV) system of the diesel engine with EGR and variable geometry turbocharger (VGT), a 2D map estimation method with a simple structure and low computational load is designed to facilitate the algorithm implementation. This paper is organized as follows. In [Section 2](#sec2-sensors-15-27142){ref-type="sec"}, the 2D map is expressed as the dot product between the regression vector and the unknown parameter vector, and the estimation problem for a class of LPV systems with an unknown parameter vector is given. In [Section 3](#sec3-sensors-15-27142){ref-type="sec"}, the LPV adaptive observer is proposed, as well as the convergence analysis. Simulation results from enDYNA are presented in [Section 4](#sec4-sensors-15-27142){ref-type="sec"}, and the conclusions are summarized in [Section 5](#sec5-sensors-15-27142){ref-type="sec"}. 2. Problem Formulation {#sec2-sensors-15-27142} ====================== 2.1. A Diesel Engine Air Path LPV Model {#sec2dot1-sensors-15-27142} --------------------------------------- [Figure 1](#sensors-15-27142-f001){ref-type="fig"} shows the model structure of a diesel engine with EGR and VGT, and the model can be expressed as \[[@B16-sensors-15-27142]\]: $$\begin{array}{ll} {\overset{˙}{p}}_{im} & {= \frac{R_{a}T_{im}}{V_{im}}\left( {W_{c} + W_{egr} - W_{ei}} \right)} \\ {\overset{˙}{p}}_{em} & {= \frac{R_{e}T_{em}}{V_{em}}\left( {W_{f} + W_{ei} - W_{t} - W_{egr}} \right)} \\ {\overset{˙}{\omega}}_{t} & {= \frac{P_{t}\eta_{m} - P_{c}}{J_{t}\omega_{t}}} \\ \end{array}$$ where $W_{c}$ is the compressor mass air flow, $W_{egr}$ is the EGR mass flow, $W_{ei}$ is the cylinder mass flow, $W_{f}$ is the fuel rate injected to cylinder, $W_{t}$ is the turbine mass flow, $P_{t}$ is the turbine power, $P_{c}$ is the compressor power, $\eta_{m}$ is the turbocharger mechanical efficiency, $p_{im}$ is the intake manifold pressure, $p_{em}$ is the exhaust manifold pressure and $\omega_{t}$ is the turbine speed. {#sensors-15-27142-f001} Meanwhile, $W_{c}$, $W_{egr}$, $W_{ei}$, $W_{t}$, $W_{f}$, $P_{c}$ and $P_{t}\eta_{m}$ in Equation ([1](#FD1-sensors-15-27142){ref-type="disp-formula"}) can be obtained as follows: $$\begin{array}{l} {W_{c} = \frac{p_{amb}\pi R_{c}^{3} \cdot \Phi_{c}\left( {p_{im},\omega_{t}} \right)}{R_{a}T_{amb}}\omega_{t},W_{egr} = \frac{A_{egr}\left( u_{egr} \right) \cdot \Psi_{egr}\left( {p_{im},p_{em}} \right)}{\sqrt{T_{em}R_{e}}}p_{em}} \\ {W_{ei} = \frac{\eta_{vol}\left( {p_{im},n_{e}} \right) \cdot n_{e}V_{d}}{120R_{a}T_{im}}p_{im},W_{t} = \frac{A_{vgt\max} \cdot f_{\Pi_{t}}\left( p_{em} \right) \cdot f_{vgt}\left( u_{vgt} \right)}{\sqrt{T_{em}R_{e}}}p_{em}} \\ {W_{f} = \frac{10^{- 6}}{120}n_{cyl}n_{e}u_{\delta},P_{c} = \frac{W_{c}\left( {p_{im},\omega_{t}} \right) \cdot c_{pa}T_{amb} \cdot \left( {\Pi_{c}^{1 - {1{{/\phantom{1\gamma_{a}}}}\gamma_{a}}} - 1} \right)}{\eta_{c}\left( {p_{im},\omega_{t}} \right)}} \\ {P_{t}\eta_{m} = \eta_{tm}\left( {p_{em},\omega_{t}} \right) \cdot W_{t}\left( {p_{em},u_{vgt}} \right) \cdot c_{pe}T_{em} \cdot \left( {1 - \Pi_{t}^{1 - {1{{/\phantom{1\gamma_{e}}}}\gamma_{e}}}} \right)} \\ \end{array}$$ However, it is difficult to estimate the measurement error of the MAF sensor based on the complicated nonlinear model Equation ([1](#FD1-sensors-15-27142){ref-type="disp-formula"}). In order to simply present the state space equation and the error estimation, define variables:$$\begin{array}{l} {\rho_{1} = \frac{p_{amb}\pi R_{c}^{3} \cdot \Phi_{c}\left( {p_{im},\omega_{t}} \right)}{R_{a}T_{amb}},\rho_{2} = \frac{A_{egr}\left( u_{egr} \right) \cdot \Psi_{egr}\left( {p_{im},p_{em}} \right)}{\sqrt{T_{em}R_{e}}}} \\ {\rho_{3} = \frac{\eta_{vol}\left( {p_{im},n_{e}} \right) \cdot n_{e}V_{d}}{120R_{a}T_{im}},\rho_{4} = \frac{A_{vgt\max} \cdot f_{\Pi_{t}}\left( p_{em} \right) \cdot f_{vgt}\left( u_{vgt} \right)}{\sqrt{T_{em}R_{e}}}} \\ {\rho_{5} = \frac{c_{pa}p_{amb}\pi R_{c}^{3} \cdot \Phi_{c}\left( {p_{im},\omega_{t}} \right) \cdot \left( {\Pi_{c}^{1 - {1{{/\phantom{1\gamma_{a}}}}\gamma_{a}}} - 1} \right)}{J_{t}\omega_{t}R_{a} \cdot \eta_{c}\left( {p_{im},\omega_{t}} \right)}} \\ \end{array}$$ According to Equation ([3](#FD3-sensors-15-27142){ref-type="disp-formula"}), the variables $\rho_{i}\mspace{600mu}\left( i = 1,2,3,4,5 \right)$ are available in real-time since $p_{im}$, $p_{em}$, $\omega_{t}$, $u_{egr}$, $u_{vgt}$, $u_{\delta}$ and $n_{e}$ can be measured or estimated online. Therefore, the nonlinear model Equation ([1](#FD1-sensors-15-27142){ref-type="disp-formula"}) can be cast into an LPV system:$$\begin{array}{r} {\overset{˙}{x} = A\left( \rho \right)x + E} \\ \end{array}$$ where:$$\begin{array}{l} {\rho = \begin{pmatrix} \rho_{1} & \rho_{2} & \rho_{3} & \rho_{4} & \rho_{5} \\ \end{pmatrix}} \\ {x = \begin{pmatrix} p_{im} \\ p_{em} \\ \omega_{t} \\ \end{pmatrix},A\left( \rho \right) = \begin{pmatrix} {- a_{1}\rho_{3}} & {a_{1}\rho_{2}} & {a_{1}\rho_{1}} \\ {a_{2}\rho_{3}} & {- a_{2}\rho_{2} - a_{2}\rho_{4}} & 0 \\ 0 & 0 & {- \rho_{5}} \\ \end{pmatrix}} \\ {E = \begin{pmatrix} 0 \\ {a_{2}W_{f}} \\ {{P_{t}\eta_{m}}{{/\phantom{{P_{t}\eta_{m}}{J_{t}\omega_{t}}}}}{J_{t}\omega_{t}}} \\ \end{pmatrix},a_{1} = \frac{R_{a}T_{im}}{V_{im}},a_{2} = \frac{R_{e}T_{em}}{V_{em}}} \\ \end{array}$$ In order to determine the bounds on the parameter vector *ρ*, a simulation study is performed using a 1.9 L four-cylinder common rail turbo diesel engine of enDYNA provided by Tesis \[[@B17-sensors-15-27142],[@B18-sensors-15-27142]\]. The bounds of the parameter vector *ρ* are found using the simulation data from enDYNA over the European Transient Cycle (ETC), Federal Test Procedure 75 (FTP75) and New European Drive Cycle (NEDC) \[[@B19-sensors-15-27142],[@B20-sensors-15-27142],[@B21-sensors-15-27142]\]. Then, the results are listed in [Table 1](#sensors-15-27142-t001){ref-type="table"}. It follows that each parameter $\rho_{i}$ from parameter vector *ρ* is bounded by a minimum and maximum value ${\underline{\rho}}_{i}$ and ${\overline{\rho}}_{i}$. sensors-15-27142-t001_Table 1 ###### Bounds on the parameter vector *ρ* under three conditions. ETC, European Transient Cycle; FTP75, Federal Test Procedure 75; NEDC, New European Drive Cycle. Parameter ETC FTP75 NEDC ------------ ----------------------------------------------------------- ------------------------------------------------------------ ------------------------------------------------------------ $\rho_{1}$ $\left\lbrack {19,150} \right\rbrack \times 10^{- 7}$ $\left\lbrack {146,110} \right\rbrack \times 10^{- 7}$ $\left\lbrack {49,130} \right\rbrack \times 10^{- 7}$ $\rho_{2}$ $\left\lbrack {- 1.65,1.6} \right\rbrack \times 10^{- 7}$ $\left\lbrack {- 0.56,1.15} \right\rbrack \times 10^{- 7}$ $\left\lbrack {- 0.003,1.0} \right\rbrack \times 10^{- 7}$ $\rho_{3}$ $\left\lbrack {1.09,6.4} \right\rbrack \times 10^{- 7}$ $\left\lbrack {1.18,6} \right\rbrack \times 10^{- 7}$ $\left\lbrack {1.1,5.4} \right\rbrack \times 10^{- 7}$ $\rho_{4}$ $\left\lbrack {0.61,6.01} \right\rbrack \times 10^{- 7}$ $\left\lbrack {0.75,5.4} \right\rbrack \times 10^{- 7}$ $\left\lbrack {0.62,4.7} \right\rbrack \times 10^{- 7}$ $\rho_{5}$ $\left\lbrack {0,3.15} \right\rbrack$ $\left\lbrack {0,2.78} \right\rbrack$ $\left\lbrack {0,2.67} \right\rbrack$ 2.2. 2D Map Description for the MAF Sensor Error {#sec2dot2-sensors-15-27142} ------------------------------------------------ The intake manifold pressure $p_{im}$, turbine speed $\omega_{t}$ and compressor mass air flow $W_{c}$ are the outputs of interest to analyze the MAF sensor error, which is:$$\begin{array}{r} {y = \begin{pmatrix} y_{1} \\ y_{2} \\ \end{pmatrix} = \begin{pmatrix} p_{im} \\ \omega_{t} \\ W_{c} \\ \end{pmatrix}} \\ \end{array}$$ where $y_{1} = \begin{pmatrix} p_{im} & \omega_{t} \\ \end{pmatrix}^{T},y_{2} = W_{c}$. Due to the existence of MAF sensor error, the output Equation ([6](#FD6-sensors-15-27142){ref-type="disp-formula"}) becomes: $$\begin{array}{r} {y_{m} = \begin{pmatrix} y_{1m} \\ y_{2m} \\ \end{pmatrix} = \begin{pmatrix} y_{1} \\ {y_{2} + \Delta W_{c}} \\ \end{pmatrix}} \\ \end{array}$$ where $y_{m}$ is the measured value from sensors. ${\Delta W}_{c}$ is the measurement error of the MAF sensor, which depends on the engine operating point (fuel mass injection quantity $u_{\delta}$ and engine speed $n_{e}$), *i.e.*, $\Delta W_{c}\left( {u_{\delta}.n_{e}} \right)$. Since it is difficult to accurately build an analytical model for $\Delta W_{c}\left( {u_{\delta}.n_{e}} \right)$, a 2D map is adopted in this paper to describe $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$. Therefore, define the partition of the 2D map input $\upsilon = \left( {u_{\delta},n_{e}} \right)$ as: $$\begin{matrix} {a = u_{\delta}^{1} < u_{\delta}^{2} < \ldots < u_{\delta}^{p_{1}} = b} \\ {c = n_{e}^{1} < n_{e}^{2} < \ldots < n_{e}^{p_{2}} = d} \\ \end{matrix}$$ where $a,b \in \mathbb{R}$ are the minimum and maximum values of $u_{\delta}$ and $p_{1}$ is the number of the grid points in $\left\lbrack {a,b} \right\rbrack$. $c,d \in \mathbb{R}$ are the minimum and maximum values of $n_{e}$, and $p_{2}$ is the number of the grid points in $\left\lbrack {c,d} \right\rbrack$. Assume that the measurement error of the input grid points $\left( {u_{\delta}^{i},n_{e}^{j}} \right)$ is $\theta^{i,j}$, *i.e.*, $$\begin{matrix} {\theta^{i,j} = \Delta W_{c}\left( {u_{\delta}^{i},n_{e}^{j}} \right)} \\ {i = 1,2,\cdots p_{1};j = 1,2,\cdots p_{2}} \\ \end{matrix}$$ Then, for $\forall\upsilon \in \left\lbrack {u_{\delta}^{i},u_{\delta}^{i + 1}} \right\rbrack \times \left\lbrack {n_{e}^{j},n_{e}^{j + 1}} \right\rbrack$, $\forall i \in \left\lbrack {1,2,\cdots,p_{1} - 1} \right\rbrack$ and $\forall j \in \left\lbrack {1,2,\cdots,p_{2} - 1} \right\rbrack$, we can hold the $n_{e}$ value fixed and apply one dimensional (1D) linear interpolation in the $u_{\delta}$ direction. Using the Lagrange form, the result is: $$\begin{matrix} {q_{u_{\delta}}^{j}\left( \theta^{i,j} \right) = \frac{u_{\delta}^{i + 1} - u_{\delta}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i,j} + \frac{u_{\delta} - u_{\delta}^{i}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i + 1,j}} \\ {u_{\delta} \in \left\lbrack {u_{\delta}^{i},u_{\delta}^{i + 1}} \right\rbrack,i = 1,2,\cdots p_{1} - 1} \\ \end{matrix}$$ Equation ([10](#FD10-sensors-15-27142){ref-type="disp-formula"}) can then be used to linearly interpolate along the $n_{e}$ dimension to yield the piecewise bilinear interpolation model of the measurement error $\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right)$ as:$$\begin{matrix} {\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right) = \frac{n_{e}^{j + 1} - n_{e}}{n_{e}^{j + 1} - n_{e}^{j}}q_{u_{\delta}}^{j}\left( \theta^{i,j} \right) + \frac{n_{e} - n_{e}^{j}}{n_{e}^{j + 1} - n_{e}^{j}}q_{u_{\delta}}^{j + 1}\left( \theta^{i,j} \right)} \\ {\upsilon \in \left\lbrack {u_{\delta}^{i},u_{\delta}^{i + 1}} \right\rbrack \times \left\lbrack {n_{e}^{j},n_{e}^{j + 1}} \right\rbrack,i = 1,\cdots p_{1} - 1;j = 1,\cdots p_{2} - 1} \\ \end{matrix}$$ For the undefined region $\upsilon \in \left( {\mathbb{R} \times \mathbb{R}} \right)\backslash\left( {\left\lbrack {a,b} \right\rbrack \times \left\lbrack {c,d} \right\rbrack} \right)$, we extend Equation ([11](#FD11-sensors-15-27142){ref-type="disp-formula"}) to the final result: $$\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right) = \left\{ \begin{matrix} {q_{u_{\delta}}^{1}\left( \theta^{i,j} \right)} & {,u \in \mathbb{R} \times \mathbb{R}_{n_{e}}^{0}} \\ {\frac{n_{e}^{j{ +}1} - n_{e}}{n_{e}^{j{ +}1} - n_{e}^{j}}q_{u_{\delta}}^{j}\left( \theta^{i,j} \right) + \frac{n_{e} - n_{e}^{j}}{n_{e}^{j{ +}1} - n_{e}^{j}}q_{u_{\delta}}^{j{ +}1}\left( \theta^{i,j} \right)} & {,u \in \mathbb{R} \times \mathbb{R}_{n_{e}}^{j}} \\ {q_{u_{\delta}}^{p_{2}}\left( \theta^{i,j} \right)} & {,u \in \mathbb{R} \times \mathbb{R}_{n_{e}}^{p_{2}}} \\ \end{matrix} \right.$$ where: $$\begin{matrix} {q_{u_{\delta}}^{j}\left( \theta^{i,j} \right) = \left\{ \begin{matrix} \theta^{1,j} & {,u_{\delta} \in \mathbb{R}_{u_{\delta}}^{0}} \\ {\frac{u_{\delta}^{i + 1} - u_{\delta}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i,j} + \frac{u_{\delta} - u_{\delta}^{i}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i + 1,j}} & {,u_{\delta} \in \mathbb{R}_{u_{\delta}}^{i}} \\ \theta^{p_{1},j} & {,u_{\delta} \in \mathbb{R}_{u_{\delta}}^{p_{1}}} \\ \end{matrix} \right.} \\ {i = 1,2,\cdots p_{1} - 1;j = 1,2,\cdots p_{2} - 1} \\ \end{matrix}$$ and: $$\mathbb{R}_{u_{\delta}}^{k} = \begin{cases} {\left( {- \infty,u_{\delta}^{1}} \right\rbrack,} & {k{ = 0}} \\ {\left( {u_{\delta}^{k},u_{\delta}^{k + 1}} \right\rbrack,} & {k{ =}1,\ldots,p_{1} - 1} \\ {\left( {u_{\delta}^{p_{1}}, + \infty} \right),} & {k{ =}p_{1}} \\ \end{cases}$$ $$\mathbb{R}_{n_{e}}^{l} = \begin{cases} {\left( {- \infty,n_{e}^{1}} \right\rbrack,} & {l{ = 0}} \\ {\left( {n_{e}^{l},n_{e}^{l + 1}} \right\rbrack,} & {l{ =}1,\ldots,p_{2} - 1} \\ {\left( {n_{e}^{p_{2}}, + \infty} \right),} & {l{ =}p_{2}} \\ \end{cases}$$ For the purposes of estimating unknown parameter $\theta^{i,j}$ in $\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right)$ expediently, Equation ([12](#FD12-sensors-15-27142){ref-type="disp-formula"}) in vector-vector form is needed. According to the input interval Equations ([14](#FD14-sensors-15-27142){ref-type="disp-formula"}) and ([15](#FD15-sensors-15-27142){ref-type="disp-formula"}), we define membership function as: $$\begin{matrix} {\delta_{u_{\delta}}^{k} = \left\{ \begin{matrix} {1,} & {u_{\delta} \in \mathbb{R}_{u_{\delta}}^{k}} \\ {0,} & {other} \\ \end{matrix} \right.} \\ {k = 0,1,\cdots,p_{1}} \\ \end{matrix}$$ and: $$\begin{matrix} {\delta_{n_{e}}^{l} = \left\{ \begin{matrix} {1,} & {n_{e} \in \mathbb{R}_{n_{e}}^{l}} \\ {0,} & {other} \\ \end{matrix} \right.} \\ {l{ = 0,}1,\ldots,p_{2}} \\ \end{matrix}$$ Using membership function Equations ([16](#FD16-sensors-15-27142){ref-type="disp-formula"}) and ([17](#FD17-sensors-15-27142){ref-type="disp-formula"}), Equation ([12](#FD12-sensors-15-27142){ref-type="disp-formula"}) becomes: $$\begin{array}{cl} {\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right)} & {= \delta_{n_{e}}^{0}q_{u_{\delta}}^{1}\left( \theta^{i,j} \right) + \sum\limits_{j = 1}^{p_{2} - 1}{\delta_{n_{e}}^{j}\left( {\frac{n_{e}^{j + 1} - n_{e}}{n_{e}^{j + 1} - n_{e}^{j}}q_{u_{\delta}}^{j}\left( \theta^{i,j} \right) + \frac{n_{e} - n_{e}^{j}}{n_{e}^{j + 1} - n_{e}^{j}}q_{u_{\delta}}^{j + 1}\left( \theta^{i,j} \right)} \right)} + \delta_{n_{e}}^{p_{2}}q_{u_{\delta}}^{p_{2}}\left( \theta^{i,j} \right)} \\ & {= \Psi_{n_{e}} \cdot q_{u_{\delta}}\left( \theta^{i,j} \right)} \\ \end{array}$$ where: $$\begin{array}{r} {\Psi_{n_{e}} = \begin{pmatrix} {\delta_{n_{e}}^{0} + \delta_{n_{e}}^{1}\frac{n_{e}^{2} - n_{e}}{n_{e}^{2} - n_{e}^{1}}} \\ {\delta_{n_{e}}^{1}\frac{n_{e} - n_{e}^{1}}{n_{e}^{2} - n_{e}^{1}} + \delta_{n_{e}}^{2}\frac{n_{e}^{3} - n_{e}}{n_{e}^{3} - n_{e}^{2}}} \\ \vdots \\ {\delta_{n_{e}}^{p_{1} - 2}\frac{n_{e} - n_{e}^{p_{2} - 2}}{n_{e}^{p_{2} - 1} - n_{e}^{p_{2} - 2}} + \delta_{n_{e}}^{p_{1} - 1}\frac{n_{e}^{p_{2}} - n_{e}}{n_{e}^{p_{2}} - n_{e}^{p_{2} - 1}}} \\ {\delta_{n_{e}}^{p_{1} - 1}\frac{n_{e} - n_{e}^{p_{2} - 1}}{n_{e}^{p_{2}} - n_{e}^{p_{2} - 1}} + \delta_{n_{e}}^{p_{1}}} \\ \end{pmatrix}^{T},q_{u_{\delta}}\left( \theta^{i,j} \right) = \begin{pmatrix} {q_{u_{\delta}}^{1}\left( \theta^{i,j} \right)} \\ {q_{u_{\delta}}^{2}\left( \theta^{i,j} \right)} \\ \vdots \\ {q_{u_{\delta}}^{p_{2} - 1}\left( \theta^{i,j} \right)} \\ {q_{u_{\delta}}^{p_{2}}\left( \theta^{i,j} \right)} \\ \end{pmatrix}} \\ \end{array}$$ and: $$\begin{array}{cl} {q_{u_{\delta}}^{j}\left( \theta^{i,j} \right)} & {= \delta_{u_{\delta}}^{0}\theta^{1,j} + \sum\limits_{i = 1}^{p_{1} - 1}{\delta_{u_{\delta}}^{i}\left( {\frac{u_{\delta}^{i + 1} - u_{\delta}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i,j} + \frac{u_{\delta} - u_{\delta}^{i}}{u_{\delta}^{i + 1} - u_{\delta}^{i}}\theta^{i + 1,j}} \right)} + \delta_{u_{\delta}}^{p_{1}}\theta^{p_{1},j}} \\ & {= \Psi_{u_{\delta}} \cdot \theta_{u_{\delta}}^{j}} \\ \end{array}$$ where: $$\begin{array}{r} {\Psi_{u_{\delta}} = \begin{pmatrix} {\delta_{u_{\delta}}^{0} + \delta_{u_{\delta}}^{1}\frac{u_{\delta}^{2} - u_{\delta}}{u_{\delta}^{2} - u_{\delta}^{1}}} \\ {\delta_{u_{\delta}}^{1}\frac{u_{\delta} - u_{\delta}^{1}}{u_{\delta}^{2} - u_{\delta}^{1}} + \delta_{u_{\delta}}^{2}\frac{u_{\delta}^{3} - u_{\delta}}{u_{\delta}^{3} - u_{\delta}^{2}}} \\ \vdots \\ {\delta_{u_{\delta}}^{p_{1} - 2}\frac{u_{\delta} - u_{\delta}^{p_{1} - 2}}{u_{\delta}^{p_{1} - 1} - u_{\delta}^{p_{1} - 2}} + \delta_{u_{\delta}}^{p_{1} - 1}\frac{u_{\delta}^{p_{1}} - u_{\delta}}{u_{\delta}^{p_{1}} - u_{\delta}^{p_{1} - 1}}} \\ {\delta_{u_{\delta}}^{p_{1} - 1}\frac{u_{\delta} - u_{{}_{\delta}}^{p_{1} - 1}}{u_{\delta}^{p_{1}} - u_{\delta}^{p_{1} - 1}} + \delta_{u_{\delta}}^{p_{1}}} \\ \end{pmatrix}^{T},\theta_{u_{{}_{\delta}}}^{j} = \begin{pmatrix} \theta^{1,j} \\ \theta^{2,j} \\ \vdots \\ \theta^{p_{1} - 1,j} \\ \theta^{p_{1},j} \\ \end{pmatrix}} \\ \end{array}$$ and $\Psi_{n_{e}} \in \mathbb{R}^{1 \times p_{1}},\Psi_{n_{e}} \in \mathbb{R}^{1 \times p_{2}},\theta_{u_{{}_{\delta}}}^{j} \in \mathbb{R}^{p_{1} \times 1},q_{u_{\delta}}\left( \theta^{i,j} \right) \in \mathbb{R}^{p_{2} \times 1}$. Now, following Equations ([18](#FD18-sensors-15-27142){ref-type="disp-formula"})--([21](#FD21-sensors-15-27142){ref-type="disp-formula"}), $\Delta W_{c,T}\left( {\theta^{i,j},\upsilon} \right)$ can be written as a dot product between regression vector $\Psi\left( \upsilon \right)$ and unknown parameter vector *θ* as follows: $$\begin{array}{r} {\Delta W_{c,T}\left( {\theta,\upsilon} \right) = \Psi\left( \upsilon \right) \cdot \theta,\forall\upsilon \in \mathbb{R} \times \mathbb{R}} \\ \end{array}$$ where: $$\begin{array}{r} {\Psi\left( \upsilon \right) = \Psi_{n_{e}} \cdot \begin{pmatrix} \Psi_{u_{\delta}} & 0 & \cdots & 0 \\ 0 & \Psi_{u_{\delta}} & \cdots & 0 \\ \vdots & \vdots & \ddots & \vdots \\ 0 & 0 & \cdots & \Psi_{u_{\delta}} \\ \end{pmatrix},\theta = \begin{pmatrix} \theta_{u_{\delta}}^{1} \\ \theta_{u_{\delta}}^{2} \\ \vdots \\ \theta_{u_{\delta}}^{p_{2}} \\ \end{pmatrix}} \\ \end{array}$$ and $\Psi\left( \upsilon \right) \in \mathbb{R}^{1 \times p},\theta \in \mathbb{R}^{p \times 1},p = p_{1} \cdot p_{2}$. With the combination of Equations ([4](#FD4-sensors-15-27142){ref-type="disp-formula"}), ([7](#FD7-sensors-15-27142){ref-type="disp-formula"}) and ([22](#FD22-sensors-15-27142){ref-type="disp-formula"}), the diesel engine air path LPV model can be described by the following state space equation: $$\begin{aligned} \overset{˙}{x} & {= A\left( \rho \right)x + E} \\ y_{m} & {= C\left( \rho \right)x + G\Psi\left( \upsilon \right)\theta} \\ \end{aligned}$$ where: $$\begin{array}{r} {C\left( \rho \right) = \begin{pmatrix} 1 & 0 & 0 \\ 0 & 0 & 1 \\ 0 & 0 & \rho_{1} \\ \end{pmatrix},G = \begin{pmatrix} 0 \\ 0 \\ 1 \\ \end{pmatrix}} \\ \end{array}$$ Equation ([24](#FD24-sensors-15-27142){ref-type="disp-formula"}) indicates that the estimation of the MAF sensor error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ becomes joint estimation of state *x* and parameter *θ* for LPV system Equation ([24](#FD24-sensors-15-27142){ref-type="disp-formula"}). 3. Adaptive Observer Design {#sec3-sensors-15-27142} =========================== The observer to estimate state *x* and parameter *θ* jointly for the LPV system Equation ([24](#FD24-sensors-15-27142){ref-type="disp-formula"}) is given: $$\begin{array}{ll} \overset{˙}{\hat{x}} & {= A\left( \rho \right)\hat{x} + E + L\left( {y_{m} - C\left( \rho \right)\hat{x} - G\Psi\left( \upsilon \right)\hat{\theta}} \right)} \\ \overset{˙}{\hat{\theta}} & {= \Gamma\Psi\left( \upsilon \right)^{T}\left( {y_{m2} - C_{2}\left( \rho \right)\hat{x} - \Psi\left( \upsilon \right)\hat{\theta}} \right)} \\ \end{array}$$ where $C_{2}\left( \rho \right) = \begin{pmatrix} 0 & 0 & \rho_{1} \\ \end{pmatrix}$, $\hat{x} \in \mathbb{R}^{3 \times 1}$ is the state estimate, $\hat{\theta} \in \mathbb{R}^{p \times 1}$ is the parameter estimate, gain $\Gamma \in \mathbb{R}^{p \times p}$ is the positive definite diagonal matrix and $L \in \mathbb{R}^{3 \times 3}$ is the feedback gain matrix. The asymptotical stability of the proposed algorithm Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) is analyzed in the following theorem. *If the following Conditions (1) and (2) hold, then LPV adaptive observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) is asymptotically stable, i.e., for any initial conditions* $x\left( 0 \right),\hat{x}\left( 0 \right),\hat{\theta}\left( 0 \right)$ *and parameter vector θ, the errors $\hat{x} - x$ and $\hat{\theta} - \theta$ tend to zero asymptotically when $\left. t\rightarrow\infty \right.$*. *(1) There exist matrices L, $P = P^{T} > 0$, $Q = Q^{T} > 0$ and constant $\varepsilon_{1},\varepsilon_{2} > 0$, such that the following set of linear matrix inequalities (LMIs) is feasible for $\forall\rho_{i} \in \left\lbrack {{\underline{\rho}}_{i},{\overline{\rho}}_{i}} \right\rbrack$, $i = 1,2,3,4,5$:* $$M\left( \rho \right) = \begin{bmatrix} {A_{cl}\left( \rho \right)^{T}P + PA_{cl}\left( \rho \right) + Q} & {PLG} & {C_{2}^{T}\left( \rho \right)} \\ {G^{T}L^{T}P^{T}} & {- \varepsilon_{1}I} & 0 \\ {C_{2}\left( \rho \right)} & 0 & {- \varepsilon_{2}I} \\ \end{bmatrix} < 0$$ $$\begin{array}{r} {2 - \varepsilon_{1} - \varepsilon_{2} > 0} \\ \end{array}$$ *where $A_{cl}\left( \rho \right) = A\left( \rho \right) - LC\left( \rho \right)$* . *(2) There exists map input υ, such that the regression vector $\Psi\left( \upsilon \right)$ is persistently exciting, i.e., $\exists\delta_{1},\delta_{2} > 0;\exists T > 0;\forall t \geq 0$:* $$\begin{array}{r} {\delta_{1}I_{p} \leq \int_{t}^{t + T}{\Psi\left( {\upsilon\left( \tau \right)} \right)^{T}\Psi\left( {\upsilon\left( \tau \right)} \right)d\tau} \leq \delta_{2}I_{p}} \\ \end{array}$$ Set the estimation error $\widetilde{x} = \hat{x} - x,\widetilde{\theta} = \hat{\theta} - \theta$. Notice that $\overset{˙}{\theta} = 0$; the error dynamic system between Equations ([24](#FD24-sensors-15-27142){ref-type="disp-formula"}) and ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) is: $$\begin{aligned} \overset{˙}{\widetilde{x}} & {= \left( {A\left( \rho \right) - LC\left( \rho \right)} \right)\widetilde{x} - LG\Psi\left( \upsilon \right)\widetilde{\theta}} \\ \overset{˙}{\widetilde{\theta}} & {= - \Gamma\Psi\left( \upsilon \right)^{T}\left( {C_{2}\left( \rho \right)\widetilde{x} + \Psi\left( \upsilon \right)\widetilde{\theta}} \right)} \\ \end{aligned}$$ A valid Lyapunov function candidate is considered as $V = \eta^{T}P\eta + {\widetilde{\theta}}^{T}\Gamma^{- 1}\widetilde{\theta}$. For $\forall\widetilde{x} \neq 0$, the derivative of *V* along with the error dynamic system Equation ([30](#FD30-sensors-15-27142){ref-type="disp-formula"}) is: $$\begin{array}{cl} \overset{˙}{V} & {= 2{\widetilde{x}}^{T}P\overset{˙}{\widetilde{x}} + 2{\widetilde{\theta}}^{T}\Gamma^{- 1}\overset{˙}{\widetilde{\theta}}} \\ & {= 2{\widetilde{x}}^{T}PA_{cl}\left( \rho \right)\widetilde{x} - 2{\widetilde{\theta}}^{T}\Psi\left( \upsilon \right)^{T}\Psi\left( \upsilon \right)\widetilde{\theta} - 2{\widetilde{x}}^{T}PLG\Psi\left( \upsilon \right)\widetilde{\theta} - 2{\widetilde{\theta}}^{T}\Psi\left( \upsilon \right)^{T}C_{2}\left( \rho \right)\widetilde{x}} \\ \end{array}$$ There exist $\varepsilon_{1},\varepsilon_{2} > 0$, such that the following inequalities hold: $$\begin{matrix} {- 2{\widetilde{x}}^{T}PLG\Psi\left( \upsilon \right)\widetilde{\theta} \leqslant {\parallel {\frac{1}{\sqrt{\varepsilon_{1}}}{\widetilde{x}}^{T}PLG} \parallel}^{2} + {\parallel {\sqrt{\varepsilon_{1}}\Psi\left( \upsilon \right)\widetilde{\theta}} \parallel}^{2}} \\ {- 2{\widetilde{\theta}}^{T}\Psi\left( \upsilon \right)^{T}C_{2}\left( \rho \right)\widetilde{x} \leqslant {\parallel {\frac{1}{\sqrt{\varepsilon_{2}}}C_{2}\left( \rho \right)\widetilde{x}} \parallel}^{2} + {\parallel {\sqrt{\varepsilon_{2}}{\widetilde{\theta}}^{T}\Psi\left( \upsilon \right)^{T}} \parallel}^{2}} \\ \end{matrix}$$ According to Condition (1) and Equation ([31](#FD31-sensors-15-27142){ref-type="disp-formula"}), the following inequality holds: $$\begin{array}{cl} \overset{˙}{V} & {\leq {\widetilde{x}}^{T}\left( {A_{cl}\left( \rho \right)^{T}P + PA_{cl}\left( \rho \right) + \frac{1}{\varepsilon_{1}}PLGG^{T}L^{T}P + \frac{1}{\varepsilon_{2}}C_{2}^{T}\left( \rho \right)C_{2}\left( \rho \right)} \right)\widetilde{x}} \\ & {- \left( {2 - \varepsilon_{1} - \varepsilon_{2}} \right){\widetilde{\theta}}^{T}\Psi\left( \upsilon \right)^{T}\Psi\left( \upsilon \right)\widetilde{\theta}} \\ & {< - {\widetilde{x}}^{T}Q\widetilde{x} < 0} \\ \end{array}$$ That is $\overset{˙}{V} < - \omega\left( t \right) < 0$, for $\forall\rho_{i} \in \left\lbrack {{\underline{\rho}}_{i},{\overline{\rho}}_{i}} \right\rbrack,i = 1,2,3,4,5$, where $\omega\left( t \right) = {\widetilde{x}}^{T}Q\widetilde{x}$. Based on the Lyapunov stability theory, we know that the equilibrium $\widetilde{x} = 0$ and $\widetilde{\theta} = 0$ are stable. Now, integrating $\overset{˙}{V} < - \omega\left( t \right)$ from zero to *t* yields: $$\begin{array}{r} {V\left( t \right) + \int_{0}^{t}{\omega\left( \tau \right)d\tau} < V\left( 0 \right)} \\ \end{array}$$ and this means that $\int_{0}^{t}{\omega\left( \tau \right)d\tau} < V\left( 0 \right)$ since $V > 0$. Therefore, we have $\lim\limits_{t\rightarrow\infty}\int_{0}^{t}{\omega\left( \tau \right)d\tau} \leq V\left( 0 \right)$, and this implies that $\lim\limits_{t\rightarrow\infty}\int_{0}^{t}{\omega\left( \tau \right)d\tau}$ exists and is finite. By Barbalat's Lemma \[[@B22-sensors-15-27142]\], we know that $\lim\limits_{t\rightarrow\infty}\omega\left( t \right) = 0$, and this leads to $\lim\limits_{t\rightarrow\infty}\widetilde{x}\left( t \right) = 0$. Under Condition (2), the vector $\Psi\left( \upsilon \right)$ is persistently exciting, that is we have $\lim\limits_{t\rightarrow\infty}\widetilde{\theta}\left( t \right) = 0$ \[[@B22-sensors-15-27142]\]. ☐ *With the concept of multi-convexity \[[@B23-sensors-15-27142]\], the solution of the infinite LMI Equation ([27](#FD27-sensors-15-27142){ref-type="disp-formula"}) can be reduced to be a solution of the finite LMIs for the vertex set, that is:* $$\begin{matrix} {{M\left( w \right) < 0},{\forall w \in \mathcal{V} = \left\{ {\left. \left( {w_{1},\cdots,w_{5}} \right) \right|w_{i} \in \left\{ {{\underline{\rho}}_{i},{\overline{\rho}}_{i}} \right\},i = 1,2,3,4,5} \right\}}} \\ \end{matrix}$$ *Therefore, feedback gain L can be obtained by the solution of inequality Equations ([28](#FD28-sensors-15-27142){ref-type="disp-formula"}) and ([33](#FD35-sensors-15-27142){ref-type="disp-formula"})*. *With the membership function $\delta_{u_{\delta}}^{k},\delta_{n_{e}}^{l}$ in Equations ([16](#FD16-sensors-15-27142){ref-type="disp-formula"}) and ([17](#FD17-sensors-15-27142){ref-type="disp-formula"}), we know that $\delta_{u_{\delta}}^{k}$ = $\delta_{n_{e}}^{l}$ = 1 when $\upsilon \in \mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$ and $\delta_{u_{\delta}}^{k}{ =}\delta_{n_{e}}^{l}{ = 0}$ when $\upsilon \notin \mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$. Therefore, the regression vector $\Psi\left( \upsilon \right)$ is a sparse vector*. According to the partition of the map input $\upsilon = \left( {u_{\delta},n_{e}} \right)$ defined in Equation ([8](#FD8-sensors-15-27142){ref-type="disp-formula"}) and the piecewise bilinear interpolation model Equation ([12](#FD12-sensors-15-27142){ref-type="disp-formula"}), the input *υ* (engine operating point) moves in only one region $\mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$ at any time, and only the parameters ${\hat{\theta}}^{i,j}$ corresponding to the region $\mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$ can participate in the interpolation. That is, for $\forall\upsilon \in \mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$: Case 1: $\left( {k,l} \right) \in \left\{ {0,p_{1}} \right\} \times \left\{ {0,p_{2}} \right\}$. Only one parameter ${\hat{\theta}}^{i,j},\left( {i,j} \right) \in \left\{ {1,p_{1}} \right\} \times \left\{ {1,p_{2}} \right\}$ takes part in the interpolation, *i.e.*, $\Delta W_{c,T}\left( {{\hat{\theta}}^{i,j},\upsilon} \right) = {\hat{\theta}}^{i,j}$. Case 2: $\left( {k,l} \right) \in \left\{ {1,2,\cdots p_{1} - 1} \right\} \times \left\{ {0,p_{2}} \right\}$. Two parameters ${\hat{\theta}}^{i,j},{\hat{\theta}}^{i + 1,j},\left( {i,j} \right) \in \left\{ {1,2,\cdots p_{1} - 1} \right\} \times \left\{ {1,p_{2}} \right\}$ take part in the interpolation, *i.e.*, Δ W c , T θ \^ i , j , υ = u δ i \+ 1 − u δ u δ i \+ 1 − u δ i θ \^ i , j \+ u δ − u δ i u δ i \+ 1 − u δ i θ \^ i \+ 1 , j Case 3: $\left( {k,l} \right) \in \left\{ {0,p_{1}} \right\} \times \left\{ {1,2,\cdots p_{2} - 1} \right\}$. Two parameters ${\hat{\theta}}^{i,j},{\hat{\theta}}^{i,j + 1},\left( {i,j} \right) \in \left\{ {1,p_{1}} \right\} \times \left\{ {1,2,\cdots p_{2} - 1} \right\}$ take part in the interpolation, *i.e.*, Δ W c , T θ i , j , υ = n e j \+ 1 − n e n e j \+ 1 − n e j θ \^ i , j \+ n e − n e j n e j \+ 1 − n e j θ \^ i , j \+ 1 Case 4: $\left( {k,l} \right) \in \left\{ {1,2,\cdots p_{1} - 1} \right\} \times \left\{ {1,2,\cdots p_{2} - 1} \right\}$. Four parameters ${\hat{\theta}}^{i,j}$, ${\hat{\theta}}^{i + 1,j}$, ${\hat{\theta}}^{i,j + 1}$, ${\hat{\theta}}^{i + 1,j + 1}$, $\left( {i,j} \right) \in \left\{ {1,2,\cdots p_{1} - 1} \right\} \times \left\{ {1,2,\cdots p_{2} - 1} \right\}$ take part in the interpolation, *i.e.*, Δ W c , T θ \^ i , j , υ = n e j \+ 1 − n e n e j \+ 1 − n e j u δ i \+ 1 − u δ u δ i \+ 1 − u δ i θ \^ i , j \+ n e j \+ 1 − n e n e j \+ 1 − n e j u δ − u δ i u δ i \+ 1 − u δ i θ \^ i \+ 1 , j \+ n e − n e j n e j \+ 1 − n e j u δ i \+ 1 − u δ u δ i \+ 1 − u δ i θ \^ i , j \+ 1 \+ n e − n e j n e j \+ 1 − n e j u δ − u δ i u δ i \+ 1 − u δ i θ \^ i \+ 1 , j \+ 1 In order to expediently discuss the convergence of the parameter estimate ${\hat{\theta}}^{i,j}$ corresponding to different regions $\mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$, a local regression vector $\Psi_{l}\left( \upsilon \right)$ is defined based on the above four classifications of the region partition as follow: $$\Psi_{l}\left( \upsilon \right) = \begin{cases} 1 & {{if}~{\upsilon \in \left( {\mathbb{R}_{u_{\delta}}^{0} \cup \mathbb{R}_{u_{\delta}}^{p_{1}}} \right) \times \left( {\mathbb{R}_{n_{e}}^{0} \cup \mathbb{R}_{n_{e}}^{p_{2}}} \right)}} \\ \begin{pmatrix} {1 - \eta_{1}} & \eta_{1} \\ \end{pmatrix} & {{if}~{\upsilon \in \mathbb{R}_{u_{\delta}}^{i} \times \left( {\mathbb{R}_{n_{e}}^{0} \cup \mathbb{R}_{n_{e}}^{p_{2}}} \right)}} \\ \begin{pmatrix} {1 - \eta_{2}} & \eta_{2} \\ \end{pmatrix} & {{if}~{\upsilon \in \left( {\mathbb{R}_{u_{\delta}}^{0} \cup \mathbb{R}_{u_{\delta}}^{p_{1}}} \right) \times \mathbb{R}_{n_{e}}^{j}}} \\ \begin{pmatrix} {\left( {1 - \eta_{1}} \right)\left( {1 - \eta_{2}} \right)} \\ {\eta_{1}\left( {1 - \eta_{2}} \right)} \\ {\left( {1 - \eta_{1}} \right)\eta_{2}} \\ {\eta_{1}\eta_{2}} \\ \end{pmatrix}^{T} & {{if}~{\upsilon \in \mathbb{R}_{u_{\delta}}^{i} \times \mathbb{R}_{n_{e}}^{j}}} \\ \end{cases}$$ where: $$\begin{matrix} {\eta_{1} = \frac{u_{\delta} - u_{\delta}^{i}}{u_{\delta}^{i + 1} - u_{\delta}^{i}},\eta_{2} = \frac{n_{e} - n_{e}^{j}}{n_{e}^{j + 1} - n_{e}^{j}}} \\ {i = 1,2,\cdots p_{1} - 1;j = 1,2,\cdots p_{2} - 1} \\ \end{matrix}$$ When $\upsilon \in \mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$, regression vector $\Psi\left( \upsilon \right)$ in Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) can be replaced by local regression vector $\Psi_{l}\left( \upsilon \right)$; then, observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) can be replaced by: $$\begin{aligned} \overset{˙}{\hat{x}} & {= A\left( \rho \right)\hat{x} + E + L\left( {y_{m} - C\left( \rho \right)\hat{x} - G\Psi_{l}\left( \upsilon \right){\hat{\theta}}_{l}^{i,j}} \right)} \\ {\overset{˙}{\hat{\theta}}}_{l}^{i,j} & {= \Gamma_{l}\Psi_{l}\left( \upsilon \right)^{T}\left( {y_{m2} - C_{2}\left( \rho \right)\hat{x} - \Psi_{l}\left( \upsilon \right){\hat{\theta}}_{l}^{i,j}} \right)} \\ \end{aligned}$$ where ${\hat{\theta}}_{l}^{i,j}$ is the local parameter estimate of appropriate size and $\Gamma_{l}$ is a local positive definite diagonal matrix of appropriate size. According to Theorem 1, the local parameter estimate ${\hat{\theta}}_{l}^{i,j}$ is convergent if local regression vector $\Psi_{l}\left( \upsilon \right)$ is persistently exciting. Meanwhile, the parameter estimate $\hat{\theta}$ is also convergent if the trajectory of the map input *υ* passes through all of the interpolation regions $\mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{u_{2}}^{l}$. There are heavy matrices calculated in real time for the covariance matrix equation of EKF in \[[@B9-sensors-15-27142],[@B10-sensors-15-27142],[@B11-sensors-15-27142]\], preventing it from being implemented in commercial electronic control units (ECUs) for map adaptation. Nevertheless, the computational burden of the proposed observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) without the additional matrix equation is lower. Moreover, the number of parameter estimates $\hat{\theta}$ updated in Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) is no more than four at any time; then, the computational load can be further reduced by stopping estimating ${\hat{\theta}}^{i,j}$ corresponding to $\upsilon \notin \mathbb{R}_{u_{\delta}}^{k} \times \mathbb{R}_{n_{e}}^{l}$. *For the area S where the trajectory of the map input υ does not move, the parameters ${\hat{\theta}}^{i,j}$ corresponding to the interpolation region belonging to S cannot be estimated by observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}). In order to get the map parameters corresponding to S, an extrapolation model can be taken as follows:* $$\begin{array}{r} {\Delta W_{c,e}\left( {u_{\delta},n_{e}} \right) = a_{2}u_{\delta}^{2}{ +}a_{1}u_{\delta} + b_{2}n_{e}^{2} + b_{1}n_{e} + c_{2}u_{\delta}n_{e} + c_{1}} \\ \end{array}$$ *where $a_{2},a_{1},b_{2},b_{1},c_{2},c_{1}$ are polynomial parameters. Based on the data from the estimated map parameters, extrapolation model Equation ([37](#FD39-sensors-15-27142){ref-type="disp-formula"}) can be fitted by polynomial fitting approach, and then map parameters corresponding to S can be obtained*. 4. Simulation Results {#sec4-sensors-15-27142} ===================== In this section, the simulation study of 2D map estimation is presented in the environment of a 1.9 L four-cylinder common rail turbo diesel engine of enDYNA, in which the ETC and FTP75 are used as test conditions, respectively. The observer architecture is illustrated in [Figure 2](#sensors-15-27142-f002){ref-type="fig"}, where $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ is the additive reference error as the true measurement error from enDYNA. {#sensors-15-27142-f002} Bounds on the parameter vector *ρ* are presented in [Table 1](#sensors-15-27142-t001){ref-type="table"}. When the inequality Equations ([28](#FD28-sensors-15-27142){ref-type="disp-formula"}) and ([33](#FD35-sensors-15-27142){ref-type="disp-formula"}) are solved with $\varepsilon_{1} = 0.25$ and $\varepsilon_{2} = 0.11$, the gain matrix *L* can be given by: $$\begin{array}{r} {L = \begin{pmatrix} \text{1635.83} & {- \text{124.95}} & {\text{6.52} \times \text{10}^{- 7}} \\ {- \text{16,063.4}} & \text{3135.43} & {\text{1.67} \times \text{10}^{- 7}} \\ \text{4.2} & \text{29.7} & {\text{4.55} \times \text{10}^{- 20}} \\ \end{pmatrix}} \\ \end{array}$$ The initial values of observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) used in the simulation are $\hat{x}\left( 0 \right) = \begin{bmatrix} {9.8 \times 10^{5}} & {9.8 \times 10^{5}} & 0 \\ \end{bmatrix}^{T},\hat{\theta}\left( 0 \right) = 0$, and the parameter gain is $\Gamma = 200I$. Here, the reference error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ assumed as MAF sensor measurement error is depicted in [Figure 3](#sensors-15-27142-f003){ref-type="fig"}, which is superimposed on the signal $W_{c}$ in enDYNA as the measured value $y_{m2}$ in the simulation. {#sensors-15-27142-f003} 4.1. 2D Map Estimation under ETC {#sec4dot1-sensors-15-27142} -------------------------------- There are three parts of the ETC representing three different driving conditions, including urban, rural and motorway driving. Due to the engine speed range from ETC Part 1 covering the other two parts, ETC Part 1 is employed as the test condition in this section. Accordingly, the fuel mass injection quantity $u_{\delta}$ and engine speed $n_{e}$ from ETC Part 1 are plotted in [Figure 4](#sensors-15-27142-f004){ref-type="fig"}a, and the trajectory of the operating point $\upsilon = \left( {u_{\delta},n_{e}} \right)$ is depicted in [Figure 4](#sensors-15-27142-f004){ref-type="fig"}b, in which the trajectory does not move in area $S = \left\lbrack {\left( {0,56} \right) \times \left( {0,800} \right)} \right\rbrack \cup \left\lbrack {\left( {40,56} \right) \times \left( {0,2000} \right)} \right\rbrack$. According to the range $u_{\delta} \in \left\lbrack {0,56} \right\rbrack$ and $n_{e} \in \left\lbrack {0,3100} \right\rbrack$ from [Figure 4](#sensors-15-27142-f004){ref-type="fig"}a, an average partition can be respectively given as \[0:4:56\] and \[0:250:3000\]. {#sensors-15-27142-f004} The estimation results of the MAF sensor error using observer Equation ([26](#FD26-sensors-15-27142){ref-type="disp-formula"}) under ETC Part 1 are shown in [Figure 5](#sensors-15-27142-f005){ref-type="fig"}a, in which the map parameters have been estimated, except area *S*. According to Remark 3, the map parameters corresponding to area *S* can be obtained from the extrapolation model Equation ([37](#FD39-sensors-15-27142){ref-type="disp-formula"}). Based on the estimated map parameters from [Figure 5](#sensors-15-27142-f005){ref-type="fig"}a, the polynomial parameters in Equation ([37](#FD39-sensors-15-27142){ref-type="disp-formula"}) can be fitted as follows: $$\begin{array}{l} {a_{2} = 9.12 \times 1\text{0}^{- 6},a_{1} = - 4.39 \times 1\text{0}^{- 4},b_{2} = 1.30 \times 1\text{0}^{- 9}} \\ {b_{1} = - 5.74 \times 1\text{0}^{- 6},c_{2} = - 5.37 \times 1\text{0}^{- 8},c_{1} = 0.0012887} \\ \end{array}$$ The parameters corresponding to area *S* obtained from Equation ([37](#FD39-sensors-15-27142){ref-type="disp-formula"}) are presented in [Figure 5](#sensors-15-27142-f005){ref-type="fig"}b, which can roughly reflect the trend of the map. In order to evaluate the accuracy of the estimated 2D map shown in [Figure 5](#sensors-15-27142-f005){ref-type="fig"}a, the comparison between the reference error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ and the estimated 2D map during the ETC segment is presented in [Figure 6](#sensors-15-27142-f006){ref-type="fig"}a. Accordingly, the true mass air flow $y_{2}$, measured mass air flow $y_{m2}$ and map compensation are shown in [Figure 6](#sensors-15-27142-f006){ref-type="fig"}b. The mean relative error between reference error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ and estimated 2D map is $10.41\%$, which demonstrates that the measured output $y_{m2}$ of the MAF sensor after map correction can approximate the true value of $W_{c}$ acceptably. {#sensors-15-27142-f005} {#sensors-15-27142-f006} 4.2. 2D Map Estimation under FTP75 {#sec4dot2-sensors-15-27142} ---------------------------------- In order to verify the effectiveness of the proposed method under different conditions, the cold start transient phase of the FTP75 is used in this section. Accordingly, $u_{\delta}$ and $n_{e}$ are plotted in [Figure 7](#sensors-15-27142-f007){ref-type="fig"}a, and the trajectory of *υ* is depicted in [Figure 7](#sensors-15-27142-f007){ref-type="fig"}b, in which the trajectory does not move in area $S = \left\lbrack {\left( {0,56} \right) \times \left( {0,650} \right)} \right\rbrack \cup \left\lbrack {\left( {25,37} \right) \times \left( {0,2000} \right)} \right\rbrack \cup \left\lbrack {\left( {37,56} \right) \times \left( {0,3500} \right)} \right\rbrack$. {#sensors-15-27142-f007} {#sensors-15-27142-f008} The estimation results of the MAF sensor error under the cold start transient phase of the FTP75 are shown in [Figure 8](#sensors-15-27142-f008){ref-type="fig"}a, and the polynomial parameters in Equation ([37](#FD39-sensors-15-27142){ref-type="disp-formula"}) are fitted as follows: $$\begin{array}{l} {a_{2} = 2.10 \times 1\text{0}^{- 5},a_{1} = - 8.62 \times 1\text{0}^{- 4},b_{2} = 1.46 \times 1\text{0}^{- 9}} \\ {b_{1} = - 6.40 \times 1\text{0}^{- 6},c_{2} = - 1.84 \times 1\text{0}^{- 8},c_{1} = 0.0032316} \\ \end{array}$$ The map added the parameters corresponding to area *S* are shown in [Figure 8](#sensors-15-27142-f008){ref-type="fig"}b, which can also roughly reflect the trend of the map. Under the FTP75 segment, the comparison between the reference error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ and the estimated 2D map from [Figure 8](#sensors-15-27142-f008){ref-type="fig"}a is shown in [Figure 9](#sensors-15-27142-f009){ref-type="fig"}a. Accordingly, the MAF sensor measured value $y_{m2}$ using map compensation is presented in [Figure 9](#sensors-15-27142-f009){ref-type="fig"}b. The mean relative error between reference error $\Delta W_{c}\left( {u_{\delta},n_{e}} \right)$ and the estimated 2D map is $5.28\%$, demonstrating that the measured output $y_{m2}$ after map correction can approximate the true value of $W_{c}$ acceptably. {#sensors-15-27142-f009} 5. Conclusions {#sec5-sensors-15-27142} ============== A method for updating and storing sensor bias from different operating points is developed and investigated. This method achieves simultaneous estimation of model states and map parameters and applies to updating the MAF sensor error 2D map in the engine. The map in the form of a vector-vector dot product is given to conveniently analyze and design the parameter estimation method. An LPV adaptive observer to estimate map parameters is designed, which has the advantage of a simple structure and low computational load. Under ETC Part 1 and the cold start transient phase of the FTP75, the effectiveness of the presented algorithm is verified and validated in the engine software enDYNA. The results demonstrate that the proposed method can estimate the MAF sensor error acceptably. The authors would like to acknowledge the support of the National Nature Science Foundations of China (No. 61034001). Changhui Wang contributed to the theoretical analysis, algorithm design, simulation verification, and the writing. Zhiyuan Liu supervised the research, and participated in writing the paper. The authors declare no conflict of interest. p a m b ambient pressure (Pa) p i m intake manifold pressure (Pa) p e m exhaust manifold pressure (Pa) ω t turbine speed (rad/s) n e engine speed (rpm) W c compressor mass air flow (kg/s) W e g r EGR mass flow (kg/s) W e i cylinder mass flow (kg/s) W f fuel rate injected to cylinder (kg/s) W t turbine mass flow (kg/s) P c compressor power (W) P t turbine power (W) Φ c volumetric flow coefficient Ψ e g r energy transfer coefficient η v o l volumetric efficiency η t m turbine efficiency η m turbocharger mechanical efficiency η c compressor efficiency T a m b ambient temperature (K) T i m intake manifold temperature (K) T e m exhaust manifold temperature (K) V i m intake manifold volume (m 3 ) V e m exhaust manifold volume (m 3 ) V d displaced volume (m 3 ) R a air gas constant (J/(kg·K)) R e exhaust gas constant (J/(kg·K)) R c compressor blade radius (m) γ a air specific heat capacity ratio γ e exhaust specific heat capacity ratio Π c compressor pressure quotient Π t turbine pressure quotient J t turbine inertia (kg·m 2 ) n c y l number of cylinders A e g r EGR valve effective area (m 2 ) A v g t m a x VGT nozzle maximum effective area (m 2 ) f Π t choking function f v g t effective area ratio function u e g r EGR valve opening percentage (%) u v g t VGT vane opening percentage (%) u δ injected amount of fuel (mg/cycle) c p a air specific heat capacity at constant pressure (J/(kg·K)) c p e exhaust specific heat capacity at constant pressure (J/(kg·K))

Main Text {#sec1} ========= (Structure *20*, 101--112, January 11, 2012) The original article unfortunately has an author error in the units for *k*~*cat*~ and *k*~*cat*~*/K*~*m*~ on page 108. In Table 2, row 2, all *k*~*cat*~ and *k*~*cat*~*/K*~*m*~ units should be "min^−1^" and "min^−1^μM^−1^," respectively, rather than be "s^−1^" and "s^−1^μM^−1^." The scientific interpretations remain the same, only the units change. Our conclusions about the active site structure based on the kinetic analysis of the mutants do not change. [^1]: These authors contributed equally to this work

Background {#Sec1} ========== We present the complete genome sequence and analysis of a novel *Serratia* species that, in conjunction with the nematode *Caenorhabditis briggsae*, forms a putative entomopathogenic association lethal to *Galleria mellonella* larvae \[[@CR2]\]. This *Serratia* species was isolated from *C. briggsae* nematodes recovered from three separate *Galleria* traps baited in soil in the Kawa Zulu Natal province in South Africa and resembles the other entomopathogenic nematode (EPN) associations. EPNs are mutualistic associations between a bacterium and a nematode that enables them to kill insects and benefit both partners with nutrients and breeding sites \[[@CR16], [@CR41]\]. Although all three players in the EPN life cycle - pathogenic bacteria, nematode and host insect larvae -- are ancient and abundant taxa in nature, only two independently evolved entomopathogenic partnerships are well studied. One is the association between bacteria in the genus *Photorhabdus* and Heterorhabditid nematodes \[[@CR29], [@CR100]\] and the other is the association between bacteria in the genus *Xenorhabdus* with Steinernematid nematodes \[[@CR46]\]. EPN associations involve complex interactions between the pathogens and the nematode worms. In typical EPN associations the nematode is responsible for locating suitable host, penetrating the host insect and releasing the bacteria into the hemocoel while the bacteria are responsible for killing the host, bioconversion of complex compounds and protection of the insect cadaver from scavenging competitors thus ensuring nutrition for itself and its nematode partner \[[@CR29], [@CR100]\]. Significant bacterial adaptations to the EPN lifestyle include the regulation of the switch between mutualism and pathogenicity, accelerated insect killing, cadaver bioconversion, and re-association with infective juveniles \[[@CR23], [@CR48]\]. Recent studies have revealed that in both canonical EPN bacterial species L-proline in the insect hemolymph is the main trigger that initiates a metabolic shift from a quasi-dormant state in the nematode gut to a dramatic increase in secondary metabolite production in the insect hemocoel \[[@CR33]\]. Following this L-proline-induced metabolic shift, major regulatory events take place. In the *Photorhabdus*/*Heterorhabdus* association, two global regulators, HexA \[[@CR58]\] and Ner \[[@CR69]\], control the switch between mutualism and pathogenesis, while the *phoP*/*phoQ* and the *astS*/*astR* two-component systems \[[@CR38], [@CR39]\] and the *pgbPE* operon \[[@CR12]\] regulate pathogenicity and mutualism genes. Furthermore, Heterorhabditid nematodes fail to grow and reproduce normally when grown with *Photorhabdus* mutants defective in *ngrA*, suggesting that its phosphopantetheinyl (Ppant) transferase product is required for nematode growth and reproduction \[[@CR27]\]. Finally, the reassociation of infective juveniles and their cognate bacteria as well as the retention of the bacteria in the nematode gut seems to be mediated by the expression of adhesion fimbriae encoded in one of the many genomic islands rich in phage remnants \[[@CR46], [@CR68], [@CR86]\]. By comparison, in the *Xenorhabdus*/*Steinernema* association, a similar, but non-homologous, mechanism operates in which the global regulator Lrp and the two component system *cpxRA* and the *lysR* regulator *lrhA* \[[@CR32]\] orchestrate all three major stages of the life cycle: infection, reproduction and transmission. Whereas many compounds are implicated in insect killing and sanitization of the insect cadaver \[[@CR84], [@CR85]\] transmission in *X. nematophila* seems to require the *nilABC* operon which encodes three surface-localized colonization factors whose mutations invariantly lead to defective recolonization of Steinernematid worms by *X. nematophila* bacteria \[[@CR77], [@CR78]\]. The absence of the *nilABC* genes in *X. bovienii* and in *Photorhabdus* species suggest that bacteria-nematode recolonization is realized by different mechanisms in these two well-studied EPN systems. To summarize, the genetic mechanisms by which *Photorhabdus* and *Xenorhabdus* achieve entomopathogenicity are quite distinct and evidence of independent evolution of a similar phenotype. The bacterium we describe here, *Serratia* sp. SCBI, belongs to the genus *Serratia* that consists of several species with diverse lifestyles that include free soil dwellers \[[@CR49]\], plant associates in the rhizosphere \[[@CR11], [@CR34], [@CR88], [@CR98]\], opportunistic pathogens \[[@CR66], [@CR96], [@CR97]\] and obligate intracellular endosymbionts \[[@CR22]\]. Most *Serratia* spp. secrete an array of active extracellular enzymes such as nucleases, proteases \[[@CR17], [@CR25]\], lipases \[[@CR64]\] and hemolysin and have swarming and swimming mobility \[[@CR4], [@CR65], [@CR70]\]. These features may enable them to colonize a wide variety of niches and contribute to their success as opportunistic pathogens. Of the sequenced *Serratia* spp., the closest species to our isolate, *S. marcescens* DB11, is a confirmed pathogen of invertebrates \[[@CR49], [@CR63]\], a function that is likely required for evolution of an EPN complex. Although *Serratia* sp. SCBI was initially isolated as an associate of *Caenorhabditis briggsae*, this bacterium will also associate with strains of the well-studied model eukaryote *Caenorhabditis elegans* and allow these bacteriovorus nematodes to kill insects \[[@CR2]\]. Both *C. briggsae* and C. *elegans* are well known associates of invertebrates \[[@CR79], [@CR83]\], a likely pre-adaptation to an EPN life cycle. Recent discoveries of *Serratia* spp. in EPN associations \[[@CR89], [@CR105]\] suggest the possibility that *Serratia* sp. SCBI may belong to a unique lineage within the genus *Serratia* that has evolved the capacity to confer an EPN lifestyle to diverse insect-associated nematodes. The *Serratia* sp. SCBI and Caenorhabditid EPN complex represents a unique opportunity to explore the evolution of symbiosis in a third EPN lineage. Here we present the complete genome sequence of *Serratia* sp. SCBI and explore how it is unique with respect to other closely related *Serratia* and how the predicted functional proteome compares to features found in the well-studied EPN-associated bacteria, *Xenorhabdus* and *Photorhabdus*. Results and discussion {#Sec2} ====================== Overview of the *Serratia* sp. SCBI Genome Structure and Annotation {#Sec3} ------------------------------------------------------------------- The genome of *Serratia* sp. SCBI is comprised of a single circular 5.04 Mb chromosome with an overall GC content of 59.7 %, 4599 predicted protein coding genes, 84 tRNA genes and seven sets of rRNA genes (Table [1](#Tab1){ref-type="table"}, Fig. [1](#Fig1){ref-type="fig"}). In addition, *Serratia* sp. SCBI contains a single 64.8 Kb conjugative plasmid with 74 putative protein coding genes, 28 of which lack similarity to known proteins. (Additional file [1](#MOESM1){ref-type="media"}: Table S1: Plasmid ORFs). Blast analysis (data not shown) has shown that the *Serratia* sp. SCBI plasmid shows no obvious homology to the other known *Serratia* plasmid found in *S. proteamaculans* 568. The annotated genome is available at NCBI under accession numbers CP003424 and CP003425.Table 1Comparison of physical parameters of Serratia sp. SCBI with three of the sequenced Serratia genomesGenome featureOrganismS_SCBIS_DB11†S_AS12S_568‡Genome size (MB)5.045.125.445.45GC content (%)59.359.155.955Predicted ORFs4599473649524891Protein coding DNA (KB)4.44.494.734.75Protein coding DNA GC content (%)61.160.9357.356.35Genes with assigned functions3736380439944111Genes without assigned functions863932958780rRNA sets7777tRNA genes84888785Plasmid Size(KB)64.8N/AN/A46.8Plasmid GC content (%)54.92N/AN/A49Plasmid encoded ORFs74N/AN/A51\*SMAR gene prediction: 4736 ORF by FgeneSB, 4763 SANGER prediction† EBI, Sanger; ‡DOE, JGI*S_SCBI Serratia* sp. SCBI; *S_DB11 S. marcescens* DB11; *S_AS12 Serratia* sp. AS12; *S_568 S. proteamaculans* 568Fig. 1Circular representation of the *Serratia* sp. SCBI genome. Circular representation of Genomic features in *Serratia* sp. SCBI. From outer to innermost: First and fourth circles, genes in the plus and minus strands, respectively, by COG category (COG category color Scheme A, side panel); second circle, genes shared with other *Serratia* and EPN species (see color scheme B); third circle, genomic Islands (GIs) (Color Scheme C); fifth circle, GC content, sixth, innermost, circle, GC skew (Color Scheme A Side panel) Among complete *Serratia* genomes, the SCBI genome was the smallest in size and encoded fewer genes (Table [1](#Tab1){ref-type="table"}). All the compared *Serratia* genomes shared a highly conserved genomic architecture as inferred from synteny of protein coding orthologs, tRNA genes, rRNA modules and their origins of replication. These genomes shared 3094 genes by MAUVE progressive alignment \[[@CR35], [@CR36]\] at 70 % coverage and 30 % identity. An additional 809 protein coding genes were shared by *S. marcescens* DB11 and *Serratia* sp. SCBI (Fig. [2](#Fig2){ref-type="fig"}). The *Serratia* sp. SCBI, *S. marcescens* DB11, *Serratia* sp. AS12 and *S. proteamaculans* 568 genomes had 519, 587, 1015 and 1011 unique genes, respectively (Fig. [2](#Fig2){ref-type="fig"}). An additional 1267 genes were shared between two or three species. The patterns of shared and unique genes are consistent with the evolutionary history of the species defined by 16S rDNA phylogeny (Fig. [3](#Fig3){ref-type="fig"}).Fig. 2Venn diagram of shared and unique genes found in four *Serratia* genomes. The unique and shared genome among the compared genomes was determined by a dual cutoff of 30 % or greater amino acid identity and sequence length coverage of at least 70 %. Analysis was done using the MAUVE genome alignment tool \[[@CR42]\]. SCBI: *Serratia* sp. SCBI; SMAR: *S. marcescens*DB11; SPRO: *S. proteamaculans*568, SAS12, *Serratia* sp. AS12Fig. 3Evolutionary relationships of *Serratia* and representative bacteria from the entomopathogenic genera *Photorhabdus* and *Xenorhabdus.* Evolutionary relationships of *Serratia* and representative bacteria from the entomopathogenic genera *Photorhabdus* and *Xenorhabdus.* **a** Phylogenetic relationships inferred from the alignment of 1500 bp of 16S rDNA using the Maximum Likelihood \[[@CR90]\]; **b** Phylogenetic relationships inferred from the alignment of 2623 bp of concatenated DNA from four housekeeping genes: *atpD* (634 bp), *gyrB* (742 bp), *ifnB* (613 bp) and *rpoB* (634 bp) using the Maximum Likelihood \[[@CR90]\]. Numbers on internal branches are the results of Bootstrap analysis where the test was done with 1000 replicates \[[@CR43]\]. Where applicable the trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA6 \[[@CR91], [@CR92]\] Phylogenetic placement of SCBI {#Sec4} ------------------------------ To place *Serratia* sp. SCBI in an evolutionary framework we compared the near complete 16S rRNA genes from diverse *Serratia* species and other relevant entomopathogenic bacteria (Fig. [3](#Fig3){ref-type="fig"}) as well as concatenated sequences from four housekeeping genes: *atpD, gyrB, ifnB* and *rpoB*. The phylogenetic tree from the concatenated housekeeping genes suggest that *Serratia* sp. SCBI is found in a clade of five *Serratia* which are all known insect associates and SCBI was closest to *S. ureilytica*. Among the well-studied and completely sequenced species included in the 16S phylogeny, *Serratia* sp. SCBI was most closely related to *S. marcescens* DB11, which is an insect pathogen \[[@CR49], [@CR50], [@CR63]\] and antagonist of *C. elegans* \[[@CR61], [@CR76], [@CR82]\]. This close relationship provides an ideal opportunity to identify candidate genes critical to the evolution of the EPN lifestyle. Another closely related species is *S. nematodiphila*, which was described as part of an EPN association with a Rhabditid nematode \[[@CR104], [@CR105]\]. Other close relatives include *S. entomophila* \[[@CR50]\], which is pathogenic to New Zealand grass grubs, and *S. ureilytica* which is a nickel resistant *Serratia* isolated from the River Torsa in West Bengal, India \[[@CR13]\]. This phylogeny suggests that *Serratia* sp. SCBI may represent a novel, independently evolving EPN association from within a lineage of *Serratia* commonly associated with insects. Genomic alignment and comparative genomic analysis {#Sec5} -------------------------------------------------- Whole genome alignment of SCBI with three of the sequenced *Serratia* genomes show extensive synteny (Fig. [4](#Fig4){ref-type="fig"}) where large colinear blocks of genes are interrupted by insertions that resulted in genomic islands, deletions relative to SCBI and a small number of rearrangements involving relatively small genomic regions. In the context of this study, a genomic island (GI) was broadly defined as any stretch of five or more consecutive protein coding genes that: 1) are not present in the other members of the genus, and 2) show a divergent %GC content compared to the core genome. Our analysis predicts at least 29 GIs in *Serratia* sp. SCBI. Based on these criteria 326 of 519 (62.8 %) of unique genes are located in the genomic islands of SCBI and as discussed below most of these GIs encode functions of potential biological relevance to the EPN lifestyle (Table [2](#Tab2){ref-type="table"}). To ascertain the significance of the *Serratia* sp. SCBI-*C. briggsae* association in relation to other *Serratia* and the established EPN bacteria, we conducted comparative analysis of the predicted proteome against the respective groups using the MAUVE \[[@CR35]\] genome alignment tool. The cutoff values of 60 % identity and 70 % coverage, i.e. similarity of 60 % of the compared residues covering 70 % of the total length of the shorter sequence in the comparison, were used to include only hits with high confidence levels and exclude spurious hits (Table [3](#Tab3){ref-type="table"}).Fig. 4Genomic alignment of the four compared *Serratia* spp. Alignment statistics were generated and rendered by MAUVE progressive alignment software \[[@CR35], [@CR36]\]. SMAR: *S. marcescens* DB11, SCBI: *Serratia* sp. SCBI, and SPRO: *S. proteamaculans*586, SAS12: *Serratia* sp. AS12. Color schemes represent blocks of contiguous genes interrupted by colorless patches where the genomes differ from each other significantly and identified as GIs are locatedTable 2Serratia sp. SCBI Genomic Islands and their putative/predicted phenotypesGI\#Locus tagsGenomic locationPutative/Predicted roleGI-1SCBI_0047-005246958..52205UnknownGI-2SCBI_0298-0302358841..362952AdhesionGI-3SCBI_0465-0482533080..558686UnknownGI-4SCBI_0809-00817928240..935735Symbiosis/MetabolicGI-5SCBI_0963-09811080968..1096217VirulenceGI-6SCBI_1013-10191130653..1141194Virulence, DefenseGI-7SCBI_1042-10611164353..1197133Virulence, SymbiosisGI-8SCBI_1205-12091349465..1355183DefenseGI-9SCBI_1258-12631405041..1410636SymbiosisGI-10SCBI_1467-14781614753..1632962MetabolicGI-11SCBI_1483-14981639797..1656736MetabolicGI-12SCBI_1540-15471701960..1709609UnknownGI-13SCBI_1814-18191993067..1996355UnknownGI-14SCBI_1883-18882062882..2068451DefenseGI-15SCBI_1970-19752150551..2156393SymbiosisGI-16SCBI_2176-21802367813..2374480UnknownGI-17SCBI_2225-22322418731..2436006SymbiosisGI-18SCBI_2295-23002513930..2520831MetabolicGI-19SCBI_2738-27942980185..3019378Defense, virulence, AdhesionGI-20SCBI_2953-30113191699..3264229Defense, virulence, AdhesionGI-21SCBI_3036-30413289235..3297687MetabolicGI-22SCBI_3184-32033460520..3479027Adhesion, DefenseGI-23SCBI_3369-33733652152..3658481SymbiosisGI-24SCBI_3376-33803662181..3666951UnknownGI-25SCBI_3539-35393835598..3842153DefenseGI-26SCBI_3778-37944112710..4132272Defense, SymbiosisGI-27SCBI_3962-39664314901..4319983MetabolicGI-28SCBI_4358-43664751221..4757933MetabolicGI-29SCBI_4408-44124806030..4811621Unknown(see Additional file [2](#MOESM2){ref-type="media"}: Table S2 for the full/expanded version)Table 3The distribution of Virulence/defense/symbiosis factors in Serratia and the EPN bacteriaSCBISMARSODOSPROPASYPLUMXBOVXNEMToxicity/VirulenceGroEL1111111Hemolysin66666855LopT12Mcf1211MtpB1311MtpD1211MtpE1211NRPS-PKS1716131242373346PirA1111121PirB-JHE121RtxA3411TC11119712Symbiosis/RecolonizationCipA22CipB11ExbD22222222HexA1111LrhA1111NgrA11211111NilA1NilB1NilC1NilR1122PbgPE66667767PhoP22221111PhoQ11111111Immune_evasion/BioconversionBacillolysin11111112Cif11CpxA11111111CpxR22221111FlhC11111111FlhD11111111FliA11111111LPS21121111Lrp22222211MalP22221111MalQ11111111MalT11111111SctC11Serralysin44221111XlpA1111*SCBI Serratia* sp. SCBI; *SMAR S. marcescens* DB11; S_AS12, *Serratia* sp. AS12; *SPRO S. proteamaculans* 568; *PASY P. asymbiotica*; *Plum P. luminescens*; *Xbov X. bovienii*; Xnem, *X. nematophila* COG analysis among *Serratia* {#Sec6} ----------------------------- Analysis of the functional categories and genome wide distribution of all unique genes with assigned Clusters of Orthologous Groups (COG) functions among the sequenced *Serratia* genomes revealed that the unique genes in *Serratia* sp. SCBI were biased towards categories that have direct bearing on the symbiosis and/or pathogenesis life style (Fig. [5](#Fig5){ref-type="fig"}). Specifically, the gains were in COG categories: \[M\]- Cell wall/membrane/envelope biogenesis; \[N\]- Cell motility; \[Q\]- Secondary metabolites biosynthesis, transport and catabolism; \[U\] - Intracellular trafficking and secretion and \[V\]- Defense mechanisms.Fig. 5Relative COG category abundance in the core proteome in comparison with unique proteins in *Serratia*. The relative abundance of COG categories between the core and unique gene pools was calculated as follows: the number of proteins in each COG category was determined and the unique pools normalized to their respective total predicted protein numbers. Then the percentage of each COG category in the core proteome was subtracted from the corresponding COG percentage in the unique category and the difference plotted. COG functional categories descriptions are: \[A\] RNA processing and modification; \[B\] Chromatin structure and dynamics; \[C\] Energy production and conversion; \[D\] Cell cycle control and mitosis; \[E\] Amino acid metabolism and transport; \[F\] Nucleotide metabolism and transport; \[G\] Carbohydrate metabolism and transport; \[H\] Coenzyme metabolism; \[I\] Lipid metabolism; \[J\] Translation; \[K\] Transcription; \[L\] Replication and repair; \[M\] Cell wall/membrane/envelope biogenesis; \[N\] Cell motility; \[O\] Post-translational modification\] protein turnover\] chaperone functions; \[P\] Inorganic ion transport and metabolism; \[Q\] Secondary metabolites biosynthesis, transport and catabolism; \[T\] Signal transduction; \[U\] Intracellular trafficking and secretion; \[R\] General functional prediction only ; \[S\] Function unknown; \[V\] Defense mechanisms. \[X\] No cog category Functional properties of *Serratia* sp. SCBI shared with other entomopathogens {#Sec7} ------------------------------------------------------------------------------ To evaluate the *Serratia* sp. SCBI in the context of its role in an EPN complex, we searched the SCBI genome for homologs or functional analogs of all genes previously shown or implicated to be involved in host immune defense, host killing and cadaver protection, pathogenesis and symbiosis and reassociation in the canonical EPN species *Xenorhabdus* and *Photorhabdus* (Table [3](#Tab3){ref-type="table"}). Although most of these genes appear in GIs in *Serratia* SCBI, we focused the comparison on three functions with well documented roles in EPN lifestyle but not confined to putative Genomic Islands: evading host defense, toxicity to host and competitors and recolonization. Functional studies on the *Serratia* sp. SCBI-*C. elegans* complex has shown that hemolysin, NRPS proteins (Petersen, LaCourse et al. submitted) and extracellular proteases are crucial to cytotoxicity and virulence in this putative EPN symbiosis \[[@CR72]\]. The dynamics of mRNA expression of the alkaline metalloproteases (prtA1-prtA4) compared to the serine metalloproteases which peak before and after the death of the host, respectively, suggest a complex regulatory mechanism in killing the host and bioconversion of the cadaver \[[@CR72]\]. Furthermore, inactivation of the hemolysin gene in *Serratia* sp. SCBI, which resulted in loss of hemolysis, failed to attenuate insecticidal activity but significantly increased motility and antimicrobial activity (Petersen et. al., submitted). Furthermore comparative study on the physiology of three sequenced strains, *Serratia* sp. SCBI, *S. marcescens* DB11 and *S. proteamaculans* 568, revealed that DB11 and SCBI were similar in insect virulence and cytotoxicity consistent with their phylogenetic proximity, but motility and lipase and hemolytic activities differed significantly between them \[[@CR71]\]. The implication of these functional studies is that the *Serratia* sp. SCBI-*C. briggsae* association is a promising model to study the dynamics of gene expression during the transition from starved, inactive stay in nematode gut to active state during pathogenesis --killing and bioconversion-- of insect hosts. Breaching host defense {#Sec8} ---------------------- Breaching the insect host's defense --both the humoral and cellular components- is crucial for the establishment of infection. EPNs neutralize humoral response elements like lysozyme and cecropins with serralysin-like proteases such as the PrtA \[[@CR42], [@CR67]\] and by haemolysins (XhlA) and lipases (XlpA) and the FhlDC regulator operon \[[@CR47], [@CR67]\]. Cellular response evasion is directed mainly at suppressing the phenoloxidase pathway to prevent hemocyte aggregation and melanization. Several mechanisms are employed to suppress the insects' cellular immune response. These include: inhibition of phospholipase A2 (PLA2) using stilbene and MalPQT operon products in *Photorhabdus* and expression of LPS in *Xenorhabdus,* the suppression of phagocytosis by Type III secretion system-mediated deposition of LopT and SctC directly into hemocytes \[[@CR19], [@CR20]\] and the production of cytotoxins such as Cif and MrxA that lead to apoptotic cell death. With the exception of the *xlpA* gene which is absent from *Serratia* spp. and the *cif* and *stc* genes that are absent not only from *Serratia* spp. but also from *Xenorhabdus* spp., all other genes identifies as instrumental in the toxicity of EPNS are present in *Serratia* spp. as well including SCBI. The presence of more serralysins in *Serratia spp.,* by comparison to the EPN genomes, suggests that the SCBI *C. briggsae* association may heavily rely on the expression of multiple serralysin genes and extracellular lipases and hemolysin production to breach the host immune system. Toxicity {#Sec9} -------- Toxicity --both for killing the insect host and warding off bacterial and fungal competitors- is mediated by a plethora of insecticidal and bactericidal gene products \[[@CR44]\]. These include the *Photorhabdus* Toxin Complex (TC) operons *tcaABCD* and their counterparts in *Xenorhabdus xptABCD*, as well as other toxicity genes like the *pirAB, xaxB*, *sepA*, *groEL*, *lopT*, *mcf*, *mcf2*, *rtxA*, *mptBDE*. Various drug efflux systems and numerous nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) genes \[[@CR15], [@CR37], [@CR45], [@CR53], [@CR55]\] are also part of the overall toxicity caused by EPN systems. The redundancy evident in the toxin gene repertoire of *Photorhabdus* and *Xenorhabdus* is astounding -- at the time of its sequencing in 2003, *P. luminescens* was described as having more genes encoding toxins than any other genome sequenced to date \[[@CR40]\], however, *Serratia sp.* SCBI also has over 200 genes encoding toxins, NRPS genes, multiple drug efflux systems and assorted virulence factors. Nematode recolonization {#Sec10} ----------------------- Nematode recolonization involves two key elements: acquisition of partner bacteria by horizontal transmission via endotokia matricida \[[@CR28]\], in which eggs hatch within the sacrificial mother to gain access to bacteria before emerging from the maternal corpse and the retention of bacteria in the gut of the infective juvenile. The latter seems to be mediated by *ngrA* \[[@CR27], [@CR59]\], the Type I fimbriae *mrxA* \[[@CR24]\] and at least in *X. nematophila*, by the products of the *nilABC* and *nilR* genes \[[@CR30], [@CR31], [@CR52]\]. Both of these requirements seem to be met in SCBI since *Caenorhabditids*, when grown on SCBI, almost exclusively reproduce by endotokia matricida \[[@CR1]\] and SCBI possess homologs of both genes implicated in recolonization. Furthermore, the absence of *the nilABC* genes of *X. nematophila* in *Xenorhabdus boveinii* as well as *Photorhabdus* spp. show that colonization, like other aspects of EPN biology, can be produced by different mechanisms in EPNs. From this comparison three patterns emerge: 1) *Serratia* lack homologs of the toxicity genes *lopT, rtxA, prtA, pirB, cif, mcf*, and *mcf*2 and the *TC* operons but have substantial number of secreted proteases and lipases, and hemolysins and 2) the majority of the *Photorhabdus/Xenorhabdus* virulence, symbiosis and regulatory genes are equally represented in *Serratia* (Table [3](#Tab3){ref-type="table"}), and 3) the presence of key genes is not uniform among the different species --*nilABC* absent in *Photorhabdus* spp. and c*if, stc, cipAB* and *lopT* missing from *Xenorhabdus* spp.-suggesting that there is no single possible path to pathogenicity, cadaver bioconversion, repelling of competitors and bacterial re-association. Unique genes and Genomic Islands (GI) of SCBI {#Sec11} --------------------------------------------- Apart from those summarized in Table [3](#Tab3){ref-type="table"}, many other genes with well-documented pathogenicity/symbiosis functions in other microbial systems are found in the genome of SCBI more than half of which are located on genomic islands. Of the 29 genomic islands identified in *Serratia* sp. SCBI genome comparison (Fig. [1](#Fig1){ref-type="fig"}, third circle, Additional file [2](#MOESM2){ref-type="media"}: Table S2), many were enriched in defense and virulence genes and in - phage remnants which, in many bacteria including those in EPN associations, have been diverted into novel toxin/virulence factor delivery vehicles \[[@CR8], [@CR21], [@CR56], [@CR62]\]. Perhaps the most notable feature among the genomic islands is the urea pathway genes located on GI-4. The *hoxN/hupN/nixA* family nickel/cobalt transporter and the urea metabolism pathway proteins found in GI-4, are absent from other members of the genus *Serratia* but interestingly present in all the EPN bacteria. The *hoxN* transporter is necessary for urea hydrolysis \[[@CR60], [@CR103]\] making it an integral part of the pathway. While there is no evidence so far of a direct role for urea metabolism in the EPN lifestyle, a potential role for these pathways can be exemplified by current research on diatoms where the urea pathway is a key to facilitating rapid recovery from prolonged nitrogen limitation followed by rapid growth under nutrient rich conditions \[[@CR6]\]. The EPN life cycle has a similar pattern in that the symbionts that survive in nutrient-limited conditions while in transit to the next cycle of infection face a sudden abundance of organic compounds upon entering the insect hemocoel, thus requiring rapid transitioning from starved state to exponential growth of the bacteria. Furthermore, the regulation of the urea pathway in the diatoms has been linked to proline \[[@CR5]\] which has also been identified as the cue for the transition from starved to metabolically active state in *Photorhabdus* and *Xenorhabdus*. Among the unique genes of SCBI that are relevant to its association with *C. briggsae* as a putative EPN are the O-antigen biosynthesis protein(SCBI_1044), NRPS proteins(SCBI_1017, SCBI_1055-1059, SCBI_2978), colicins (SCBI_1883-1887), Type IV secretion systems (SCBI_2992, SCBI_2984, SCBI_2994), and iron acquisition proteins(SCBI_1973-1975). These genes have been shown to be required for both symbiotic and pathogenic properties \[[@CR12], [@CR101], [@CR102]\] and may play a similar role in the *Serratia*-*Caenorhabditis* EPN association. The list of genes unique to SCBI also include efflux system proteins (SCBI_1013, SCBI_1208) that are specific for macrolide- a class of antibiotics that inhibit the growth of bacteria- and the resistance-nodulation- division (RND) efflux proteins (SCBI_3788-3791) that are known to actively scavenge antimicrobial compounds released by competitors or the host immune system \[[@CR3], [@CR14], [@CR73], [@CR74], [@CR87], [@CR93], [@CR94]\], a predicted HtpX protease, a heat shock/stress inducible membrane bound zinc metalloprotease \[[@CR81]\], the DinI family protein also known as MsgA (macrophage survival protein) (SCBI_3186) and prophage lysozyme proteins (SCBI_2756-2758) an endolysin/autolysin system known to degrade the peptidoglycan structures in bacterial cell walls \[[@CR18], [@CR51], [@CR99]\]; Microcin H47(SCBI_2968, SCBI_3536), a bactericidal peptide antibiotic related to the Colicin V family secretion protein \[[@CR10]\] and associated secretion ATPase required for its export \[[@CR95]\]; several fimbrial proteins with functional annotations ranging from pilus assembly to anchoring which, in light of recent findings that implicate *Photorhabdus* and *Xenorhabdus* adherence to the gut walls of their respective worm associates via fimbrial proteins, \[[@CR46], [@CR68], [@CR86]\] and two homologs of the HigB toxin protein/HigA protein (antitoxin to HigB)( SCBI_0907-0908), which in *Vibrio*, have been shown to inhibit cell growth in *Escherichia coli* upon ectopic expression \[[@CR26]\]. Conclusions {#Sec12} =========== EPN associations are complex tripartite interactions between bacterial pathogens, symbiotic entomophagous nematodes and susceptible insect/insect larval hosts. Thus far our understanding of the mechanisms of EPN associations is limited to two superficially similar and convergent systems. Here we report the complete genome sequence of the bacterium involved in a novel independently evolving putative EPN association between a species of *Serratia* and nematodes in the genus *Caenorhabditis*. This *Serratia* is most closely related to another recently discovered EPN association, *Serratia nematodiphila,* and the well-studied insect pathogen *Serratia marcescens* DB11. Based on a comparison to the two well studies EPN systems, the genome of *Serratia* sp. SCBI contains a large number of genes that are potential candidates for EPN adaptations. Among the most notable shared functions with other EPN associates are the O-antigen, the *syrP* protein, several non-ribosomal peptide synthetases, bacteriocins, many fimbrial biogenesis and ushering proteins, secondary metabolite and toxin secretion systems and multiple drug resistance/efflux systems. However, the SCBI genome carries neither the TC complexes nor the *mcf* genes, which implies its use of different mechanisms of insect killing. The presence of several ORFs encoding putative virulence factors in horizontally acquired genomic islands suggest that EPN associations can be established by dissimilar sets of mechanisms for killing, bioconversion, sanitization and colonization. The presence of these sets of genes in many bacteria further suggests that the major hurdle in EPN complex formation may be the initial development of co-tolerance between potential partners. This complete genome sequence of one of the partners in a nascent EPN association should enable future analysis of the *Serratia/Caenorhabditis* EPN complex. Methods {#Sec13} ======= Bacterial isolation and identification {#Sec14} -------------------------------------- The bacterium was isolated from a *Galleria mellonela* trap laid in soil in the Kawa Zulu Natal province of South Africa. The detailed procedure has been previously described elsewhere \[[@CR2]\]. Following isolation bacterial identity was determined by 16S rDNA PCR which shows 99 % identity with *Serratia marcescens* rDNA sequences at NCBI (CP003959.1). Genomic DNA isolation {#Sec15} --------------------- Genomic DNA (gDNA) was isolated from overnight cultures grown in LB medium \[10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaCL, H2 O to 1 liter, pH 7.5\] (BD, Sparks, MD) using the Qiagen Genomic DNA isolation kit (Qiagen, Germantown, MD) and following the procedure outlined for bacterial gDNA isolation. Precipitated DNA was collected by spooling the DNA using flame-sterilized and cooled glass rod. The spooled gDNA was immediately transferred to a microcentrifuge tube containing 1.5 ml sterile, nuclease free water. The DNA was dissolved on a shaker at 55 °C for 2 h. The yield, purity, and length of the DNA was determined for 1-5 μl samples by electrophoresis on 0.8 % agarose gel and by spectrophotometry on a NanoDrop 1000 (Thermo Scientific, USA). Fosmid Library construction {#Sec16} --------------------------- The fosmid library was constructed from the genomic DNA (gDNA) using the CopyControl pCC1FOS™ Vector that contains both the *E. coli* F-factor single-copy origin of replication and the inducible high-copy *oriV* according to the manufacturer's protocol (Epicentre, Madison, U.S.A.). Briefly, gDNA was mechanically sheared by passing through a narrow gage sterile syringe then it was separated by pulse field gel electrophoresis (PFGE) overnight. A gel slice was excised from the 36-40 KB size window and the DNA extracted by gel extraction. The gel extracted gDNA was subsequently ligated into the fosmid vector exactly as described in the Epicenter protocol. The ligated mixture was then packaged into lambda phages using MaxPlax Lambda Packaging Extracts (Epicentre, Madison, U.S.A.). The packaged library was then transduced into *E. coli* EPI300™ Plating Strain, and transformants were selected on LB agar supplemented with 34 mg/ml Chloramphenicol. The library clones were picked by the Genetix Q-bot colony picking robot (Genetix Ltd, UK) and inoculated into 384 well plates and allowed to grow for 24 h at 37oC in humidified chamber. Colonies were stored in -80oC freezers until they were retrieved for downstream processing. To isolate fosmid DNA, randomly selected individual clones were grown overnight in Chloramphenicol supplemented LB broth and plasmid copy number were amplified by adding 5ul induction solution and incubating further for 4 hours. Fosmids were isolated by alkaline lysis method using Qiagen plasmid isolation buffers. The presence of recombinant DNA in the isolated fosmids and the polymorphism of the insert DNA were evaluated by agarose gel electrophoresis of BamHI (NEB, USA) digestion of the purified plasmid DNA. PCR primer design and amplicon sequencing {#Sec17} ----------------------------------------- The genome sequencing gaps were closed using PCR based amplicon sequencing. To this end batches of PCR primer were designed by the Primer3 software driven by an in-house Perl script. Primers were synthesized by IDG (IDG Inc., MA, USA) and they were used to both amplify and sequence the amplicons after cleaning the PCR product from any unused primers. PCR fragments were purified using magnetic beads and SPRI solution. The primers were designed to have a melting point of 60 °C or above for increased specificity and simultaneous amplification at a single annealing temperature. Cycling conditions were as follows: Initial denaturation of plasmid, 5 min at 95 °C; 30 cycles or denaturation, annealing and primer extension at 95 °C 30 s, 60 °C 30 s and 2 min at 72 °C, respectively; a final synthesis hold at 72 °C for 10 min and a 4 °C hold until reactions were removed from the iCycler PCR machine (Biorad Inc. CA, USA). Long PCR for amplification of 3-8 KB fragments were performed the same way except that the 10X buffer was supplemented with additional MgCl~2~ to bring the final concentration to 25 mM and the extension time was increased to 6 min. All PCR amplifications were done using Finnzymes PCR kit (NEB, MA, USA) and the 15 mM 10X buffer was used for fragments between 100 to 2000 bases long. Fosmid end sequencing and quality monitoring {#Sec18} -------------------------------------------- DNA isolated from 209 plasmids was end sequenced using the pCC1FOS™ forward and reverse primers designed by Epicenter. Half of the clones were sent to a commercial sequencing facility (Genewiz Inc, NJ, USA) the other half were sequenced in house by the ABI 3130 genetic analyzer. Efficiency of Fosmid library construction and the quality of the library were monitored by selecting at random 209 clones and subjecting them to different analyses. A subset of these (46 clones) were tested by Bam HI digestion and, except for a failed plasmid isolation in one clone, all 45 (100 %) of them gave distinct digestion patterns and one common band at 8 KB, representing the plasmid backbone. BLAST results showed that except for 4 clones that failed to sequence, probably due to sample cross over contamination, all fosmid end sequences hit the assembled genome at 34 to 45 KB apart in the right orientation giving an overall high efficiency of library construction and quality. Genome sequencing by 454 technology and assembly of contigs {#Sec19} ----------------------------------------------------------- An aliquot of the *Serratia* sp. SCBI gDNA was sent to the genome sequencing center at the University of Indiana for genome sequencing by 454 pyrosequencing technology. Two plates of genomic sequencing and one plate of 2 KB paired end sequencing were done to generate the complete genomic sequence of one circular chromosome and one large, circular plasmid of 68 KB size. The 454 sequencing resulted in ten contigs ranging in size from 2 KB to 3.5 MB and harbored 236 gaps of ranging from 4 nucleotides to 2 kb in length which were closed by PCR amplicon sequencing as detailed in the PCR primer design and amplicon sequencing section. The genome sequencing output from the 2 plates of 454 genomic reads and one plate of 2 KB paired end sequencing yielded 40 and 21 MB data, giving an overall 12X coverage of the genome. The main chromosome was resolved in seven genomic regions separated by seven sets of rDNA assemblies of 5 KB length as outlined below. Sequence gap filling, genome assembly and bioinformatics analysis {#Sec20} ----------------------------------------------------------------- The 10 scaffolds assembled by the Newbler Assembler sequence initially included 236 gaps ranging from 4 to 2050 bases long. PCR based amplicon end sequencing resolved all the gaps bringing the sequence assembly to a satisfactory level of completion. PCR primers were designed on regions flanking gaps by the Primer3 program \[[@CR80]\] after orientation and order of scaffolds were determined by a combination of bioinformatics and laboratory based methods. Briefly, truncated scaffold sequences consisting of 500 bp from the start and end of each scaffold were generated by a Perl script. These truncated sequences were then blasted against the *S. marcescens* DB11 genome which identified their counterparts unambiguously showing a great deal of synteny between the two genomes. This finding was then confirmed by long PCR performed using primers pointing outward from the pairs of gap ends predicted by the bioinformatics analysis. All of the predicted combinations resulted in the amplification of the expected 6 KB fragment confirming both the order and orientation of the clones as well as their size. The remaining two fragments were resolved into a circular plasmid by blast hit with two fosmid clones which hit the two scaffolds unambiguously. This was also confirmed by PCR that amplified predicted size of amplicons from primers designed at the ends of the two scaffolds and individual fosmids used as template. Long PCR and amplicon sequencing were performed as described above and the resultant amplicon sequences were then added to the 454 reads and reassembled by the Newbler to generate the complete genome sequence. Comparative genomic analysis of *Serratia* sp. SCBI genome with the two other completed genomic sequences in the genus, *S. marcescens*DB11 and *S. proteamaculans*568 was done to determine the genes shared between the three genomes and the three way paired genomes (*Serratia* sp. SCBI-DB11, *Serratia* sp. SCBI-SPRO, DB11-SPRO) and the genes unique to each of them using MAUVE \[[@CR35], [@CR36]\], MURASAKI \[[@CR75]\] and Blast \[[@CR7], [@CR57]\] bioinformatics tools, the RAST Annotation Server \[[@CR9]\] and in house developed Perl scripts. A comparison was also done with *P. luminescens* and *X. nematophilia* to shed light on the entomopathogenic aspects of the *Serratia* sp. SCBI-*Caenorhabditis* relationship and to determine whether it resembles the *Photorhabdus*/*Heterorhabdus* or the *Xenorhabdus*/*Steinernema* type association or it constitutes a novel class of EPN association with a different entomopathogenic signature driven by a novel set of genes and pathways. Accession numbers {#Sec21} ----------------- The genome sequence and its annotations are available at NCBI under the accession numbers CP003424 and CP003425. Additional files {#Sec22} ================ Additional file 1: Table S1.*Serratia* sp. SCBI predicted plasmid encoded genes.Additional file 2: Table S2.*Serratia* sp. SCBI Genomic Island genes with putative/Predicted functions relevant to EPN life style. **Competing interests** The authors declare that they have no competing interests. **Authors' contribution** FAA, WKT, VSC, LST and EA conceived of and provided project oversight. FAA conducted the molecular biology and comparative genomics analysis and manuscript draft. PJH provided key scripts for the analysis. All authors edited, read and approved manuscript. This work was supported in part by Hatch grant NH496, and by the College of Life Sciences and Agriculture, The University of New Hampshire, Durham, NH. Partial funding was provided by the New Hampshire Agricultural Experiment Station. This is Scientific Contribution Number 2571. We thank Heidi-Goodrich-Blair and Jonathan Ewbank for access to the *Xenorhabdus* and *Serratia* genome databases, respectively. We thank Jobriah Anderson for technical help.

1. Introduction {#sec1-polymers-12-00289} =============== The environmental problems caused by textile printing and dyeing wastewater have become the focus of attention. Typical textile printing and dyeing wastewater usually contains dyes, polyvinyl alcohol (PVA), starch, and so on \[[@B1-polymers-12-00289],[@B2-polymers-12-00289]\]. Some dyes may be further degraded into toxic and carcinogenic substances, which will affect human health \[[@B3-polymers-12-00289]\]. The efficient and complete removal of dyes from effluents is necessary. Starch and PVA in textile printing and dyeing wastewater have a great influence on the reoxygenation behavior of bodies of water. As a result of the high chemical oxygen demand (COD) values and lower biodegradability, PVA is difficult to remove from textile wastewater by simple treatment facilities \[[@B4-polymers-12-00289],[@B5-polymers-12-00289]\]. A lot of technologies, including flocculation, membrane separation, chemical oxidation, and degradation, have been studied for treating dye wastewater \[[@B6-polymers-12-00289],[@B7-polymers-12-00289],[@B8-polymers-12-00289],[@B9-polymers-12-00289]\] or for treating PVA-containing wastewater \[[@B5-polymers-12-00289],[@B10-polymers-12-00289],[@B11-polymers-12-00289],[@B12-polymers-12-00289],[@B13-polymers-12-00289]\], but so far, there have been no reports on the simultaneous removal of dyes, PVA, and starch from textile printing and dyeing wastewater. These methods also have many difficulties for industrial application, due to the high cost of investment and operation, complicated processes, and inability to remove pollutants completely \[[@B5-polymers-12-00289],[@B12-polymers-12-00289],[@B14-polymers-12-00289]\]. By contrast, adsorption is one of the most efficient and reliable textile wastewater treatment techniques \[[@B15-polymers-12-00289],[@B16-polymers-12-00289],[@B17-polymers-12-00289]\], and fixed-bed adsorption is often desired from an industrial point of view, because of its advantages of its 100% pollutant removal ability from wastewater, lower floor space, and lower cost of equipment investment and operation \[[@B18-polymers-12-00289],[@B19-polymers-12-00289],[@B20-polymers-12-00289],[@B21-polymers-12-00289],[@B22-polymers-12-00289]\]. Many studies have been conducted on the dye adsorption properties of various adsorbents used in fixed beds, such as agricultural waste, chitosan-based materials, zeolites, and industrial by-products, etc. \[[@B18-polymers-12-00289],[@B23-polymers-12-00289],[@B24-polymers-12-00289],[@B25-polymers-12-00289]\]; however, these adsorbents usually have defects with a low adsorption capacity. Up until now, activated carbon (AC), although its removal efficiencies for organic dyes are still relatively low \[[@B26-polymers-12-00289],[@B27-polymers-12-00289]\], is still the most commonly used adsorbent for industrial wastewater treatment \[[@B28-polymers-12-00289],[@B29-polymers-12-00289]\]. Therefore, in order to remove dye, polyvinyl alcohol, and starch from textile wastewater simultaneously, it is necessary to develop an efficient and low-cost fixed-bed adsorbent alternative. Starch and its derivatives are of increasing interest because of their higher biodegradability and renewability \[[@B30-polymers-12-00289],[@B31-polymers-12-00289]\]. Cationic starch derivatives with quaternary ammonium groups are commonly prepared through the reaction of starch with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) \[[@B32-polymers-12-00289],[@B33-polymers-12-00289]\]. As a result of the strong quaternary ammonium groups, cationic starch derivatives can adsorb anionic compounds, and have been widely utilized for removing various ions and organic pollutants from wastewater \[[@B34-polymers-12-00289],[@B35-polymers-12-00289],[@B36-polymers-12-00289]\]. PVA possesses hydrophilic, biocompatible, and nontoxic characters. PVA derivatives are important functional polymers, and are widely used in paper, medicine, and water treatment \[[@B37-polymers-12-00289],[@B38-polymers-12-00289],[@B39-polymers-12-00289]\]. Nevertheless, so far, there have been no reports on the synthesis of modified starch/PVA composite microspheres and their applications in textile wastewater treatment. In this paper, the cationic cross-linked starch/PVA composite microspheres (CCSP) were synthesized for the purpose of removing the starch, PVA, and dyes from the textile wastewater concurrently, and their properties were determined. Combined with fixed-bed adsorption techniques, the adsorption capacities of CCSP for treating textile wastewater were investigated. Most importantly, the CCSP used exhibited great sustainable reutilization by converting into regenerated AC, thereby avoiding secondary solid-waste pollution. It is expected that the strategies shown in this work will provide new inspiration and ideas for the development of environmentally-friendly textile wastewater treatment techniques. 2. Materials and Methods {#sec2-polymers-12-00289} ======================== 2.1. Materials {#sec2dot1-polymers-12-00289} -------------- Corn starch containing approximately 71% amylopectin and 29% amylose was supplied by Tianjin Tingfung Starch Development Co., Ltd., Tianjin, China. Polyvinyl alcohol (PVA; 1700 degrees of polymerization, 99% hydrolyzed) was obtained from China Petroleum and Chemical Corporation, Shanghai, China. 3-Chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) was obtained from Sigma-Aldrich Chemie Gmbh, Steinheim, Germany. Colour Index (C.I.) Reactive Black 5 and C.I. Reactive Orange 131 were supplied by BASF SE, Ludwigshafen, Germany. Their structures are displayed in [Figure 1](#polymers-12-00289-f001){ref-type="fig"}. The commercial activated carbon (AC) was purchased from Ningxia Huahui Activated Carbon Company (Yinchuan, China), and its methylene blue number was 225.3 mg/g. All of the other chemicals were of analytical grade, obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), without further purification. 2.2. Preparation of Modified Starch/PVA Composites {#sec2dot2-polymers-12-00289} -------------------------------------------------- Cross-linked cationic PVA (CCP) was obtained by a two-stage process. First, the cationic PVA (CP) was prepared by PVA with CHPTAC, in the presence of sodium hydroxide, at 40 °C for 4 h \[[@B40-polymers-12-00289]\]. In the second step, 7.2 g of Span-80 was added into 100 g of cyclohexane and was stirred, and was then added into 50 g of a cationic PVA aqueous solution (5 wt%). The mixture was homogenized at 6000 r/min by a high-shear dispersion homogenizer for 30 min to form a stable W/O emulsion system. Then, 12 g of a borax solution (2.5 wt%) was dropwise added into the mixture within 30 min, and continued to react for another 4 h at 35 °C. Finally, it was centrifuged, washed with acetone, and dried in an oven at 40 °C. Thus, the CCP microspheres were obtained. The degree of substitution of synthesized CP was 0.24 (2.99 mmol/g of the cationic group content), which was calculated based on the content of the element nitrogen. Cross-linked cationic starch (CCS) and cationic starch (CS) were prepared using borax and CHPTAC as a cross-linking agent and cationic reagent, respectively, in the same way as the above methods. The degree of substitution of the synthesized CS was 0.39 (1.76 mmol/g of the cationic group content), calculated from the nitrogen content. The modified starch/PVA composite sample (denoted as CCSP1) was prepared based on the preparation procedures of CCP, by replacing only the CP with a mixture of CS and CP (CS/CP with a mass ratio of 2:1). The same treatments were repeated in order to obtain the samples of CCSP2 and CCSP3, by changing only the mass ratios of CS/CP from 2:1 to 1:1 and 1:2, respectively. 2.3. Characterization {#sec2dot3-polymers-12-00289} --------------------- The FT-IR spectra of the samples were measured using a Nicolet IS 10 FT-IR spectrophotometer (Nicolet, Madison, Wis., USA). The particle size analyses were carried out using a laser light-scattering-based particle sizer (Rise-2028, Jinan, China), and ethyl alcohol and distilled water were used as the dispersion medium. The sample microtopography was observed using a scanning electron microscope (SEM; JSM-6400, Tokyo, Japan), and the morphology was observed using a transmission electron microscopy (TEM; JEOL JEM-2100F, Tokyo, Japan). The samples were treated with supersonic dispersion before the TEM measurement \[[@B41-polymers-12-00289]\]. The crystalline structure of the samples was examined using powder X-ray diffraction (XRD; DX2700, Dandong, China). The surface charges of the samples were obtained by a Zeta potential analyzer (Malvern-Zetasizer nano ZS, Malvern, UK) under neutral conditions. 2.4. Adsorption Studies {#sec2dot4-polymers-12-00289} ----------------------- The adsorption properties of the samples were investigated using a fixed-bed column (1.2 cm in diameter × 30 cm in height) connected to a spectrophotometer detector. The fixed-bed column was prepared according to the method of Zhang et al. \[[@B42-polymers-12-00289]\]. In view of this, the microsphere samples (CCS, CCP, and the CCSPs) had some of the swelling properties of water absorption, and diatomite or activated carbon were chosen as the filter aid to increase the space velocity of the fixed-bed column. The column was filled with a mixture adsorbent composed of microsphere samples (5 g) and diatomite (5 g; as the filter aid), or a mixture adsorbent composed of microsphere samples (5 g) and activated carbon (5 g), or only 10 g of activated carbon. The microsphere samples, diatomite, and commercial activated carbon were all sieved into a particle size range of 200--300 mesh. The simulated wastewater was continuously fed to the top of the column at a flow rate of 2 mL/min, which was controlled by a peristaltic pump at room temperature. The absorbance readings were taken at set time intervals to analyze the solutions leaving the column. Among these, iodine and potassium iodide solutions and iodine-boric acid solutions were used as color developing agents for solutions containing starch and PVA, respectively. The simulated wastewater used in this study included dye wastewater (0.5 g/L), starch solution (0.1 g/L), PVA solution (0.1 g/L), and simulated textile wastewater (0.5 g/L of Reactive Orange 131, 0.1 g/L of starch, and 0.1 g/L of PVA). The volumes of the completely purified effluent solution were recorded. The adsorption capacities of the adsorbents were calculated based on the volumes of the completely purified effluent. As for the simulated textile wastewater, the adsorption capacity was the sum of the adsorption amount for each component. The effect of the bed height (9.5, 7.6, and 5.7 cm) and flow rate (2, 3, and 4 mL/min) on the column adsorption capacity of the mixture adsorbent (CCSP2 and AC) to treat the textile wastewater was investigated. The effluent solution was analyzed to yield output concentration breakthrough curves. 2.5. The Recycle of Disabled Adsorbent {#sec2dot5-polymers-12-00289} -------------------------------------- After adsorption, the disabled mixture adsorbent was recycled as follows. First, the disabled adsorbent was dried at 110 °C for 12 h under an air atmosphere, and then carbonized at 350 °C for 3 h under an N~2~ atmosphere. Next, the carbon spheres were impregnated with the activating agent KOH \[[@B43-polymers-12-00289]\], and the activation process was carried out at 850 °C for 2 h under an N~2~ atmosphere. The obtained material, signed as regenerated activated carbon (RAC), was washed with diluted HCl and deionized water separately, and finally dried. The schematic representation of the adsorption-recycle process is shown in [Figure 2](#polymers-12-00289-f002){ref-type="fig"}. 3. Results {#sec3-polymers-12-00289} ========== 3.1. FT-IR Analysis {#sec3dot1-polymers-12-00289} ------------------- The FT-IR spectra of samples are shown in [Figure 3](#polymers-12-00289-f003){ref-type="fig"}(1). It can be seen that the main absorption bands for CCSP2, as depicted in curve c, were found at 3396, 2931, 1444, and 1027 cm^−1^. The peak at 3396 cm^−1^ was attributed to the stretching vibration of hydroxyl. The peak at 2931 cm^−1^ was caused by the stretching vibration of C--H. The peak at 1027 cm^−1^ was attributed to the stretching vibration of C--O--C. The peak at 1444 cm^−1^ was assigned to the bending vibration of C--N \[[@B32-polymers-12-00289],[@B44-polymers-12-00289]\]. For the FT-IR spectra of (a) CCS, the peaks at 3404, 2926, and 1018 cm^−1^ were attributed to the stretching vibration of O--H, C--H, and C--O--C, respectively. The peak at 1430 cm^−1^ in curve b (CCP) was similarly assigned to the bending vibration of C--N. The FT-IR spectra of the mixture adsorbent composed of CCSP and AC before and after treating the textile wastewater are shown in [Figure 3](#polymers-12-00289-f003){ref-type="fig"}(2); it can be seen that the characteristic peak of the hydroxyl group (3419 cm^−1^) was enhanced relatively after treating the textile wastewater, and the peak at 1475 cm^−1^ confirmed the existence of azo groups in the dye molecules. 3.2. XRD Analysis {#sec3dot2-polymers-12-00289} ----------------- As shown in [Figure 4](#polymers-12-00289-f004){ref-type="fig"}, it can be clearly seen that corn starch exhibited a typical A-type XRD pattern \[[@B45-polymers-12-00289]\], and strong diffraction bands centered at about 15°, 17°, 18°, and 23° (2*θ* degrees). However, the XRD pattern of CCS had only a dispersed broad peak, and the crystallization peaks of starch disappeared (see curve c), indicating that the modification could inhibit the crystallization of starch molecules \[[@B45-polymers-12-00289]\]. As for PVA, the intensity of its crystallization peak also significantly decreased after cross-linking and cationic modification. The XRD pattern of CCSP2 was similar to that of CCP. 3.3. SEM and TEM Analysis {#sec3dot3-polymers-12-00289} ------------------------- From the SEM images ([Figure 5](#polymers-12-00289-f005){ref-type="fig"}a--e), the particle sizes of CCS were larger than those of the other four samples. The morphology of most of the CCP was shaped with a good sphericity, although some particles congregated together. It can be obviously seen that the particle sizes of the CCSPs were all larger than that of CCP, but smaller than that of CCS, and gradually increased with the dosage increase of cationic starch in its preparation. This result was in good agreement with the analysis of the TEM images (see [Figure 5](#polymers-12-00289-f005){ref-type="fig"}f--h). 3.4. Particle Size Analysis {#sec3dot4-polymers-12-00289} --------------------------- The particle size distributions of CCS, CCP, and the CCSPs are shown in [Figure 6](#polymers-12-00289-f006){ref-type="fig"}. From [Figure 6](#polymers-12-00289-f006){ref-type="fig"}, no matter whether using ethyl alcohol or distilled water as the dispersion medium, the average diameter of CCS was the largest, while the average diameter of CCP was the smallest out of the five samples. This result was in good agreement with the morphology analyses of the samples (see [Figure 5](#polymers-12-00289-f005){ref-type="fig"}). The average diameters of the five kinds of microsphere samples all increased obviously when the dispersion medium was changed from ethyl alcohol to distilled water, indicating that CCS, CCP, and the CCSPs all had some of the swelling properties of water absorption. This result meant that the microsphere samples should be used together with filter aids (diatomite or activated carbon) in the column adsorption process. 3.5. Application in Wastewater Treatment {#sec3dot5-polymers-12-00289} ---------------------------------------- The adsorption properties of the samples were investigated using the fixed-bed column. Previous research has shown that the cationic group built-in CCS molecule could interact with dye molecules in wastewater via charge neutralization \[[@B46-polymers-12-00289]\]. The results of the fixed-bed column adsorption experiments are shown in [Table 1](#polymers-12-00289-t001){ref-type="table"}. Among these, the adsorption capacities of CCS, CCP, the CCSPs were calculated by subtracting the adsorption capacity of diatomite from that of the corresponding mixture adsorbents. From [Table 1](#polymers-12-00289-t001){ref-type="table"}, CCS, CCP, and the CCSPs all had a much larger adsorption capacity than that of AC when treating simulated dye wastewater, and CCP had the largest dye adsorption capacity, owing to its highest cationic group content. Meanwhile, they all had a smaller adsorption capacity than that of AC when treating a starch or PVA solution. CCS had a better adsorption capacity than that of CCP and the CCSPs when treating a starch solution, but CCP had a better adsorption capacity than that of CCS and CCSPs when treating a PVA solution. All of the CCSPs had a larger adsorption capacity than that of CCS, and had a smaller adsorption capacity than that of CCP when treating the simulated dye wastewater and PVA solution. Inversely, all of the CCSPs had a smaller adsorption capacity than that of CCS, and had a larger adsorption capacity than that of CCP when treating a starch solution. The composition of the CCSPs had a great influence on their adsorption capacity. The adsorption capacities of the CCSPs for Reactive Black 5, Reactive Orange 131, and PVA all gradually increased with the dosage increase of cationic PVA in their preparation, but decreased for starch. These results indicated that the adsorption capacities of the CCSPs could be developed based on the respective advantages of CCS and CCP when they were used to treat practical textile wastewater. Compared with CCS and CCP, the adsorption capacity of the CCSPs increased significantly when treating simulated textile wastewater. The adsorption capacities of the CCSPs were over 10 times larger than that of the commercial activated carbon (AC) when treating dye wastewater. According the zeta potential analysis (see [Table 2](#polymers-12-00289-t002){ref-type="table"}), the surfaces of the CCSPs all possessed a high positive charge, whereas the dye molecules had negative charges in the solutions. This means an electrostatic interaction between the cationic group built-in CCSP and dye molecules there should exist \[[@B46-polymers-12-00289]\]. In addition, CCSP had quite a similar molecular structure, with both the starch and PVA molecules, indicating that a hydrogen bonding mechanism might also exist. Therefore, the electrostatic interaction and hydrogen-binding interaction might be the main adsorption mechanisms of CCSP for treating textile wastewater. It is worthwhile to note that the mixture adsorbent composed of CCSP2 and AC (1:1 in weight) had the largest adsorption capacity compared with the other adsorbents when treating the simulated textile wastewater, which indicated that the combination of AC and CCSP2 could show an obvious synergetic effect on each other for adsorption, and that the starch, PVA, and dyes could be removed effectively from the simulated textile wastewater simultaneously. The effect of the bed height and flow rate on the adsorption capacity of the mixture adsorbent (CCSP2 and AC) to treat the textile wastewater was investigated, and the results are shown in [Table 3](#polymers-12-00289-t003){ref-type="table"}. It can be found that the column adsorption performance of the mixture adsorbent could be enhanced at a higher bed height and lower flow rate. The reason for this could be that the residence time of the adsorbate molecules in fixed-bed columns could be longer at the higher bed height and at the lower flow rate, and more adsorbate molecules would establish the adsorption equilibrium in the column \[[@B18-polymers-12-00289],[@B25-polymers-12-00289]\]. As shown in [Figure 7](#polymers-12-00289-f007){ref-type="fig"}, the breakthrough curves of the dye, starch, and PVA were diverse from each other, and the breakthrough point, defined as the point at which the pollutant concentration (any one of starch, or PVA, or dyes) of the effluent reached to above zero, of the starch first appeared, while at this point, the mixture adsorbent still had a high adsorption capacity to the dye and PVA. This result indicated that it could obtain the higher adsorption ability for treating textile wastewater by adjusting the composition of the mixture adsorbent based on the breakthrough curves of the adsorbates. 3.6. The Recycle of Disabled Adsorbent {#sec3dot6-polymers-12-00289} -------------------------------------- After treating the simulated textile wastewater, the particle surfaces of the mixture adsorbent composed of CCSP and AC would gather a large amount of starch, PVA, and dyes. It was difficult to be regenerated by the conventional desorption methods. In order to avoid producing solid waste, a chemical activation process was carried out to convert these disabled adsorbents into regenerated activated carbon. The SEM images of the original activated carbon (AC) and the regenerated activated carbon (RAC) that came from the disabled mixture adsorbent composed of CCSP2 and AC (mass ratio of 1:1) are shown in [Figure 8](#polymers-12-00289-f008){ref-type="fig"}. As can be seen from [Figure 8](#polymers-12-00289-f008){ref-type="fig"}, significant differences in the morphology of the RAC and AC were observed. The average particle size of the RAC was obviously smaller than that of the AC, and some honeycomb-shaped porous carbons were developed after carbonization. The Brunauer-Emmett-Teller (BET) data (see [Table 4](#polymers-12-00289-t004){ref-type="table"}) showed that the RAC had a larger surface area and pore volume than that of AC. This result indicated that the starch and PVA in CCSP2 should be converted into biomass carbon and porous carbon after carbonization \[[@B47-polymers-12-00289],[@B48-polymers-12-00289]\]. It is worth mentioning that the adsorption capacity of RAC on the simulated textile wastewater was higher than that of the original activated carbon, no matter whether used alone or in combination with CCSP2 (see [Figure 9](#polymers-12-00289-f009){ref-type="fig"}). This result indicated that the mixture adsorbent composed of CCSP2 and AC exhibited the advantages of a high adsorption capacity, recycling ability, and no solid waste when it was used as the adsorbent of the fixed-bed to treat the simulated textile wastewater. 4. Conclusions {#sec4-polymers-12-00289} ============== A novel modified starch/polyvinyl alcohol composite (CCSP) was prepared and employed as a highly efficient adsorbent for textile wastewater treatment. The CCSP was shaped with sphericity, and had some of the swelling properties of water absorption and a lower crystallinity. The dye adsorption capacity of CCSP was much larger than that of AC. The mixture adsorbent composed of CCSP and AC could make full use of the respective advantages of its components, and could remove starch, PVA, and dyes from the textile wastewater simultaneously, combined with the fixed-bed adsorption technique. The fixed-bed column adsorption process could be influenced by the bed height and flow rate. After adsorption, the disabled mixture adsorbent was converted into regenerated activated carbon for sustainable use by the chemical activation process, and the regenerated activated carbon had a higher adsorption capacity to the textile wastewater than AC. The developed method, through fixed-bed adsorption using CCSP, would be an excellent green textile wastewater treatment, on account of the high adsorption capacity, recycling ability, and no solid waste. Methodology, W.W. and X.Y.; formal analysis, X.L.; investigation, K.X.; data curation, K.X., W.W., and X.Y.; writing (original draft preparation), K.X.; writing (review and editing), X.Z. All authors have read and agreed to the published version of the manuscript. This research was financially supported by the Major Science and Technology Innovation Project of Shandong (no. 2019JZZY010507), the National Natural Science Foundation of China (no. 21808115), and the China Postdoctoral Science Foundation (no. 2019T120571 and no. 2018M632623). The authors declare no conflict of interest. {#polymers-12-00289-f001} {#polymers-12-00289-f002} {#polymers-12-00289-f003} {#polymers-12-00289-f004} {#polymers-12-00289-f005} {#polymers-12-00289-f006} {#polymers-12-00289-f007} {#polymers-12-00289-f008} {#polymers-12-00289-f009} polymers-12-00289-t001_Table 1 ###### The results of the fixed-bed column adsorption experiments. Simulated Wastewater Solutions The Adsorption Capacity (mg/g) -------------------------------- -------------------------------- ------- ------- ------- ------- ------ ------- Reactive Black 5 572.0 693.5 605.0 629.5 661.0 54.0 378.0 Reactive Orange 131 494.0 605.5 539.0 560.0 573.5 48.0 340.5 Starch solution 12.4 9.3 11.4 10.6 9.9 16.2 14.4 PVA solution 7.7 16.2 10.1 13.4 15.1 19.6 17.7 Textile wastewater 40.6 52.5 68.6 73.5 60.9 56.7 93.1 polymers-12-00289-t002_Table 2 ###### The zeta potential of the prepared microspheres. Adsorbents Zeta Potential (mV) ------------ --------------------- CCS 20.3 CCP 30.4 CCSP1 22.7 CCSP2 26.2 CCSP3 28.1 polymers-12-00289-t003_Table 3 ###### Effect of bed height and flow rate on the column adsorption capacity of the mixture adsorbent (CCSP2 and AC) to treat the textile wastewater. Bed Height (cm) Flow Rate (mL/min) The Adsorption Capacity (mg/g) ----------------- -------------------- -------------------------------- 9.5 (10g) 2 93.1 7.6 (8g) 2 88.9 5.7 (6g) 2 80.5 9.5 (10g) 3 86.8 9.5 (10g) 4 77.0 polymers-12-00289-t004_Table 4 ###### Characterization of the regenerated activated carbon (RAC) and AC. Characterization BET Surface Area (m^2^/g) Pore Volume (cm^3^/g) Average Pore Diameter (nm) ------------------ --------------------------- ----------------------- ---------------------------- AC 386 0.39 4.12 RAC 581 0.53 3.13

Background {#Sec1} ========== Dumbo, the famous Disney cartoon character, is an elephant with oversized ears that enable it to fly. Some real-life animals with abnormal external ears are also named "dumbo" such as the *dumbo* mouse and *dumbo* rat \[[@CR1], [@CR2]\]. Almost all mammals have outer ears (pinna) of variable sizes. The main function of the pinna is to collect sound waves and direct them into the ear. In some species, pinna also serve functions such as dissipating heat and signaling mood. In humans, different congenital pinna malformations are observed. Microtia (OMIM 600674) is an external ear developmental malformation characterized by a small, abnormally shaped pinna \[[@CR3]\]. It ranges from mild structural abnormalities to complete absence of the ear, affects one or both ears, and occurs as isolated or syndromic birth defects. The concha-type microtia is considered a mild form of microtia, with remnant ear lobule, concha, acoustic meatus, tragus, and incisura intertragica \[[@CR4]\]. Because of the variety of severity and forms, it is hard to estimate microtia prevalence---reported data varies from 0.83 to 17.4 in 10,000 live births worldwide \[[@CR3]\]. The causes of microtia among most patients are unknown, although some risk factors have been reported, such as gestational exposure to teratogens, maternal diabetes, and higher maternal parity \[[@CR3]\]. Genetic studies have made great progress in understanding ear development and function by identifying underlying genetic defects of certain diseases, especially in hearing loss, but less is known about genetic control of external ear morphogenesis. To date, only *HOXA2* mutations have been reported as responsible for isolated bilateral microtia with or without hearing loss in humans \[[@CR5]--[@CR7]\]. Single-gene defects and chromosomal aberrations have also been reported in different microtia-associated syndromes \[[@CR3], [@CR8]\]. Nevertheless, efforts in finding coding region mutations in genes responsible for microtia-associated syndromes have failed for isolated microtia. Among these, the H6 family homeobox 1 transcription factor gene (*HMX1*) in 4p16.1 deserves special attention. It plays an important role downstream of embryonic patterning genes in lateral facial mesenchyme differentiation \[[@CR1]\]. In human, recessive loss of function mutations in *HMX1* have been associated with oculoauricular syndrome (OAS, OMIM 142992) characterized by malformation of the external ear and eyes \[[@CR9], [@CR10]\]. A linkage locus of 10-Mb encompassing 4p16 has been reported in a five-generation Chinese family with isolated bilateral microtia \[[@CR11]\]. In the human genome, 98% of sequences are non-coding but harbor many regulatory elements that direct the precise spatial and temporal expression of coding genes. Comparing genomic sequences from diverse vertebrate species has revealed numerous highly conserved non-coding regions near developmental regulatory genes, particularly transcription factors and these regions are considered to have potential regulatory functions \[[@CR12]\]. These functional regions are collectively referred to conserved non-coding elements (CNEs). The general features of CNEs were noticed, including their non-random distribution in line with key developmental regulatory target genes across genomes, the distinguished sequence features with AT-rich and runs of identical nucleotides, the overlapping with transcription factor binding sites and known function as developmental enhancers in many cases \[[@CR12]\].Human diseases and phenotypic changes have been associated with alterations in CNEs \[[@CR13]--[@CR18]\]. One of the well-characterized example is the *SHH* ZRS enhancer, in which point mutations and copy number variations could result in limb malformation in both human and other species \[[@CR19]--[@CR21]\]. In wild populations of animals, a CNE proximal to the Hmx1 was also noticed and proved to be associated with external ear development \[[@CR22]\].Structural variants (SVs) such as deletions, duplications, insertions and inversions can disrupt or rearrange functional genomic elements \[[@CR23], [@CR24]\]. The genetic etiology of many diseases such as limb malformation and autism has been proven to relate to rare inherited SVs in coding gene cis-regulatory elements \[[@CR25]--[@CR27]\]. For ear development, evidence in mice implicated an evolutionarily conserved enhancer region (ECR) downstream of *Hmx1* as an important regulatory element driving ear development. Hoxa2, Meis and Pbx can act cooperatively on a 32 bp core sequence within the ECR to regulate *Hmx1* expression \[[@CR28]\]. Mutations in *Hmx1* coding region and SVs involving the *Hmx1*-ECR region have been found in animals with dysmorphic external ears, including 'dumbo' or 'misplaced ears' in mice, 'dumbo' in rats, 'crop ear' in highland cattle, and 'short ear' in Altay sheep (Table [1](#Tab1){ref-type="table"}) \[[@CR1], [@CR2], [@CR10], [@CR29], [@CR30]\]. In human genome, \~ 600 bp conserved sequence homologous to mouse *Hmx1*-ECR was also observed. However, whether genetic changes affecting this region are associated with human ear malformations is unknown.Table 1Genomic changes and dysmorphic outer ear phenotypes across speciesPhenotype descriptionPhenotype/disease entrySpeciesGenomic changesInheritanceReferencesEnlarged ear pinnae with a distinctive ventrolateral shift, microphthalmic anomaliesDumbo (*dmbo*)MouseNonsense mutation in *Hmx1* exon1RecessiveMunroe et al. \[[@CR1]\]Laterally-protruding ears and microphthalmic anomaliesMisplaced ears (*mpe*)Mouse8 bp deletion in *Hmx1* exon2RecessiveMunroe et al. \[[@CR1]\]Congenital malformations of the pinna and modest reduction in ocular sizeDumbo (dmbo)Rat5777 bp deletion encompassing Hmx1-ECRRecessiveQuina et al. \[[@CR2]\]Moderately to severely truncated earCrop earHighland cattle76 bp Hmx1-ECR duplicationDominantKoch et al. \[[@CR29]\]Shorter and thicker earShort earAltay sheep76 bp Hmx1-ECR duplicationDominantHe et al. \[[@CR30]\]Ophthalmic anomalies and external ear abnormalitiesOculoauricular syndrome (OAS)Human26 bp deletion in HMX1 coding regionRecessiveSchorder et al. \[[@CR10]\]Bilateral external ear malformation/cup earConcha type microtiaHumanDuplications involving HMX1-ECRDominantThis study In the present study, we show that duplications involving *HMX1*-ECR are associated with human isolated bilateral concha-type microtia. A \~ 600 bp human ECR sequence may function as a tissue-specific enhancer regulating *HMX1* expression and response to *HOXA2* in the lateral facial mesenchyme that contributes to outer ear development. Materials and methods {#Sec2} ===================== Subjects {#Sec3} -------- Five Han Chinese families with isolated bilateral microtia were included in the present study (Fig. [1](#Fig1){ref-type="fig"}). All patients were clinically evaluated, and digital photographs were taken to document ear phenotypes in affected individuals. Family 1 (F1) consisted of 25 individuals including 10 affected individuals with microtia in four generations. Family 2 (F2) and Family 3 (F3) each had six affected individuals in four generations. Family 4 (F4) and Family 5 (F5) are nuclear families with an affected child and affected mother. The ear malformations are consistent within the five families (Fig. [1](#Fig1){ref-type="fig"}b--q). We also recruited 53 patients with oculoauricular syndrome, six patients with severe bilateral isolated microtia and two patients with bilateral syndromic microtia. Blood samples from all available family members were collected following informed consent. The study was reviewed and approved by the institutional review board of the Chinese Academy of Medical Sciences.Fig. 1Five families with isolated bilateral Concha-Type Microtia. **a** Pedigree of five families (F1-F5). Individuals with available blood samples are indicated with an asterisk. **b**--**q** Identical pinna phenotypes in five families. All patients have identical bilateral concha-type microtia phenotype, and representative individuals from each family are shown. IV-4 (**b**), III-8 (**c**), IV-6 (**d**), IV-5 (**e**) in F1; III-4 (**f**, **g**), III-6 (**h**), IV-7 (**i**) in F2; IV-1 (**j**, **k**), III-1 (**l**), IV-2 (**m**) in F3; II-1 in F4 (**n**, **o**), II-1 in F5 (**p**, **q**) Genotyping, whole genome linkage and haplotype analysis {#Sec4} ------------------------------------------------------- Affymetrix Genome-Wide Human SNP array 5.0 was used to perform whole genome linkage analysis in the four-generation F1 family. Genomic DNA samples from 10 individuals were genotyped following the manufacturer's instructions. Genotype calling and quality control were performed with the Affymetrix Genotyping Console 2.1 package. Parametric multipoint linkage analysis was performed using MERLIN v.1.1.2 under the assumptions of autosomal-dominant inheritance with 99% penetrance, a disease allele frequency of 0.1%, and equal SNP allele frequency (50%). Genotyping and data analysis were accomplished at the CapitalBio Corporation (Beijing, China). Selected polymorphic micro-satellite markers within candidate disease loci were genotyped. Polymorphic micro-satellite markers and amplification primers are summarized in Additional file [1](#MOESM1){ref-type="media"}: Table S1. Whole genome sequencing {#Sec5} ----------------------- Ten individuals from F1 (III2, III3, III4, III5, III8, III9, IV1, IV4, IV6, IV7) underwent whole genome sequencing (WGS) using the NEBNext Ultra II DNA Library Prep kit for Illumina (New England Biolabs, Ipswich, MA, USA) and a HiSeq X Ten sequencer (Illumina, San Diego, CA, USA). Reads were aligned to the GRCh37/hg19 human reference sequence using the Burrows-Wheeler Aligner (BWA, v.0.7.8-r455) and variant calling was performed with SAMtools (v.1.0) and annotated using ANNOVAR (v.2015Dec14). Picard (v.1.111) was used to merge BAM files of the same sample and filter out duplicate reads marked. SNP/Indel, CNV, and SV variants were called and classified by SAMtools (v.1.0), Control-FREEC (v.V7.0), and CREST (v.V0.0.1), respectively. WGS was performed and bioinformatic analysis accomplished at the Novogene Corporation (Beijing, China). Microarray analysis {#Sec6} ------------------- Genomic copy number changes at locus 4p16.1 in F2-5 were further tested by the Laboratory of Clinical Genetics of Peking Union Medical College Hospital using a High-Resolution Array CGH analysis (SurePrint G3 Human 1x1M; Agilent Technologies, Santa Clara, CA, USA). One patient from each family was selected to undergo microarray analysis. The experiment and data analysis were performed according to the manufacturer's instructions. In brief, patient and control DNA were labeled and combined to hybridize to the 60mer oligonucleotide-based microarray. The resulting fluorescent signals were automatically scanned by the Agilent SureScan Microarray Scanner. Agilent CytoGenomics software was then used to extract and translate the signal into log ratios for further analysis of copy number changes. Real-time quantitative PCR (qPCR) and Gap-PCR {#Sec7} --------------------------------------------- We performed qPCR to confirm the *HMX1*-ECR duplication and determine the extent of duplications in different families. qPCR primer sequences and amplicon positions are given in Additional file [2](#MOESM2){ref-type="media"}: Table S2. qPCR assays were performed using SYBR premix Ex Taq (TaKara Bio., Dalian, China), and reactions were run in a Rotor-gene 6000 real-time rotary analyzer (Qiagen, Hilden, Germany) as previously reported \[[@CR19]\]. Data were analyzed by Rotor Gene Q series software (Qiagen, Hilden, Germany). The relative copy number (RCN) of the target sequence was determined by the comparative ΔΔCt method where ΔCt = (mean Ct~Target~) − (mean Ct~Reference~) and ΔΔCt = ΔCt~patient~ − ΔCt~control~. An RCN of \~ 1.5 indicated a heterozygous duplication. For F1 and F2, Gap-PCR was designed according to the extent of duplication implicated by qPCR assays. q20 forward and q3 reverse primers were used for Gap-PCR in F1, while q35 forward and q8 reverse primers were used for Gap-PCR in F2 (Additional file [2](#MOESM2){ref-type="media"}: Table S2). Breakpoint junctions were detected by direct Sanger sequencing of Gap-PCR products. Dual-luciferase activity assay {#Sec8} ------------------------------ hECR and mECR fragments were PCR amplified from genomic DNA and inserted into the pGL4.23 firefly luciferase vector (Promega, Madison, WI, USA) using either a restriction digest strategy or the In-Fusion cloning kit (TaKaRa Bio, Beijing, China). The human HOXA2 cDNA sequence was inserted into the multiple cloning site of the pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) using *HindIII* and *BamHI*. All plasmids were sequenced to confirm correct fragment insertion. Primers for plasmid construction are summarized in Additional file [3](#MOESM3){ref-type="media"}: Table S3. COS-1 cells were plated into 24-well plates 1 day before transfection and grown until 70--90% confluent. For each well, 500 ng luciferase reporter vector was transfected into the cells using Lipofectamin™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA) with or without the pcDNA3.1 expression vector, with 25 ng of the pRL-TK Renilla luciferase vector used as an internal control to normalize transfection efficiency. 24 h post-transfection, cells were harvested and lysed with 100 μl passive lysis buffer (Promega, Madison, WI, USA). The firefly and renilla luciferase activities for each 20 μl cell lysate were measured by the Microplate Luminometer Centro LB 960 (Berthold, Germany). Relative luciferase activity was calculated by the ratio of firefly luciferase activities/renilla luciferase activities as fold change compared to pGL4.23. Assays were conducted as indicated in the dual luciferase reporter assay system manual (Promega, Madison, WI, USA). Normalized luciferase activity fold change (mean ± SD) of three experiments with six duplicates each is reported. Results {#Sec9} ======= Mapping of a susceptibility locus on 4p16.1 {#Sec10} ------------------------------------------- Genome-wide linkage analysis in F1 suggested three candidate loci: a 20 Mb interval on 4p16.1, a 25 Mb interval on 4q, and a 2 Mb interval on 5q. Genotyping of selected polymorphic microsatellite markers within candidate regions of 4q and 5q showed no co-segregation status in F1. However, one polymorphic microsatellite marker (CHLC.GATA151E03) on 4p16.1 co-segregated with phenotype in F1. Fine mapping using Affymetrix SNP 5.0 microarray probe-sets refined the critical region to 1.9 Mb between rs4696668 to rs16891285 (chr4:8061832--9954880, hg19) with a HLOD score of 1.8. The interval includes 13 protein-coding genes including *HMX1*, yet we found no potential coding region mutations in these genes by sanger sequencing. Identification of the *HMX1*-ECR duplication in five families with isolated bilateral concha-type microtia {#Sec11} ---------------------------------------------------------------------------------------------------------- We further performed WGS in 10 members of F1. Consistent with the previous sanger sequencing result, no potential mutations were identified in the coding region. However, WGS implicated a \~ 95 Kb duplication in the critical interval in six patients, but not in two unaffected members or two unrelated members in the family (Fig. [2](#Fig2){ref-type="fig"}a). The duplication encompasses a partial intragenic region between *CPZ* and *HMX1*, and involves the \~ 600 bp evolutionarily conserved region downstream of *HMX1* (*HMX1*-ECR). qPCR assays designed within a 600 bp critical region confirmed the duplication and detected full-segregation status in F1 (Fig. [2](#Fig2){ref-type="fig"}c). This finding prompted us to detect copy number changes in other families with isolated bilateral concha-type microtia. There are limited probes within the identified duplicated region designed in commercial array CGH systems, decreasing our accuracy and efficiency in CNV detection. Nevertheless, SurePrint G3 Human 1x1M microarray implicated increased copy number in a 46.2 Kb intergenic region (chr4: 8677567--8723767, hg19) between *CPZ* and *HMX1* in four additional families with the identical phenotype same as F1 (Fig. [2](#Fig2){ref-type="fig"}b). Duplications were confirmed by qPCR assay in the *HMX1*-ECR region in all four additional families (Fig. [2](#Fig2){ref-type="fig"}c).Fig. 2Detected duplications involving the long range *HMX1* Enhancer in five families. **a** Whole genome sequencing indicated duplications in F1. Red bar shows the duplicated region. **b** Duplications detected by array-CGH in F2-F5. Blue arrows show where the probes detected 3 copies. **c** qPCR assays in the *HMX1*-ECR region confirm the duplication and co-segregation status with phenotype in five families Determination of the duplication extent and critical region {#Sec12} ----------------------------------------------------------- To determine the size of the duplications in different families, multiple qPCR assays were designed to cover a region of 253 Kb (chr4:8617326--8871246, hg19) encompassing *CPZ*, *HMX1*, and their intergenic region (Additional file [2](#MOESM2){ref-type="media"}: Table S2). Extent and overlapping regions of duplications in five families were detected (Fig. [3](#Fig3){ref-type="fig"}). We performed qPCR assays on one affected individual per family to determine the extent of duplication in each family and identified duplications of 94.6 Kb, 147 Kb, 185--213 Kb, 49.8--55.9 Kb, and 67.4--104 Kb in F1, F2, F3, F4 and F5, respectively (Fig. [3](#Fig3){ref-type="fig"}a). We detected the precise duplicated segment and breakpoints by gap-PCR and sanger sequencing in F1 (chr4:8638135--8732725, hg19) and F2 (chr4: 8677560--8,824,629, hg19) (Fig. [3](#Fig3){ref-type="fig"}b). In F3, F4 and F5, multiple qPCR assays detected the boundary regions harboring the breakpoints (Fig. [3](#Fig3){ref-type="fig"}c). All identified duplications contained a 21.8 Kb overlapping region (chr4:8,684,896--8,706,719, hg19) harboring the *HMX1*-ECR.Fig. 3Extent and overlapping regions of duplications in five families. A. Schematic diagram showing detected duplications, 600 bp core hECR sequences, and CNVs in the database of genomic variants. The blue line highlights the ECR region. B. Chromatogram of the breakpoint junctions in F1 and F2. C. A series of qPCR assays detected the extent of duplications in F3, F4 and F5 A 600 bp sequence within the duplicated region shows enhancer activity increased by HOXA2 {#Sec13} ----------------------------------------------------------------------------------------- A 594 bp *Hmx1*-ECR region has been demonstrated to be a specific enhancer determining endogenous Hmx1 lateral facial expression patterns in mouse \[[@CR28]\]. Thus, a 600 bp human sequence (hECR) in the identified duplicated region homologous to the 594 bp mouse sequence (mECR) was tested for enhancer function by dual luciferase assay (Fig. [4](#Fig4){ref-type="fig"}). As a result, constructs containing hECR showed increased luciferase activity compared to the empty group (replicate = 3, *p *\< 0.0001), suggesting hECR enhancer activity (Fig. [4](#Fig4){ref-type="fig"}a). However, the induced luciferase activity was significantly lower in the hECR than in the mECR group, which is regulated by the Hox-Pbx-Meis complex (Fig. [4](#Fig4){ref-type="fig"}a). *HOXA2* mutations were reported in patients with isolated bilateral microtia without hearing loss. In the luciferase assay, co-transfection with human *HOXA2* expression vectors led to an 8.14-fold increase in enhancer activation, indicating that the hECR is responsive to *HOXA2* (Fig. [4](#Fig4){ref-type="fig"}b).Fig. 4Human ECR within the duplicated region shows an enhancer activity increased by HOXA2. **a** hECR showed increased luciferase activity. **b** hECR is responsive to *HOXA2. \*\*\*\*p *\< *0.0001* Detection of *HMX1*-ECR CNVs in patients with other types of microtia {#Sec14} --------------------------------------------------------------------- To determine whether *HMX1*-ECR CNVs associate with other ear malformations, we performed qPCR assays in the 600 bp *HMX1*-ECR in 53 patients with unilateral lobule-type microtia, six patients with isolated bilateral lobule-type microtia, one patient with bilateral concha-type microtia with preauricular sinus, and one patient with bilateral concha-type microtia with atrial septal defect. No duplications or deletions were detected in these microtia cases. In health population, some duplications involving the *HMX1*-ECR region were documented in the database of genomic variants (DGV) \[[@CR31]\], but they were relatively large in size and involved other nearby genes (at least *CPZ*) at the same time. There were also some duplications only involving the *CPZ* and *HMX1* intergenic region, but they did not contain the *HMX1*-ECR. While deletions only involving the intragenic region between *CPZ* and *HMX1*, and containing *HMX1*-ECR were also documented in the DGV database (Fig. [3](#Fig3){ref-type="fig"}a). Discussion {#Sec15} ========== Microtia is phenotypically and etiologically heterogeneous. Little is known about the genetic background underlying microtia. Among candidate loci for microtia, Chromosome 4p16 deserves special attention. A partial deletion from the short arm of chromosome 4 (4p deletion) results in Wolf-Hirschhorn syndrome (WHS, OMIM\#194190) featuring a distinct craniofacial phenotype and intellectual disability \[[@CR32]\]. WHS patients with pure and translocated forms of monosomy 4p16.1 → pter (M4p16.1) have different types of external ear malformation such as poorly rolled descending helix edge, short ear lobes, or deep or long concha \[[@CR33]\]. By studying 72 oculoauriculovertebral spectrum (OAVS) patients with highly heterogeneous phenotypes involving ears, eyes, face, neck and other organs, Bragagnolo et al. observed recurrent chromosomal imbalances predominantly in chromosome 4 in four patients \[[@CR34]\]. Balikova et al. reported on a large family with autosomal-dominantly inherited microtia, eye coloboma, and imperforation of the nasolacrimal duct, and found the phenotype linked to a cytogenetically visible alteration at 4p16 consisting of five copies of a copy-number-variable region \[[@CR35]\]. Li et al. reported a 10 Mb susceptibility locus for isolated bilateral microtia on 4p15.32--4p16.2 in a 5-generation Chinese family \[[@CR11]\]. *HMX1* harbored in 4p16.1, also known as *NKX5*-*3,* is an important transcription factor in craniofacial structure development, especially in eye and ear. Expression of *Hmx1* was observed in the external ear, lens, and retina of mice as early as E13.5. In humans, *HMX1* expression was observed in the optic vesicle in the 5--6-week embryonic period and in the developing pinna and auricular mesenchymatous cells at the 20-week fetus period \[[@CR10]\]. The different expression patterns of *HMX1* in ear and eye development suggest that there may be different regulatory elements determining strict spatial--temporal expression. Meanwhile, homozygous mutation in the human *HMX1* gene leads to abrogation of gene function causing oculoauricular syndrome (OAS, OMIM \#612109) affecting both the eye and external ear \[[@CR9], [@CR10], [@CR36]\]. Thus, isolated microtia and syndromic microtia without eye affects are unlikely to be caused by mutations in the *HMX1* coding region. Accordingly, we found no potential *HMX1* coding region mutations in 120 OAVS patients by whole exome sequencing (unpublished data). Conserved non-coding elements (CNEs) are sequences outside of protein coding regions highly conserved across diverse vertebrate species \[[@CR37]\]. They may act as cis-regulatory modules (CRMs) that interact with nearby genes to determine tissue-specific gene expression, and they are enriched near transcription factor genes expressed during embryogenesis, suggesting a possible role in regulating the expression of essential developmental genes \[[@CR38], [@CR39]\]. CNEs are required for normal development, and mutations in CNEs have been established as causal for human diseases and subtle phenotypic changes that likely lead to decreased fitness over evolutionary time \[[@CR12]\]. Dickel et al. created knock out mice with individual or pairwise deletion of four CNEs near *ARX*, the essential neuronal transcription factor \[[@CR40]\]. These knockout mice showed substantial alterations of neuron populations and structural brain defects that potentially detrimental in the wild, although they were viable and fertile in laboratory conditions. *Rosin* et al. showed that *Hmx1* has such a CNE that functions as a strong and highly dynamic lateral facial enhancer \[[@CR28]\]. The CNE is a \~ 600 bp evolutionarily conserved region (ECR) with a 32 bp core sequence containing consensus binding sites for Hoxa2, Pbx, and Meis, and it has tissue-specific enhancer function in the craniofacial mesenchyme which contributes to the pinna. Genomic structural variations disrupting the ECR enhancer role associate with loss of Hmx1 expression specifically in the first and second branchial arch (BA1 and BA2) mesenchyme, leading to dysmorphic outer ears across species (Table [1](#Tab1){ref-type="table"}). Genomic findings in human patients with isolated bilateral concha-type microtia reinforce the enhancer role of *HMX1*-ECR in conserved pinna developmental processes. We also noticed that hECR has weaker enhancer activity compared to mECR via luciferase assay. However, it remains unclear whether the difference in the relative size of the pinna between human and mice is related to the level of enhancer activity. The core sequence of hECR is highly homologous to mECR including the consensus binding sites of HOXA2, PBX and MEIS \[[@CR22], [@CR28]\]. In dual luciferase assays, co-transfection of HOXA2 and hECR resulted in increased expression level, suggesting that the hECR may also be regulated by the HOX-PBX-MEIS complex. HOX, PBX and MEIS are all homeobox proteins involved in transcriptional regulation by forming heterodimers and are essential contributors to developmental programs. Genes encoding this homeoprotein complex associate with congenital anomalies with craniofacial phenotypes. *HOXA2* is the only reported gene responsible for isolated microtia to date. Patients with homozygous mutations in HOXA2 display more severe microtia than hECR duplicated carriers, presenting middle ear deformities and hearing loss \[[@CR7]\]. *PBX1* mutations lead to congenital kidney and urinary tract anomalies with or without hearing loss, abnormal ears, or developmental delay \[[@CR41]\]. MEIS2 mutations associate with cleft palate, cardiac defects, and mental retardation \[[@CR42], [@CR43]\]. These findings suggest that *HOXA2, PBX1,* and *MEIS2* act early in patterning of the branchial arch region and transactivate *HMX1* by binding to hECR. Therefore, we speculate that any genetic changes affecting hECR regulation by the HOX-PBX-MEIS complex may lead to developmental defects involving ears and eyes. Regulatory elements and their target gene clusters often exist in the same local chromatin interaction regions, called topologically associated domains (TADs), to ensure that the regulatory elements are specific to their target genes rather than other nearby genes \[[@CR44]\]. Boundaries between TADs are required and provide an insulator function to prohibit interference between opposing activities of neighboring domains \[[@CR44], [@CR45]\]. We used the 3D Genome Browser (<http://promoter.bx.psu.edu/hi-c/>) to visualize the chromatin interaction surrounding *HMX1*. According to Hi-C profile data from human embryonic stem cells, *HMX1* and *CPZ* are in two different TADs, while *hECR* sequences appear in the same TAD with *HMX1* but not with *CPZ* (Additional file [4](#MOESM4){ref-type="media"}: Figure S1). The detected duplications in isolated bilateral concha-type microtia patients are in the same TAD with *HMX1,* and do not interrupt the TAD boundary. They contain hECR but not the HMX1 gene. Therefore, these duplications may result in overexpression of *HMX1* by increasing the number of local enhancers but not the coding gene. Meanwhile, the copy number variation (nsv1014219) in the DGV database detected in normal population involves the hECR, the CPZ gene, and the boundary between two TADs. Therefore, due to the insulator effect of TAD boundaries, the increased hECR could not interact with *HMX1*, thus it probably does not change gene expression level. Notably, the size of the hECR region (600 bp) is small and its copy number changes could be missed by chromosomal microarray analysis (CMA). Duplications detected in the present study range from \~ 50 to \~ 200 Kb. Although they were implicated in the Agilent SurePrint G3 Human CGH 1X1M microarray analysis, they could not be automatically detected in standard analysis process due to limited probes designed within the region. The genomic findings in these patients indicate the importance of checking the *HMX1*-ECR copy number status and highlight the necessity for custom designed microarrays with higher probe density covering this region. Conclusions {#Sec16} =========== In this study, we found various genomic duplications involving the *HMX1*-ECR long range enhancer in five families with isolated bilateral concha-type Microtia. The *HMX1*-ECR duplications were specifically associated with isolated bilateral concha-type microtia but not with other ear malformations or syndromic microtia. We add to evidence in humans that copy number variations in *HMX1*-ECR, a conserved non-coding elements (CNEs), associates with ear malformations, as in other species. We provide additional evidence that the dosage sensitive effects of *HMX1* may result in different types of ear malformations. Unveiling genetic causes of isolated microtia provides an entry point into understanding the regulatory network for common lateral facial birth defects and complex syndromes involving external ear malformations. Meanwhile, the results could be used for genetic counseling and screening for isolated bilateral concha-type microtia. Supplementary information ========================= {#Sec17} **Additional file 1: Table S1.** Polymorphic micro-satellite markers and primers. **Additional file 2: Table S2.** qPCR primers in 4p16.1. **Additional file 3: Table S3.** Primers for plasmid construction. **Additional file 4: Figure S1.** Visualization of chromatin interaction surrounding *HMX1*. CNVs : Copy number variations ECR : Evolutionarily conserved enhancer region HMX1 : The H6 family homeobox 1 transcription factor gene SNP : Single nucleotide polymorphism aCGH : Array comparative genomic hybridization qPCR : Quantitative real-time polymerase chain reaction OAS : Oculoauricular syndrome SVs : Structural variants WGS : Whole genome sequencing hECR : Human sequence of evolutionarily conserved enhancer region mECR : Mouse sequence of evolutionarily conserved enhancer region DGV : Database of genomic variants WHS : Wolf--Hirschhorn syndrome OAVS : Oculoauriculovertebral spectrum CNEs : Conserved non-coding elements CRMs : Cis-regulatory modules BA1 and BA2 : First and second branchial arch TADs : Topologically associated domains CMA : Chromosomal microarray analysis **Publisher\'s Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Nuo Si and Xiaolu Meng contributed equally to this work Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s12967-020-02409-6. We thank all participants in the study. NS, BP and conceived and designed the study. XL, CL, MY, YZ, CW, PG, LZ, LL, ZL, ZZ, ZC, BP and HJ carried out the study of the clinical part. NS, XM, ZL, ZQ, LW and BP performed the genetic analysis. NS, XM and BP wrote the manuscript. NS and BP revised the manuscript. XZ, HJ contributed to supervision. XZ, HJ, BP and ZL contributed to funding acquisition. All authors read and approved the final manuscript. This study was financially supported by the National Key Research and Development Program of China (2016YFC0905100), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-002), the National Natural Science Foundation of China (81571863, 81871574), the Key Laboratory of craniofacial congenital malformation of Chinese Academy of Medical Sciences (2018PT31051). All data generated or analyzed during this study were included in this published article and its additional files. The study was approved by institutional review board of Chinese Academy of Medical Sciences, and all participants signed written informed consent. Not applicable. The authors declare that they have no competing interests.

INTRODUCTION ============ Testosterone replacement therapy (TRT) for the treatment of testosterone deficiency syndrome (TDS) has been widely used in the South Korea for decades.[@B1] The aim of TRT is to establish a physiologically normal concentration of serum testosterone to correct for androgen deficiency, relieve its symptoms, and prevent long-term sequelae. The target organs of testosterone exist throughout the whole body and include the skin, muscle, liver, kidney, brain, bone marrow, and male reproductive organs.[@B2] However, in terms of the prostate, TRT has possible side effects including development of prostate cancer and worsening symptoms of benign prostatic hyperplasia (BPH), because prostate growth is dependent on the presence of androgens, and androgens play an important role in the development of BPH. Nevertheless, there is no direct correlation between serum testosterone levels in men and the risk of developing prostate cancer.[@B3],[@B4] Furthermore, there are no compelling data suggesting that TRT contributes to worsening of lower urinary tract symptoms (LUTS) or promotion of urinary retention.[@B5] Instead, an increasing body of literature with short-term follow up has recently demonstrated positive effects on LUTS and uroflow parameters.[@B6]-[@B9] We therefore evaluated the correlation between TRT and LUTS for patients who had been treated with TRT for over a year, in patients with TDS concomitant with moderate LUTS. To the best of our knowledge, this is the first data published on this issue particularly in a Korean population. MATERIALS AND METHODS ===================== 1. Institutional strategy for TRT --------------------------------- Between January 2006 and January 2011, 383 patients underwent TRT using intramuscular injection of 1,000 mg of testosterone undecanoate (Nebido™, Bayer Pharma AG, Berlin, Germany) in our institution. The institutional policy for initiation of TRT was the complaint of erectile dysfunction as the main symptom of TDS with a serum testosterone level less than 3.5 ng/ml. For all, a complete medical history regarding concomitant medical disease and medication from other departments or institutions was carefully taken and physical examination was performed. To estimate the efficacy of TRT, serum testosterone levels were routinely measured during the first visit, then 3, 6, and 12 months later; the time of blood sampling was 7 AM to 11 AM. The amount of total and free testosterone were measured separately, both by radioimmunoassay. Also, the changes in symptoms induced by TDS were assessed by a self-administered aging male symptoms (AMS) questionnaire, which was performed again routinely a year after TRT. The exclusion criteria for TRT in our institution were a prior history of treated hypogonadism, history of prostate cancer, active systemic disease, human immunodeficiency virus, psychosis, or history of sleep apnea. In case of initial prostate specific antigen (PSA) over 4.0 ng/ml, TRT was selectively performed for the pathologically confirmed BPH cases. 2. The main assessment of prostatic status ------------------------------------------ To investigate the baseline status of the prostate, patients underwent transrectal ultrasonography to determine the prostate volume, serum PSA, uroflowmetery parameters including maximal flow rate, voiding volume, and post-voiding residual urine. The International Prostate Symptom Score (IPSS) questionnaire was performed before TRT. In the IPSS questionnaire, the storage symptom score was defined as the sum of items 2, 4 and 7 and the voiding symptom score was defined as the sum of items 1, 3, 5, and 6. The serum PSA, uroflowmetry, and IPSS were routinely re-evaluated a year after TRT, except for a case of a complaint of aggravation of LUTS that required clinical assessment for the status of the prostate. According to the decision of the treating physician, BPH medications including alpha blockers or phytotherapy were additionally administered after initiation of TRT. However, 5-alpha reductase inhibitor was not used during TRT. At the beginning of this series, we did not have any regular restriction on the usage of LUTS medication during TRT, considering the potential development or aggravation of prostatic disease. 3. Patient selection and statistical analysis --------------------------------------------- Among all potential subjects, 246 patients who had completed a minimum of a year of follow up, with a normal digital rectal examination and PSA level of less than 4 ng/ml, and had undergone a prostate evaluation after a year of TRT were recruited for this study by retrospective chart review. In addition, another group of 17 patients who had moderate LUTS, which was defined as an initial IPSS over 8 points but below 19 with a maximal flow rate of at least 10 ml/s, but who did not take any BPH medication or use of phosphodiesterase type 5 inhibitors daily during testosterone replacement were selected from the 246 cases of TRT. Then the outcomes were assessed separately for the two groups. The primary end point of this study was changes in the IPSS and uroflowmetry parameters including maximal flow rate (Qmax), voiding volume, and post voiding residual urine (PVR) during a year. Secondary efficacy and safety was assessed according to changes in serum testosterone, AMS score, Body Mass Index (BMI), and serum PSA. For statistical analysis of the whole patient, to evaluate the goodness-of-fit, the Kolmogorov-Smirnov test was performed first, then Wilcoxon\'s signed rank test or a paired t-test was performed to compare several parameters before and after TRT. In the 17 selected cases with LUTS without BPH medication, the changes in each parameter were compared by Wilcoxon\'s signed rank test, assuming p\<0.05 to be significant. To compare the no-BPH medication patients and their counterparts, the Mann-Whitney U test was applied. All statistical analyses were performed using SPSS™ version 17.0 (SPSS Inc., Chicago, IL, USA). RESULTS ======= 1. Outcomes from 246 patients with a year follow up --------------------------------------------------- The median age with interquartile range (IQR) of the 246 patients with TRT was 58.5 (52\~64.2) years old and the mean follow up period for TRT was 14.7 (13\~19.3) months. Before TRT, the median (IQR) total and free testosterone levels were 2.51 (1.97\~3.06) ng/ml and 8.12 (6.46\~10.49) ng/ml, respectively. The median baseline prostate volume was 24.2 (19.7\~29.7) ml. After a year of TRT, the AMS score and serum testosterone level including total and free testosterone were significantly increased ([Table 1](#T1){ref-type="table"}). The BMI was also significantly decreased compared to baseline (p\<0.001). In contrast, the IPSS and uroflowmetry parameters did not show any significant change from the baseline status. Interestingly, the serum PSA level remained unchanged after a year of TRT (p=0.078). 2. Outcomes from the 17 patients with moderate LUTS without BPH medication -------------------------------------------------------------------------- The median (IQR) age of the 17 patients was 53 (52\~63) years old, and median follow up period during TRT was 15.1 (14\~16) months. In all of the patients, the total and free testosterone increased after TRT, and this change was statistically significant ([Table 2](#T2){ref-type="table"}). However, the change in AMS score and BMI was not significant in this patient group (p=0.154 and 1.000, respectively). Regarding the prostate status, when comparing with the counterpart group (n=229), these selected 17 patients with moderate LUTS without BPH medication had similar baseline prostate characteristics in all variables including prostate volume, IPSS, Qmax, voiding volume, and serum PSA except PVR, but increased PVR in the no-BPH medication group (21 ml vs. 10 ml, [Table 3](#T3){ref-type="table"}). All of the selected patients had a minimal Qmax over 10 (10.84\~45) ml/s. The total IPSS had decreased significantly in the no-BPH medication group (p=0.028). One patient with a baseline prostate volume of 27.2 ml had reported an increased IPSS score from 10 to 12, and one patient with a baseline prostate volume of 31 ml had an unchanged IPSS score of 11. However, the maximal flow rate from both patients was maintained above 20 ml/s during TRT. Other than these two cases, the IPSS score was diminished in the rest of the patients, both in storage symptoms (items 2, 4, and 7) and voiding symptoms (items 1, 3, 5, and 6; [Table 2](#T2){ref-type="table"}). However, uroflowmetery variables including Qmax and PVR were maintained after a year of TRT. The serum PSA level also remained unchanged (p=0.144). No patients experienced urinary retention, BPH-related surgery, or admission for urinary tract infection during follow up. DISCUSSION ========== Although contraindications for testosterone supplementation in aging men are controversial due to the lack of large-scale studies, the greatest concerns for contemporary TRT are potential side effects on the prostate. It is well-known that the presence of androgen is required for the development of BPH, and antiandrogen agents can decrease prostate volume in patients with BPH,[@B10] suggesting that TRT could result in worsening of LUTSs. However, recent research on the relationship between TRT and the progression of LUTS revealed outcomes opposite these traditional concerns. Tenover[@B6] performed a short-term clinical study using 100 mg of testosterone enanthate every 3 weeks for 3 months and reported that short-term TRT did not significantly increase prostate volume or PVR. From a double-blind controlled study design, Holmäng et al[@B7] indicated that TRT contributed to an increase of 12% in volume of the prostate gland after 8 months of treatment, but did not affect urodynamic data or IPSS. Similarly, in a double-blind placebo-controlled study, Marks et al[@B8] demonstrated that 6 months of testosterone administration normalized serum androgen levels in aging men with hypogonadism but had little effect on prostatic tissue androgen levels or on their urinary symptoms and urodynamic data. Recently, Shigehara et al[@B9] reported the outcome from a year-long randomized control study on 42 patients with TDS concomitant with LUTS, demonstrating improvement not only in IPSS but also in Qmax only by TRT. In this study, we found that TRT for hypogonadal men with moderate LUTS could contribute to improvement of subjective LUTS. IPSS sub-scores also showed improvements both in storage (2, 4, 7) and voiding (1, 3, 5, 6) symptoms. On the other hand, there was no statistical difference in terms of uroflowmetery parameters including Qmax, the relatively higher Qmax in our selected group may have affected the outcomes. Like the outcomes reported by other researchers, the serum PSA level remained unchanged, and it is noteworthy that no clinical progression of BPH including urinary retention, surgery for BPH, or admission for urinary tract infection occurred during more than a year of follow up. Possible explanations for the improvement of LUTS and bladder function by testosterone therapy have been suggested. Indeed, there are several similarities in the etiologies of erectile dysfunction and LUTS, such as metabolic syndrome, autonomic nervous activity, nitric oxide (NO) activity, and rho-kinase activity.[@B11] Testosterone has been shown to have an effect on rho-kinase activity, and rho-kinase activity in the urinary tract may at least in part depend on testosterone.[@B12] Increased rho-kinase activity coincides with the development of LUTS in aging men with BPH, and inhibition of rho-kinase in the rat model is thought to decrease prostatic smooth muscle cell proliferation and to decrease adrenergic contractions.[@B13] NO is also one of the mediators of dilatation of the urethra and bladder neck.[@B11] Chamness et al[@B14] reported that testosterone had an effect on NO synthase in the urinary tract and suggested the possibility of improving urinary symptoms by increasing NO production. By an uncontrolled prospective study, Karazindiyanoğlu and Cayan[@B15] indicated that TRT significantly increased maximal bladder capacity and compliance, and decreased detrusor pressure at maximal flow, based on the results of pressure-flow analysis.[@B15] Celayir[@B16] observed the changes in rabbit bladder tissue after bilateral orchiectomy and demonstrated that testosterone injection significantly increased bladder capacity and compliance. By observing bladders of castrated rats receiving androgen therapy for 28 days, Madeiro et al[@B17] had reported improvement in terms of the number of vessels, epithelial thickness, and quantity of muscular fibers compared to untreated controls. Cayan et al[@B18] reported that, in rats, testosterone treatment resulted in a significantly higher bladder smooth muscle/collagen ratio than controls. These findings suggest that TRT can affect bladder muscle contractility and compliance. This study had inherent limitations mainly due to its retrospective study design, including absence of urodynamic studies and follow up prostate volume after a year. Also, the small number of the patients with moderate LUTS without BPH medication lessened the power of the statistical analysis. The relatively highly maintained Qmax in the 17 selected men may bias the outcome toward a more favorable direction. In addition, the relatively high follow up loss rate of this study among 383 TRT men (29.5%) may also have biased the outcome toward positive results. Finally, due to the absence of a placebo group, we cannot draw conclusions about the subjective improvements, particularly in the Qmax without TRT, and the constitutive impact on LUTS. This is the reason that a larger prospective study design is still required on this issue, considering the lifelong impact of contemporary TRT. CONCLUSIONS =========== Over a year of TRT for selected patients with moderate LUTS and no BPH medication improved both storage and voiding symptoms, without causing clinical progression of BPH or increasing PSA. Thus, TRT can be a safe strategy even in TDS patients with moderate LUTS but maintaining a relatively high uroflow rate. ###### Comparison of parameters at baseline and after TRT in 246 patients  Values are presented as mean (range). TRT: testosterone replacement therapy, AMS: aging male symptom, BMI: body mass index, IPSS: International Prostate Symptom Score, QoL: quality of life, Qmax: maximal flow rate, PVR: post voiding residual urine, PSA: prostate specific antigen. ###### Comparison of parameters at baseline and after TRT in 17 selected patients with moderate LUTS without BPH medication  Values are presented as mean (range). TRT: testosterone replacement therapy, LUTS: lower urinary tract symptom, BPH: benign prostate hyperplasia, AMS: aging male symptom, BMI: body mass index, IPSS: International Prostate Symptom Score, Qmax: maximal flow rate, PVR: post voiding residual urine, PSA: prostate specific antigen. ###### Comparison of baseline characteristics in patients with or without BPH-related medication  Values are presented as mean (range). BPH: benign prostate hyperplasia, BMI: body mass index, AMS: aging male symptom, IPSS: International Prostate Symptom Score, Qmax: maximal flow rate, PVR: post voiding residual urine, PSA: prostate specific antigen.

Background {#Sec1} ========== Colorectal cancer (CRC) is one of the most common causes of cancer death, bringing much inconvenience to people's daily life \[[@CR1]\]. Although an increasing number of new therapy methods are applied to CRC treatment, the therapeutic efficacy for advanced CRC patients is not good. Tumor recurrence or metastasis is still the main cause of therapy failure \[[@CR2]\] and poor prognosis. The 5-year survival rates of CRC patients were about 90% for the ones with only primary tumors, 70.4% for the ones with lymph node or peripheral metastasis, and 12.5% for the ones with distal metastasis \[[@CR3]\]. Long non-coding RNAs (lncRNAs) have been discovered to act important roles in tumor development \[[@CR4]\]. Metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) is one of these functional lncRNAs, which is highly expressed in CRC patients. In our previous studies, we have successfully demonstrated that MALAT1 could promote tumor growth and metastasis in CRC by binding to SFPQ (PTB-associated splicing factor) and releasing oncogene PTBP-2 (polypyrimidine tract binding protein) from the SFPQ/PTBP-2 complex \[[@CR5]\], and increase the nuclear translocation of β-catenin from cytoplasm, thereby activating downstream genes of β-catenin signaling pathway \[[@CR6], [@CR7]\]. Ying et al also revealed that, in bladder cancer, MALAT1 promoted EMT by activating Wnt signaling in vitro \[[@CR8]\]. However, the upstream regulatory mechanism of MALAT1 is not well-elucidated. Jumonji domain-containing protein 2C (JMJD2C), also known as KDM4C, could modulate transcription factors, establish global chromatin environments and regulate gene expression \[[@CR9]--[@CR11]\]. A number of evidences have suggested the association between JMJD2C protein and various tumors \[[@CR12]--[@CR15]\]. Histone demethylase JMJD2C holds great potential of epigenetic regulating mechanism in tumor diseases \[[@CR16], [@CR17]\], especially the regulating effect on the promoter activity of targeted genes. Based on bioinformatics analysis of the MALAT1 promoter and our previous studies, we hypothesized that, JMJD2C might influence MALAT1 promoter activity, thereby activating MALAT1/β-catenin signaling pathway and leading to the promotion of CRC metastasis. Therefore, in this study, we aimed to investigate the new effect of JMJD2C on the metastasis of CRC cells in vitro and in vivo, and to detect the regulatory effect of JMJD2C on the expression of MALAT1 and β-catenin signaling pathway related genes. Most importantly, the potential epigenetic regulating mechanism of JMJD2C on MALAT1 was the focus of this research. Materials and methods {#Sec2} ===================== Human tissues collection and cell culture {#Sec3} ----------------------------------------- A total of 78 human primary CRC tissues without metastasis and 46 human primary CRC tissues with matched hepatic or lung metastasis were collected between 2006 and 2015 at Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, and Fudan University Shanghai Cancer Center. HCT116 (human colon, CRC) and LoVo (human colon, Dukes' type C, grade IV, colorectal adenocarcinoma) cells were purchased from the ATCC (Manassas, USA), and cultured in RPMI-1640 and F12K medium, respectively. Both mediums were supplemented with 10% heat-inactivated FBS, 100 U ml^− 1^ penicillin, and 100 μg ml^− 1^ streptomycin. These cells were incubated under 37 °C, 5% CO~2~ conditions. All the experimental procedures were approved by the Institutional Review Board of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine. Plasmids construction and lentivirus infection {#Sec4} ---------------------------------------------- Three shRNA fragments for human JMJD2C (Gene ID:23081) were synthesized by the Sangon Biotech company (Shanghai, China), sub-cloned into the pLKD shRNA vector, and named pLKD-CMV-G&PR-U6-JMJD2C-shRNA1/2/3, respectively. The full length of JMJD2C gene was amplified by PCR, following by gene sequencing, and the right full fragments was cloned into the plenti overexpression vector, named plenti-CMV-EGFP-P2A-JMJD2C-3FLAG. These above plasmids were used to transfect well-growing state HCT116 and LoVo cells by using Lipofectamine 3000 transfection reagent. After 48 h transfection, the transfected cells were selected with neomycin (G418, 1 mg ml^− 1^, Sigma, USA). Recombinant lentiviruses containing pLV4-shRNA/NT, pLV4-shRNA/JMJD2C, pLV4-empty vector, and pLV4-JMJD2C^+/+^ were prepared by GeneChem (Shanghai, China), respectively. HCT116 or LoVo cells were infected with 2 × 10^6^ transducing units of corresponding lentiviruses, and were selected with 2 μg/ml puromycin for 2 weeks. The efficiencies of knockdown or overexpression of JMJD2C were determined by real-time PCR and western blot. Real-time PCR {#Sec5} ------------- Total RNA was extracted using the TRIzol reagent (Takara) according to the manufacturer's instructions. The RNA concentrations were determined using a NanoDrop ND-1000 (NanoDrop). cDNA was synthesized with the PrimeScript RT Reagent Kit (TaKaRa) using 500 ng total RNA as template. Real-time PCR analyses were conducted to quantitate KDM4C mRNA and lncRNA-MALAT1 relative expression using SYBR Premix Ex Taq (TaKaRa) with GAPDH as an internal control. The Real-time PCR results were defined from the threshold cycle (Ct), and relative expression levels were calculated by using the 2^-△△Ct^ method. PCR was performed using an ABI 7500 instrument (Applied Biosystems, USA). The primers used for real-time PCR analysis were as follow: KDM4C, forward primer: 5-AGGCCTAAGGCTGATGAGGA-3, reverse primer: 5-TTGGCCATGAAAGCTCGGAT-3; MALAT1, forward primer: 5-GCTCTGTGGTGTGGGATTGA-3, reverse primer: 5-GTGGCAAAATGGCGGACTTT-3, GAPDH, forward primer: 5-GGTGGTCTCCTCTGACTTCAACA-3, reverse primer: 5-CCAAATTCGTTGTCATACCAGGAAATG-3. Luciferase reporter assay {#Sec6} ------------------------- To test MALAT1 promoter activity, HCT116 or LoVo cells were co-transfected with the recombinant plasmid pGL3-basic-MALAT1 promoter with a control positive plasmid pRL-SV40 as previously described \[[@CR18]\]. The promoter activity was analyzed using a commercial dual-luciferase assay kit (Promega, USA) according to the manufacturer's instructions. Immunofluorescence staining {#Sec7} --------------------------- To observe the expression and location of JMJD2C and β-catenin, HCT116 and LoVo cells after transfection were plated at a density of 2.0 × 10^4^/ml in 6-well plates, fixed with methanol, blocked with 5% BSA. The cells were first stained with β-catenin mouse antibody followed by Cy3-conjugated goat anti-mouse IgG (Millipore). After the cells were washed four times with PBS, the JMJD2C rabbit antibody was added, followed by FITC-conjugated goat anti-rabbit IgG (Millipore). Nuclear staining was done with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution. Cells were imaged using TCS SP2 spectral confocal system (Leica, Germany). All experiments were conducted according to instructions from the antibody manufacturer. Western blot analysis {#Sec8} --------------------- Protein lysates from cells were prepared in lysis buffer and centrifuged at 12000 rpm at 4 °C. The primary antibodies used were JMJD2C (Santa Cruz, USA), β-catenin (CST, USA), c-Myc (CST, USA), ITGBL1 (Abcam, USA), PCNA (CST, USA) and GAPDH (CST, USA). The secondary antibody used was HRP-labeled goat anti-rabbit/mouse IgG (H + L) (Beyotime, China). Each band was quantitatively analyzed using quantity one Software and normalized to the expression of GAPDH in the same lane. Chromatin immunoprecipitation assay {#Sec9} ----------------------------------- ChIP assays were performed using a ChIP assay kit (Millipore, USA). Chromatin extracts were immunoprecipitated using 10 μg anti-JMJD2C (Abcam, USA), 4 μg anti-H3K9 (Abcam, USA), and 4 μg anti-H3K36 (Abcam, USA), respectively. IgG (Merck, Germany) were used as mock ChIP controls. Fold enrichments were calculated by determining the ratios of the amount of immunoprecipitated DNA to that of the input sample, and were normalized to the level observed at a control region, which was defined as 1.0. The ChIP primers for MALAT1 promoter are as follow: forward primer: 5-GGTCAGCCTGAGACCACTTC-3, reverse primer: 5-CTGTGCCTGTTCTGGGGAAT-3. Transwell assay {#Sec10} --------------- To measure cell migration, the transfected HCT116 and LoVo cells (2 × 10^5^) were seeded to the upper chambers and cultured with 100 μl serum-free F12K or RPMI1640 medium, whereas the lower chamber was filled with 600 μl F12K or RPMI1640 medium containing 15% FBS and 10 μg ml^− 1^ fibronectin. After incubation for 48 h at 37 °C and 5% CO~2~, the chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells in the upper chamber were carefully removed with a cotton swab. The numbers of cells were analyzed by counting five independent visual fields under a DMI3000 B inverted microscope (Leica, USA) with a 20 × objective. All assays were performed in triplicate and independently repeated three times. Wound healing assay {#Sec11} ------------------- Cells (4.5 × 10^5^) were seeded on a six-well plate to form a confluent monolayer in 10% FBS-containing medium. The monolayer cells were scratched by a plastic tip and washed with PBS to remove cell debris; 0.5% FBS-containing F12K or RPMI1640 were then added to each well, and the scratched monolayer was incubated in a 37 °C incubator with 5% CO~2~ for 24 h. Wound closure was measured in five random fields at × 200 magnification using Image J software and a DMI3000B inverted microscope (Leica, USA). Percentage of wound healing was calculated as follows: migrated cell surface area/ total surface area × 100, in which, migrated cell surface area = length of cell migration (mm) × 2 × length of defined areas, total surface area = beginning width × length of defined areas. MTT assay {#Sec12} --------- Cells were cultured at a density of 2.5 × 10^3^ cells per well in a flat-bottomed 96-well plate. MTT \[3-(4,5)-dimethylthiahiazo(−z-y1)-3,5-di-phenytetrazoliumromide\] was used to determine the cell viability by measuring the absorbance at 490 nm. All assays were performed in triplicate and independently repeated three times. Flow cytometry {#Sec13} -------------- Cells were harvested by trypsinization, washed once with cold PBS. Then, the cells were stained with PI (propidium iodide) and anti-Annexin-V antibody (Becton Dickinson, USA) at 4 °C for 1 h. Subsequently, cells were washed once with PBS and analyzed by FACS (BD, USA). In vivo analysis {#Sec14} ---------------- All the animal experiments were performed under the approval of the Institutional Committee for Animal Research and were in accordance with national guidelines for the care and use of laboratory animals. HCT116-shRNA/NT, HCT116-shRNA/JMJD2C, HCT116-empty vector, and HCT116-JMJD2C^+/+^ cells (2 × 10^6^ cells in 100 μl) were injected into female BALB/c nude mice (6--8 weeks old, obtained from SLAC Laboratory Lab, Shanghai, China) by tail intravenous to establish the lung metastasis mice model, respectively. Mice were euthanized 42 days post injection, and the lung metastases images were observed and quantified by LB983 NIGHTOWL II system. The numbers of lung metastatic nodes were measured, and the metastatic tumor tissues were collected for immunohistological and western blot analysis with JMJD2C, β-catenin, c-Myc, and ITGBL1 antibody. HE staining and IHC staining {#Sec15} ---------------------------- Paraffin-embedded tissues were sectioned for HE (hematoxylin and eosin) and IHC (immunohistochemistry) staining. For IHC analysis, the experiments were performed using the first antibody, HRP-conjugated secondary antibody, and DAB (diaminobenzidine) detection reagents. The DMI3000B microscope connected to the digital imaging system was applied for taking photographs and the following analysis. All the data were evaluated and classified blindly by two investigators (ZYW and LLW) from the pathology department of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine. Statistical analysis {#Sec16} -------------------- All data are presented as means with standard deviation (SD) or median with 95% confidence interval (95% CI). Statistic comparison was performed using the Student's t-test, one-way ANOVA analysis, Mann-Whitney test, or Kruskal-Wallis test, as appropriate, with the significance level at *P*\<0.05. All statistical analyses were completed with SPSS 22 software. Results {#Sec17} ======= Overexpression of JMJD2C in CRC metastatic foci predicted poor prognosis {#Sec18} ------------------------------------------------------------------------ Firstly, the expression levels of KDM4C mRNA (encoding JMJD2C protein) were detected in 124 CRC tumor specimens. The results demonstrated that, the levels of KDM4C mRNA were significantly higher in recurrent tumors and metastatic sites than that of non-recurrent ones (Fig. [1](#Fig1){ref-type="fig"}a), and the mRNA levels were closely associated with TMN stages and distant metastasis (Table [1](#Tab1){ref-type="table"}). Survival analysis concluded that CRC patients with lower KDM4C mRNA expression had longer OS (Overall Survival) (Fig. [1](#Fig1){ref-type="fig"}b). In our previous reports, we have shown in 124 CRC tumor specimens that MALAT1 had higher expression levels in recurrent primary and metastatic sites, relative to non-recurrent primary tumors (7). In the present study, we further found that, highly matched co-expression of KDM4C mRNA and MALAT1 was found in our 124 CRC cases (Fig. [1](#Fig1){ref-type="fig"}c). In addition, a TCGA (The Cancer Genome Atlas)-dataset analysis also showed the elevated mRNA levels of KDM4C in 367 CRC primary tissues, and the KDM4C mRNA expression was inversely correlated with OS of CRC patients (Fig. [1](#Fig1){ref-type="fig"}d). Next, the immunohistochemistry staining further revealed significantly higher protein levels of JMJD2C (encoded by KDM4C) in recurrent tumors and lung/liver metastasis than the ones in non-recurrent tumors (Fig. [1](#Fig1){ref-type="fig"}e), implying that JMJD2C overexpression correlated closely with CRC progression. Fig. 1KDM4C expression in post-surgical, recurrent primary and metastatic sites, comparing to primary sites of non-recurrent CRC patients. **a** Expression levels of KDM4C mRNA in 124 CRC tissues and matched metastatic sites were analyzed by real-time PCR. The significant differences between primary tumor I (without paired metastatic tissues) and primary tumor II (with paired metastatic tissues, Metastasis II) were analyzed using the Wilcoxon signed-rank test. **b** Kaplan-Meier analyses of the correlations between KDM4C mRNA expression levels and overall survival (OS) of 124 CRC patients, and the median expression level was used as the cutoff. **c** The co-expression between KDM4C and MALAT1was showed in 124 cases from our datasets. **d** The mRNA expression of JMJD2C in 367 CRC primary tissues from TCGA dataset, and its association with CRC prognosis, including OS. **e** Immunohistochemical analysis of JMJD2C protein (encoded by KDM4C) in representative CRC and metastatic lung/liver tissues, including primary CRC tumor without paired metastatic tissues, and primary CRC tumor with paired metastatic tissues (scale bars, 100 μm, respectively). \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) Table 1Association between KDM4C expression and clinicopathological variables of CRC patientsVariablesLow KDM4C expression (*n* = 67)High KDM4C expression (*n* = 57)*P*n (%)n (%)Age \> 6513 (19.40)10 (17.54)0.8210 ≤ 6554 (80.60)47 (82.46)Gender Male39 (58.21)39 (68.42)0.2673 Female28 (41.79)18 (31.58)Tumor site Rectum37 (55.22)36 (63.16)0.4642 Colon30 (44.78)21 (36.84)Tumor differentiation Well20 (29.85)19 (33.33)0.7019 Moderate + Poor47 (70.15)38 (66.67)TNM stage Stage II35 (52.23)12 (21.05)0.0004\*\* Stage III32 (47.77)45 (78.95)Lymph vascular invasion Positive44 (65.67)42 (73.68)0.4347 Negative23 (34.33)15 (26.32)Perineural invasion Positive52 (77.61)46 (80.70)0.8253 Negative15 (22.39)11 (19.30)Liver metastasis Positive9 (13.43)22 (38.60)0.0017\*\* Negative58 (86.57)35 (51.40)Lung metastasis Positive1 (1.49)8 (14.04)0.0114\* Negative66 (98.51)49 (85.96)\*, *P* \< 0.05; \*\*, *P* \< 0.01 JMJD2C promoted the migration of CRC cell lines in vitro {#Sec19} -------------------------------------------------------- To thoroughly understand the biological function of JMJD2C, we first knocked down and overexpressed JMJD2C expression using shRNA and overexpressing vector, respectively. Real-time PCR assay was carried out to detect the mRNA expression of JMJD2C and showed efficient down-regulation or up-regulation of JMJD2C in HCT116 and LoVo cells (Fig. [2](#Fig2){ref-type="fig"}a, Additional file [1](#MOESM1){ref-type="media"}: Figure S1A). The protein expression of JMJD2C was further confirmed by western blot and quantitative assay (Fig. [2](#Fig2){ref-type="fig"}b, c, Additional file [1](#MOESM1){ref-type="media"}: Figure S1B, C). Subsequently, by transwell and wound healing assays in HCT116 and LoVo cells, we found a positive role of JMJD2C in promoting CRC cells migration (Fig. [2](#Fig2){ref-type="fig"}d-g, Additional file [1](#MOESM1){ref-type="media"}: Figure S1D, E). Additionally, we also found that, JMJD2C could promote the proliferation of CRC cells (Additional file [2](#MOESM2){ref-type="media"}: Figure S2A), but affected little on the apoptosis of CRC cells (Additional file [2](#MOESM2){ref-type="media"}: Figure S2B). Fig. 2JMJD2C promoted the metastasis of CRC cells in vitro. **a-c** Real time PCR and western blotting were performed to confirm the gene silencing and overexpressing efficiency for JMJD2C. HCT116 was transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. **d** Migration assays of HCT116 cells transfected with shRNA/NT, shRNA/JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. **e** Numbers of migrated cells are shown as mean ± SD; *n* = 3. **f-g** Wound healing assay was used to evaluate the effect of JMJD2C on migration of HCT116 cells. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) Nuclear translocation of JMJD2C lowered the histone methylation level of MALAT1 promoter in CRC {#Sec20} ----------------------------------------------------------------------------------------------- Histone demethylase JMJD2C holds great potential of epigenetic regulating mechanism in tumor diseases \[[@CR19]--[@CR27]\], especially its important regulating effect on the promoter activity of targeted genes \[[@CR28], [@CR29]\]. By immunofluorescent staining assay, we found that, knockdown of JMJD2C could significantly decrease the nuclear accumulation of JMJD2C protein in CRC cells, while overexpression of JMJD2C could effectively elevate the distribution of JMJD2C protein in the nuclei of CRC cells (Fig. [3](#Fig3){ref-type="fig"}a, b). Then, above results were further validated by the western blot detection (Fig. [3](#Fig3){ref-type="fig"}c, d). Fig. 3Translocation of JMJD2C protein from the cytoplasm into the nuclei in CRC cells in vitro. **a**-**b** Immunofluorescence detection of JMJD2C protein in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. **c**-**d** Western blot and quantitative assay of JMJD2C protein (nuclear and whole cell lysates) in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) Urged by above data, we next studied if nuclear JMJD2C could bind to the promoter of MALAT1 to affect the expression of MALAT1 by chromatin immunoprecipitation (ChIP) (Fig. [4](#Fig4){ref-type="fig"}a). Based on our ChIP assay, we found that JMJD2C could bind to the promoter of MALAT1, and regulate lower the histone methylation level of MALAT1 promoter in sites of H3K9m3 and H3K36m3, whether not only in HCT116 or but also in LoVo cells (Fig. [4](#Fig4){ref-type="fig"}b). Next, the luciferase reporter assay further confirmed that, JMJD2C could promote the promoter activity of MALAT1 gene (Fig. [4](#Fig4){ref-type="fig"}c, d). Moreover, the real-time PCR results also demonstrated that, JMJD2C could promote the transcriptional level of MALAT1 (Fig. [4](#Fig4){ref-type="fig"}e, f). All above results suggested that JMJD2C could translocate into the nuclei, regulate lower the histone methylation level of MALAT1 promoter, and elevate the expression of MALAT1. Fig. 4JMJD2C promoted MALAT1 expression by regulating the histone methylation level of MALAT1 promoter. **a** Schematic diagram of the ChIP (Chromatin Immunoprecipitation) procedure for detecting the effect of JMJD2C on the histone methylation level of MALAT1 promoter in sites of H3K9m3 and H3K36m3. Purple ovals: no-targeting protein; Yellow ovals: targeting protein A; Green ovals: protein binding to targeting protein A. **b** JMJD2C, H3K9, H3K36 antibody and MALAT1 special primers were used to investigate the interaction between JMJD2C and MALAT1 promoter, IgG was used as the negative control. **c-d** MALAT1 promoter activities assay in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. **e-f** Real time PCR assay of MALAT1 levels in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) JMJD2C enhanced nuclear translocation of β-catenin and activated β-catenin signaling in CRC {#Sec21} ------------------------------------------------------------------------------------------- Our previous studies have demonstrated that MALAT1 could enhance the β-catenin signaling pathway \[[@CR6], [@CR7]\]; thus we further investigated the role of JMJD2C on regulating the expressions of β-catenin signaling pathway related proteins including β-catenin, c-Myc, and ITGBL1 (integrin β-like 1). By immunofluorescent staining assay, we found that, knockdown of JMJD2C could significantly decrease the nuclear accumulation of β-catenin protein in CRC cells, while overexpression of JMJD2C could effectively reverse the distribution of β-catenin in the nuclei of CRC cells (Fig. [5](#Fig5){ref-type="fig"}a, b). The following western blot results further confirmed the immunofluorescent staining results (Fig. [5](#Fig5){ref-type="fig"}c, d). Fig. 5JMJD2C activated the β-catenin signaling pathway in CRC cells. **a**-**b** Immunofluorescence detection of β-catenin protein in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. **c**-**d** Western blot and quantitative assay of β-catenin protein (nuclear, cytoplasm and whole cell lysates) and β-catenin signaling downstream targets including c-Myc and ITGBL1 in HCT116 or LoVo cells transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) As we know, the nuclear β-catenin and other transcription factors such as LEF, TCF and BCL9 will function together to trans-activate downstream target genes \[[@CR30]\]. Therefore, the luciferase reporter assay was further performed to observe the effect of JMJD2C on the activity of the LEF/TCF promoter. As expected, the final results confirmed the promoting effect of JMJD2C on the activity of the LEF/TCF promoter (Additional file [3](#MOESM3){ref-type="media"}: Figure S3). In addition, in Fig. [5](#Fig5){ref-type="fig"}c, d we also showed that, JMJD2C could enhance the expression of β-catenin signaling downstream targets including c-Myc and ITGBL1. All together, above data demonstrated JMJD2C enhanced the activity of β-catenin signaling pathway in CRC. JMJD2C promoted CRC lung metastasis in vivo {#Sec22} ------------------------------------------- In order to observe the in vivo effect of JMJD2C, HCT116-shRNA/NT, HCT116-shRNA/JMJD2C, HCT116-empty vector, and HCT116- JMJD2C^+/+^ cells were injected into nude mice (into the lateral tail vein), and the lung metastases images were observed by LB983 NIGHTOWL II (IVIS) system (Fig. [6](#Fig6){ref-type="fig"}a). As shown in Fig. [6](#Fig6){ref-type="fig"}b, knockdown of JMJD2C could significantly decrease the luciferase intensity of CRC cells in lung metastasis, while overexpression of JMJD2C could increase the luciferase intensity of CRC cells in lung metastasis. Additionally, the numbers of lung metastatic lesions were in accordance with the luciferase detecting results (Fig. [6](#Fig6){ref-type="fig"}c). Then, we detected the expression of JMJD2C protein in the lung metastatic lesions by immunohistochemical assay. The results showed the positive roles of JMJD2C in promoting CRC cells metastasis in vivo (Fig. [6](#Fig6){ref-type="fig"}d, e). Fig. 6JMJD2C promoted the metastasis of CRC cells in vivo. **a-b** HCT116-shRNA/NT, HCT116-shRNA/JMJD2C, HCT116-empty vector, and HCT116-JMJD2C^+/+^ cells were respectively injected into the tail vein of nude mice (*n* = 6). After 42 days, the lung metastases images were observed and quantified by LB983 NIGHTOWL II system. **c** Quantification of lung metastatic nodules from 6 mice subjected to the indicated treatments. **d-e** Immunohistochemical and quantitative analysis of JMJD2C proteins on consecutive tissue microarray slides of lung metastatic nodules from 6 mice subjected to the indicated treatments. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) JMJD2C elevated the expression of MALAT1 and β-catenin signaling related proteins in CRC lung metastasis mice models {#Sec23} -------------------------------------------------------------------------------------------------------------------- Since the in vitro study has found the regulatory effect of JMJD2C on the expression of MALAT1 and β-catenin signaling related proteins, we next validated the results in vivo. Real-time PCR showed that, knockdown of JMJD2C could significantly decrease the expression of MALAT1 in the lung metastatic lesions, while overexpression of JMJD2C could significantly elevate the expression of MALAT1 in the lung metastatic lesions (Fig. [7](#Fig7){ref-type="fig"}a). Then, the western blot experiments further demonstrated that, knockdown of JMJD2C could significantly decrease the nuclear accumulation of β-catenin protein in the lung metastatic lesions, while overexpression of JMJD2C could effectively reverse the distribution of β-catenin in the nuclei of lung metastatic lesions (Fig. [7](#Fig7){ref-type="fig"}b, c). By immunohistochemical assay we further found that, knockdown of JMJD2C could significantly decrease the expression of β-catenin signaling downstream targets including c-Myc and ITGBL1 in the lung metastatic lesions, while overexpression of JMJD2C could significantly elevate the expression of c-Myc and ITGBL1 in the lung metastatic lesions (Fig. [7](#Fig7){ref-type="fig"}d, e). Fig. 7JMJD2C elevated the expression of MALAT1 and β-catenin signaling related proteins in CRC lung metastasis mice models. **a** Real-time PCR was performed to detect the expression of MALAT1 in lung metastatic nodules from 6 mice subjected to the indicated treatments. **b-c** Western blot and quantitative assay of β-catenin protein (nuclear, cytoplasm and whole cell lysates) in the lung metastatic tissues from 6 mice subjected to the indicated treatments. **d-e** Immunohistochemical and quantitative analysis of ITGBL1 and c-Myc proteins on consecutive tissue microarray slides of lung metastatic nodules from 6 mice subjected to the indicated treatments.\*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test) Discussion {#Sec24} ========== Histone demethylase JMJD2C has been reported to play crucial roles in the progression of colon cancer \[[@CR19], [@CR20]\], breast cancer \[[@CR21]--[@CR23]\], prostate cancer \[[@CR24], [@CR25]\], gastric cancer \[[@CR26]\], lung cancer \[[@CR27]\] and so on, indicating that JMJD2C represents a promising anti-cancer target. JMJD2C is mapped to human chromosome 9p24.1, and it has been proved to be a demethylase for H3K9 and H3K36 methylation \[[@CR28], [@CR29]\]. More evidences have shown that JMJD2C is a candidate oncogene both in normal biology and tumorigenic processes \[[@CR31]\]. Ye et al \[[@CR23]\] found that knockdown of JMJD2C inhibited the proliferation of breast cancer cells in vitro and in vivo. In addition, Kim et al firstly uncovered that JMJD2C was overexpressed in five colon cancer cell lines and especially associated closely with the growth of HCT116 cells, and JMJD2C might promote the survival of colon cancer cells and stimulate the proliferation of colon cancer cells via up-regulating the levels of Cyclin D1 and FRA1 \[[@CR19]\]. These results revealed the important roles of JMJD2C in tumors through targeting different oncogenes or tumor suppressor genes. Our previous studies have successfully demonstrated that MALAT1 could promote CRC metastasis through regulating β-catenin signaling pathway \[[@CR6], [@CR7]\]. However, the upstream regulatory mechanism of MALAT1 is not well-elucidated. Based on bioinformatics analysis of the MALAT1 promoter, we found the potential binding sites for JMJD2C in the promoter of MALAT1, and hypothesized that, JMJD2C, the important histone demethylase, could influence the activity of MALAT1 promoter, thereby regulating MALAT1/β-catenin signaling pathway and leading to the promotion of CRC metastasis. In the presented paper, we demonstrated that the mRNA expression levels of JMJD2C in CRC metastatic lesions are significantly higher than the ones in primary lesions, and there is a positive correlation between JMJD2C mRNA expression and MALAT1 expression. Our previous studies have shown the promoting effect of MALAT1 on the proliferation and migration of CRC cells \[[@CR6]\]. Currently, our results further suggested that JMJD2C can also promote the proliferation and migration of CRC cells in vitro. Nevertheless, the direct evidence about the interaction between JMJD2C and MALAT1 is not presented. Various studies have demonstrated the complicated biological function of JMJD2C. Ishimura et al has proved that overexpression of JMJD2C could up-regulate the expression of oncogene Mdm2 and lead to the decreased expression of tumor suppressor gene p53 \[[@CR32]\]. Our data suggested that JMJD2C elevated the expression of MALAT1 and regulated the β-catenin signaling pathway, as well as the downstream gene expression including c-Myc and ITGBL1. Further mechanism studies demonstrated that, JMJD2C could directly bind to the promoter region of MALAT1, lower the histone methylation level of MALAT1 promoter in the sites of H3K9me3 and H3K36me3, and enhance the promoter activity and the final expression of MALAT1. Ye et al has shown that knockdown of JMJD2C not only inhibited the breast tumor growth but also effectively blocked the lung metastasis in mice model \[[@CR23]\]. In our study, by establishing the lung metastasis mice model, we found that JMJD2C could promote CRC metastasis in vivo. Moreover, in vivo, we also found that, JMJD2C could significantly elevate the expression of MALAT1 in the lung metastatic lesions, increase the nuclear accumulation of β-catenin protein, and elevate the protein expression of downstream targets including c-Myc and ITGBL1 in the lung metastatic lesions. These results further confirmed that JMJD2C could up-regulate the MALAT1 expression and enhance the activity of β-catenin signaling pathway, which were in accordance with the results in vitro. Conclusion {#Sec25} ========== In summary, our findings imply that JMJD2C plays a vital role in the epigenetic regulation process. In details, JMJD2C could directly influence the expression of MALAT1 by regulating the histone methylation level of MALAT1 promoter in the sites of H3K9me3 and H3K36me3, enhancing the activity of β-catenin signaling pathway, and promoting CRC metastasis (Fig. [8](#Fig8){ref-type="fig"}). Thus, JMJD2C might be a useful target for the prevention and treatment of CRC metastasis, and the interaction between JMJD2C and MALAT1 could be considered as the points for drug development. Fig. 8A schematic model of JMJD2C promoted CRC metastasis by regulating the histone methylation level of the MALAT1 promoter and enhancing β-catenin signaling pathway Supplementary information ========================= {#Sec26} **Additional file 1: Figure S1.** JMJD2C promoted the metastasis of CRC LoVo cells. a-c Real time PCR and western blotting were performed to confirm the gene silencing and overexpressing efficiency for JMJD2C. LoVo was transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. d Migration assays of LoVo cells transfected with shRNA/NT, shRNA/JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. e Numbers of migrated cells are shown as mean ± SD; *n* = 3. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test). **Additional file 2: Figure S2.** JMJD2C promoted the proliferation of CRC HCT116 cells, but affected little on the apoptosis of the indicated cells. a MTT assay of HCT116 cells transfected with shRNA/NT, shRNA/JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. b Flow cytometry was performed to measure the apoptosis rates of HCT116 cells transfected with shRNA/NT, shRNA/JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. **Additional file 3: Figure S3.** JMJD2C enhanced the activity of the LEF/TCF promoter. a-b LEF/TCF promoter activity assay in HCT116 and LoVo cells transfected with shRNA/NT, shRNA/JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. \*, *P* \< 0.05; \*\*, *P* \< 0.01 (*t* test). ChIP : Chromatin immunoprecipitation; CRC : Colorectal cancer DAB : Diaminobenzidine HE : Hematoxylin and eosin; IHC : Immunohistochemistry ITGBL1 : Integrin β-like 1 JMJD2C : Jumonji domain-containing protein 2C lncRNAs : Long non-coding RNAs MALAT1 : Metastasis associated in lung adenocarcinoma transcript 1 OS : Overall survival PTBP-2 : Polypyrimidine tract binding protein SFPQ : PTB-associated splicing factor **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Xinnan Wu, Ruixiao Li and Qing Song contributed equally to this work. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s13046-019-1439-x. We thank Professor Sizhi Paul Gao from Department of Medicine, Memorial Sloan Kettering Cancer Center. QJ and QL conceived and designed the study and the experimental setup and wrote the manuscript. XW, CZ, QS, RL, ZH, RJ, YW, LZ, LY, and HS performed the experiments. QJ, QL and HZ analyzed the data. All authors read and approved the final version of the manuscript. This work was supported by National Natural Science Foundation of China (No. 81830120 to QL), National Natural Science Foundation of China (No. 81573749 to QJ), National Natural Science Foundation of China (No. 81673783, No. 81874405 to XL), National Natural Science Foundation of China (No. 81603457 to ZFH), National Natural Science Foundation of China (No. 81874399 to HRZ), Science Foundation of Shanghai Committee of Science Project (No. 16401970500 to QL), Shanghai Rising-Star Program (No. 18QA1404100 to XL). All of the data and materials in this paper are available when requested. All the procedures involving human tumor biopsies were performed with the approval of the ethics committee of the Shuguang Hospital, Shanghai University of Traditional Chinese Medicine on samples from patients who had given written informed consent. All the animal protocols were approved by the Animal Care Commission of Shanghai University of Traditional Chinese Medicine. Not applicable. The authors declare that they have no competing interests.

Introduction ============ Most protein functions associated with pathological conditions are mediated by protein conformational changes and protein-protein interactions. In terms of drug discovery, what counts is to find ways to modulate a target protein function, not to hit the protein itself. This means that partner proteins (upstream or downstream), which are able to control the function of a target protein, become themselves exciting targets for drug discovery. Understanding the complex processes of protein crosstalk becomes therefore essential, in particular to discover new individual target proteins belonging to well-known classes of therapeutic targets (enzymes, for example). {#molecules-08-00349-f001} This is the world of Signal Transduction ([Figure 1](#molecules-08-00349-f001){ref-type="fig"}), which for chemists has tremendous advantages for proposing new targets for drug discovery within the scope of current knowledge and know-how. Indeed, plenty of Signal Transduction molecular mechanisms involve protein conformational changes, which are under the control of kinases and phosphatases, two enzyme classes that are tractable targets for drug design. Protein kinases catalyse the phosphorylation of Ser, Thr or Tyr residues of proteins ([Figure 2](#molecules-08-00349-f002){ref-type="fig"}), leading to phosphoproteins which generally adopt a different conformation, allowing them to interact with different partner proteins compared to the native, un-phosphorylated ones. Phosphatases, on the contrary, catalyse the de-phosphorylation of these phosphoproteins. It is important to note that both enzyme types function through very distinct mechanisms since kinases are all characterised by a well-preserved ATP-binding site, close to the catalytic site (since kinases transfer one phosphate residue to proteins, generating ADP as a by-product) while phosphatases work in a way totally independent of ATP, generating inorganic phosphate upon the cleavage of the phospho-protein bond. {#molecules-08-00349-f002} Since the pioneering work of Ed Fisher and Ed Krebs (who were awarded the Nobel Prize in 1992), the reversible phosphorylation of proteins has been widely recognised as central to the regulation of most aspects of cell function and cell physiology. Increasing numbers of human diseases are known to involve mutations, over-expression, genetic association or malfunctioning of protein kinases and phosphatases, and their regulators and effectors. Signal Transduction, mediated by protein phosphorylation is extremely complex and our understanding of signalling mechanisms and their implication in diseases is still in its infancy. However, it represents a fantastic field of investigation to explore where small molecules will play a critical role, both in helping understand cell biology mechanisms to gain knowledge and progress, but also, hopefully, in proposing therapeutic solutions to unmet medical needs. Let's take the example of insulin, whose implication and therapeutic usefulness is well-known in diabetes type 1, while diabetes type 2 is characterised by insulin-resistance and impaired glucose tolerance with dramatic consequences for patients (blindness, kidney failure, amputations and heart diseases). Insulin resistance appears to involve an early process in insulin signal transduction which is initiated through the binding of insulin to the membrane insulin tyrosine kinase receptor (IR), which causes auto-phosphorylation of the receptor kinase and activates phosphorylation of other protein substrates, including IR substrate proteins (IRS-1, IRS-2, etc...) leading to a cascade of intracellular events which mediate the biological effect of insulin ([Figure 3](#molecules-08-00349-f003){ref-type="fig"}). {#molecules-08-00349-f003} Activation (phosphorylation) of the IR by insulin is reversed by the action of protein tyrosine phosphatases (PTP's), among which PTP1B has been clearly identified as a major player, based on *in vitro* experiments and knock-out mice models ([Figure 4](#molecules-08-00349-f004){ref-type="fig"}). {#molecules-08-00349-f004} Interestingly enough, it has also been found that PTP1B is involved in regulating the leptin-signalling pathway as well, and resistance to the leptin hormone is a hallmark of obesity. Thus, PTP1B is thought to function as a negative regulator of insulin and leptin signal transduction; PTP1B inhibitors have therefore the potential to restore insulin/leptin sensitivity and should be effective in treating type 2 diabetes and obesity, which are linked diseases. The search for PTP1B inhibitors is currently extensively pursued by several pharma and biotech companies. Our efforts in Serono, based on rational design, (screening of focused libraries around proprietary scaffold design with the help of PTP1B X-ray structures and in-house SAR studies), followed by drug-like properties optimisation led us to identify PTP1B inhibitors which increase glucose uptake in C2C12 muscle cells and decrease plasma glucose and insulin levels in genetically modified obese mice (db/db) upon oral administration in a dose dependant manner, with no sign of toxicity. Efforts are currently pursued to move the most promising PTP1B inhibitors to the clinic and evaluate their therapeutic potential in diabetic or obese patients. The insulin/PTP1B example illustrates in a powerful way that blocking phosphatases can stimulate a signal transduction cascade and therefore mimic or replace a secreted protein which action is beneficial in pathological conditions. Inhibition of kinases (which, most of the time positively relay signalling events) is, on the contrary, a powerful way to inhibit or control signal transduction cascades, which become particularly relevant in drug discovery when pathological conditions are associated with stimulation of intra-cellular signalling (see the example of MAP kinase cascade in [Figure 5](#molecules-08-00349-f005){ref-type="fig"}). This is the case in important therapeutic areas like cancer, inflammation and apoptosis-related disorders where efforts to find selective kinase inhibitors have literally exploded in the last five years. A landmark event occurred in May 2001, when Gleevec^TM^ (Glivec® outside the U.S.), the first important drug targeting specifically a protein kinase (ABL) was approved for clinical use to treat chronic myeloid leukaemia. {#molecules-08-00349-f005} The human genome encodes for around 500 different kinases, which control most cellular events, directly or indirectly. This is what makes kinases attractive as a drug target class; however this also raises immediately two important questions: What kinase for what disease? And how to design selective inhibitors for specific kinases of therapeutic interest? What kinase for what disease? ============================= Validation of individual kinases for drug discovery (which means obtaining enough convincing evidence that inhibitors of such kinases will demonstrate a therapeutic effect in patients during phase II clinical trials) is not trivial and remains one of the most challenging aspects of post-genomic drug discovery. Dissection of signal transduction mechanisms is certainly a good starting point, generally followed by knock-out animal experiments and/or pharmacological studies with known inhibitors. However, none of these approaches is fully satisfactory and new ones are currently under investigation based on chemical genetics, as for example the Analog-Sensitive Kinase Allele (ASKA) technology where chemists have engineered modified kinases and inhibitors (by generating functionally active kinase mutants which are specifically inhibited by chemically modified inhibitors), thus allowing to study specific responses *in vivo* in knock-in animals, (and therefore validate the kinase for a given disease) prior to start Med Chem. Such mouse disease models with functionally intact, specifically and pharmacologically inhibitable kinase targets will also provide invaluable information with respect to therapeutic index linked to the target kinase inhibition, new substrates identification and biomakers discovery. This is a beautiful example where a multidisciplinary approach (chemistry, together with structural biology, molecular biology, genomics an pharmacology) is providing new breakthough tools which will allow drug discovery to progress very rapidly. Designing selective kinase inhibitors ===================================== Beyond the choice of the kinase, the next challenge will be to design selective inhibitors. So far, the vast majority of kinase inhibitors reported to date are ATP competitive, which means that they bind to the ATP binding site, which is common for all kinases and which is highly conserved in terms of amino acid sequence. It is therefore not surprising that plenty of ATP-competitive inhibitors are poorly selective (as for example staurosporine). But, ... is it possible to design selective ATP-competitive inhibitors? As part of our programme directed toward the discovery of JNK inhibitors in inflammation/apoptosis related disorders, we have discovered a new chemical class of ATP-competitive inhibitors derived from the benzothiazole scaffold. First compounds from this series which were identified as potent JNK inhibitors also displayed strong activity against a few other kinases, as shown by the selectivity profile depicted in chart A below ([Figure 6](#molecules-08-00349-f006){ref-type="fig"}). {#molecules-08-00349-f006} Chemical modifications based on JNK 3D-structure, docking experiments and in-house SAR data provided second generation JNK inhibitors, which were found highly selective versus other kinases, as shown by the selectivity profile depicted in chart B. Kinetic experiments at various ATP and inhibitor concentrations demonstrate that this compound is purely ATP competitive as shown by Lineweaver-Burk representation in [Figure 7](#molecules-08-00349-f007){ref-type="fig"}. {#molecules-08-00349-f007} The best JNK inhibitors identified so far from this project are currently evaluated in pre-clinical studies, based on preliminary promising results in animal models of auto-immune diseases and neuronal apoptosis. This example shows that it is possible to design new potent selective inhibitors of kinases, which are ATP competitive. The starting point is a generic kinase scaffold (hopefully proprietary) from which selectivity can be achieved through proper substitution driven by rationale design based on the kinase specific sequence and its 3D structure. This is a powerful tool, because it can be applied to different kinases, from the same central scaffold, providing proper information is available to direct chemist's efforts in the right direction. Forthcoming kinases of therapeutic interest (among the 500 ones encoded by the human genome) will benefit from recent progresses in bioinformatics (in particular prediction of 3D structure based or primary sequence) and high-throughput production of protein constructs associated with new crystallisation technologies, which will provide new kinase 3D models or real structures for *in silico* design of selective inhibitors. What about non-ATP competitive inhibitors? ========================================== An alternative way to avoid promiscuity in the design of kinase inhibitors is to look for non-competitive inhibitors with respect to ATP, with the aim to hit interactions at kinase sites, which are not commonly found within the enzyme class. Very little is known to really define a strategy based on rationale design for such type of inhibitors and therefore high-throughput screening of highly diverse collection of compounds remains as one of the most promising approach. This is the one we have followed as part of our efforts to find new MEK inhibitors as potential anti-cancer agents. Screening of 50K compounds led to the identification of a few positives among which AS701173 was characterised as a potent (IC~50~=30nM) non ATP-competitive inhibitor since inhibition of MEK by that particular inhibitor was independent of ATP concentrations used in the assay conditions ([Figure 8](#molecules-08-00349-f008){ref-type="fig"}). {#molecules-08-00349-f008} Evaluation of AS701173 against a panel of 40 available kinases show that this compound is highly selective for MEK since none of the other kinase was inhibited by more than 20% at 10μM. {#molecules-08-00349-f009} Lead optimisation based on this new selective MEK inhibitor is in progress and promising preliminary results have recently been obtained with newly found analogs demonstrating potent activity in tumour cell proliferation assays and in *in vivo* models of cancer. From a chemist's perspective, these two examples show that combinatorial chemistry and chemical diversity (cfr MEK selective inhibitors) on one side, as well as "surgical" modifications of molecules in a precise way driven by rationale design (cfr JNK selective inhibitors design) on the other side are complementary tools in drug discovery; both of them can only lead to success if chemists master the art of organic chemistry, and if the art of organic chemistry is continuously offering new methods, reactions, reagents, strategies to solve new emerging challenges. Kinases and phosphatases inhibitors described above illustrate the potential of signal transduction in drug discovery where understanding of cell biology events has allowed to identify druggable targets which play a critical role in diseases and for which chemists are able to design potent and selective inhibitors with promising therapeutic applications. This drug discovery strategy has enormous potential since, on one hand many proteins that will be identified as key players in pathological conditions will be up-or down-regulated, directly or indirectly by kinases or phosphatases. On the other hand, the design and synthesis of selective, potent, cell permeable drug-like inhibitors of kinases and phosphatases is within the scope of chemist's current expertise. Synergy between synthetic chemistry, computational chemistry, enzymology, bioinformatics, cheminformatics, cell biology, structural biology and molecular pharmacology will provide the necessary technology platform to speed-up the discovery of inhibitors and improve the quality of drug candidates. PTP1b:Rob Hooft, Agnès Bombrun, Dominique Swinnen, Pierre-Alain Vitte.JNK:Pascale Gaillard, Christian Rommel, Montse Camps, Yolande Chvatchko, Dominique Perrin, Anthony Nichols, Jean-Pierre Gotteland.MEK:Robert Murray, Sharad Magar, Peter Blume- Jensen

Introduction {#s1} ============ Our world is facing the pandemic of influenza H1N1 viral infection with probable immense effects on daily lives and on the world economy due to the easiness of transmission and to the high virulence of the virus [@pone.0008393-Zimmer1], [@pone.0008393-Morens1]. As of August 30^th^, world health organization (WHO) has reported 254206 laboratory-confirmed cases of infection of H1N1 infection; as of August 2^nd^ 26513 of these cases have occurred in the European continent [@pone.0008393-1], [@pone.0008393-European1]. These data are considered to be an under-estimation of the situation occurring worldwide due to the existence of cases with a very mild physical course not seeking medical attention. However, a total of 2837 deaths have been reported from the confirmed cases. The main question that needs to be answered when taking care of a severely ill patient infected by the H1N1 virus is if deterioration of the human host is due to the virus per se or to impairment of the immune system created in the field of infection and to any subsequent predilection for super-infections. Data to provide a clear-cut answer are missing. The present study is aiming to evaluate if infection by the novel H1N1 virus may lead to impairment of the function of the innate and adaptive immune responses of the human host. Results {#s2} ======= Demographic Characteristics {#s2a} --------------------------- A total of 110 patients were screened; 59 were enrolled in the study. Thirty-one patients were infected by H1N1 virus and six developed H1N1-related pneumonia; their mean ± SD age was 28.5±11.6 years; 17 were male and 14 female. Fever and malaise were the predominant symptoms occurring in all patients (100%) followed by cough in seven (22.5%) patients; and diarrhea in three patients (9.8%). Mean ± SD white blood cells were 5708.0±2578.8/µl. No underlying medical history was reported in any patient. Infection convalescence was noted in all. Eighteen patients were presented with flu-like syndrome; their mean ± age was 39.6±15.2 years; 10 were male and eight female. Fever and malaise were the predominant symptoms occurring in all patients (100%) followed by cough in six (31.6%) patients; and diarrhea in three patients (15.8%). Mean ± SD white blood cells were 8722.2±4817.3/µl. No underlying medical history was reported in any patient. None of these patients developed pneumonia. Infection convalescence was noted in all. Mean ± SD age of healthy volunteers was 36.3±7.4 years. Mean ± SD white blood cells were 5170.0±1296.2/µl. Effects in the Innate Immune System {#s2b} ----------------------------------- Infection by the H1N1 virus was accompanied by significant increase of monocytes (p\<0.0001 compared with healthy volunteers). Co-expression of HLA-DR on monocytes was more than 96% in all patients ([Figure 1](#pone-0008393-g001){ref-type="fig"}) {#pone-0008393-g001} Mean rates of apoptosis of monocytes were 52.4%, 65.5% and 75.5% among healthy volunteers, among patients with flu-like syndrome and among patients infected by the H1N1 virus respectively (p non-significant between groups). Respective mean rates of apoptosis of NKT-cells were 16.6%, 34.3% and 39.1% (p non-significant between groups). Respective mean rates of apoptosis of NK-cells were 19.1%, 27.2% and 23.1% (p non-significant between groups). Concentrations of TNFα of PBMCs-stimulated supernatants are shown in [Figure 2](#pone-0008393-g002){ref-type="fig"}. Patients infected by the H1N1 virus had lower production of TNFα after stimulation with PHA (p\<0.0001 compared with healthy volunteers) and heat-killed PSSP (p\<0.0001 compared with healthy volunteers). {#pone-0008393-g002} Concentrations of IL-1β of PBMCs-stimulated supernatants are shown in [Figure 3](#pone-0008393-g003){ref-type="fig"}. No differences were found between healthy volunteers, patients with flu-like syndrome and patients infected by the H1N1 virus. {#pone-0008393-g003} In order to define if the effect of H1N1 is related with the production of T~H~-1 type cytokines or not, concentrations of IL-6, IL-18, IFNα and IFNγ were also estimated in supernatants of PBMCs stimulated with either PHA or *Streptococcus pneumoniae* ([Figure 4](#pone-0008393-g004){ref-type="fig"}). It was found that stimulation of PBMCs of both patients with flu-like syndrome and H1N1-infection produced greater concentrations of IL-6 compared with healthy volunteers (p: 0.009 for comparisons between flu-like syndrome and healthy volunteers; p: 0.009 for comparisons between H1N1 infection and healthy volunteers). With the exception of single patients, IL-18 and IFNα were below the lower detection limit. Production of IFNγ was greater by PBMCs of H1N1-infected patients after stimulation either with PHA (p: 0.010 compared with healthy volunteers) or with *S.pneumoniae* (p: 0.029 compared with healthy volunteers). {#pone-0008393-g004} Effects in the Adaptive Immune System {#s2c} ------------------------------------- Infection by the H1N1 virus was accompanied by significant decrease of CD4-lymphocyte counts (p: 0.003 compared with healthy volunteers) and of B-lymphocyte counts (p\<0.0001 compared with healthy volunteers). A significant increase of Tregs was also found compared with healthy volunteers (p: 0.001) ([Figure 5](#pone-0008393-g005){ref-type="fig"}). {#pone-0008393-g005} Mean rates of apoptosis of CD4-lymphocytes were 9.94%, 17.7% and 11.2% among healthy volunteers, among patients with flu-like syndrome and among patients infected by the H1N1 virus respectively (p non-significant between groups). Respective mean rates of apoptosis of B-lymphocytes were 16.2%, 18.4% and 17.3% (p non-significant between groups). Respective mean rates of apoptosis of CD8-lymphocytes were 36.9%, 44.1% and 39.3% (p non-significant between groups). Comparisons between H1N1-Infected Patients without and with Pneumonia {#s2d} --------------------------------------------------------------------- Any of the above estimated parameters of the innate and adaptive immune systems were compared between 25 H1N1-infected patients without pneumonia and six patients with H1N1-related pneumonia. No differences were found between them with the sole exception of Tregs counts being greater among the latter compared with the former (p\<0.0001) ([Figure 6](#pone-0008393-g006){ref-type="fig"}). {#pone-0008393-g006} Serum Cytokines {#s2e} --------------- Concentrations of TNFα and IL-1β in serum did not differ between the three groups. Those of IL-6 were higher in serum of patients with flu-like syndrome compared with healthy volunteers (p: 0.025) and in patients with H1N1 infection compared with healthy volunteers (p: 0.034) ([Figure 7](#pone-0008393-g007){ref-type="fig"}). {#pone-0008393-g007} Over-Time Changes {#s2f} ----------------- Absolute counts of monocytes and of B-lymphocytes of patients infected by the H1N1 virus at baseline and after 48 hours are shown in [Figure 8](#pone-0008393-g008){ref-type="fig"}. Counts of monocytes did not change; however absolute counts of B-lymphocytes were increased (p: 0.034 compared with baseline). {#pone-0008393-g008} Discussion {#s3} ========== The emerging flu pandemic by the H1N1 virus creates considerable dilemmas in all health-care authorities about the real threat for the human host. It is traditionally been conceived that danger to the host is created when infection by an influenza type A strain predisposes to secondary infections by bacterial pathogens [@pone.0008393-Morens1]. Estimation of that danger for the public health requires in-depth knowledge of the effects of the emerging H1N1 virus in the innate and adaptive immune responses of the host. To fully elucidate this, immune responses of laboratory-confirmed cases were studied and compared with those of healthy volunteers and those of patients with flu-like syndrome who were negative for infection by H1N1. Results revealed that within the first two days of advent of the first symptoms, considerable changes of both the innate and the adaptive immune responses were found among patients infected by the H1N1 virus. Principal changes were a) increase of absolute monocyte counts; b) selective defect of TNFα and IFNγ production from PBMCs after stimulation with *S.pneumoniae*; and c) increase of absolute counts of Tregs mainly observed among patients with H1N1-related pneumonia. Despite the increase of absolute monocyte counts, the expression of HLA-DR on monocytes remained very high. This is of considerable importance for the effect of H1N1 infection on our immune system. High level of expression of HLA-DR on monocytes is an index of the ability of the innate immune system for antigen-presentation. Suppression of the expression of HLA-DR on monocytes is characteristic of immunoparalysis that supervenes in moribund conditions like sepsis and that creates predisposition for super-infection by a variety of pathogens [@pone.0008393-Tschoeke1]. This does not seem to happen in the event of infection by H1N1 influenza. To fully clarify the effect of infection by H1N1 on the monocyte function, PBMCs of H1N1-infected patients were stimulated with a variety of stimuli representative of a broad range of pathogens like Gram-negative bacteria, Gram-positive cocci and fungi. Production of cytokines was adequate for most of the applied stimuli being similar to that of healthy volunteers. Pro-inflammatory cytokines were also estimated in serum of patients. IL-6 was increased in both patients with flu-like syndrome and patients with H1N1 infection. In an earlier study of 39 patients with either influenza A or B in Hong Kong during the first semester of 2006 i.e. well before the arrival of H1N1, concentrations of pro-inflammatory cytokines were estimated in plasma and found particularly elevated [@pone.0008393-Lee1]. Defective cytokines responses were noted after stimulation of PBMCs with PHA and with *S.pneumoniae* and involved production of TNFα and of IFNγ among the panel of estimated cytokines. This implies that infection by H1N1 creates a selective impairment of the innate immune response for *S.pneumoniae* probably mediated through the T~H~1 immune response. Infection by H1N1 was also accompanied by alterations of the adaptive immune responses. Decreases of CD4-lymphocytes and of B-lymphocytes and increase of Tregs were observed. The former decreases occurred not only in H1N1-infected patients but also in enrolled patients with flu-like syndrome. Apoptosis was not a mechanism involved in the induction of these changes. However, increase of the absolute count of Tregs was a predominant finding of H1N1 infection particularly when infection involved the lower respiratory tract. This may be a sign of an attempt of the host to counterbalance exaggerated immune responses since Tregs are acting by enhancing endogenous anti-inflammatory responses [@pone.0008393-Majlessi1]. B-lymphocytes also started to increase within the first 48 hours after baseline counting. This is a very optimistic sign for the impact of H1N1 infection on our health since B-lymphocytes are responsible for antibody production. However several limitations of this study should be addressed. Viral load of H1N1 and probable correlations with the described immune alterations were not assessed. It should also be underscored that the study was focused on circulatory effector cells whereas potent immune cells may be accumulated in the infection site. Even if this is the case, the validity of the study remains intact since results describe a qualitative derangement of circulatory effector cells within the first 48 hours of initiation of symptoms of infection by the new H1N1 virus. The presented results revealed that infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to *S.pneumoniae*. This is accompanied by alterations of the adaptive immune responses predominated by increase of Tregs. The significance of the presented results is underscored by three key-elements: a) alterations of the immune responses are shown even with a study enrolling a relative few number of patients; b) H1N1-infected cases are either of moderate severity or of considerable severity since they are all presented with fever above 38.5°C whereas pneumonia is presented in six; and c) enrolled patients infected by H1N1 influenza are young of a mean age of 28.4 years without any underlying disease. It may be postulated that since impairments of the immune responses for *S.pneumoniae* are even seen for young infected patients, these findings have considerable value for patients with underlying diseases predisposing to pneumococcal infections. It should be further underscored that reported cases of severe infection by the H1N1 virus accompanied by pneumonia and increased mortality were between 15 and 44 years old [@pone.0008393-PerezPadilla1], [@pone.0008393-Chowell1] i.e. within the age range of patients enrolled in the present study. In all cases, the results of the present study may help to better understand the life cycle of H1N1 in the human host and to improve our strategies to control it. Methods {#s4} ======= Ethics Statement {#s4a} ---------------- The study was performed during the period July-August 2009. The study protocol was approved by the Ethics Committee of the ATTIKON University Hospital of Athens. All patients admitted in the emergency department for flu-like symptoms and who provided written-informed consent were eligible. Only patients with core temperature greater than 38.5°C were further screened for enrolment. Study Design {#s4b} ------------ Inclusion criteria were: a) written informed consent; b) age more or equal to 16 years; c) symptoms compatible with infection by H1N1 as already defined [@pone.0008393-Health1]; d) core temperature above 38.5°C; and e) start of symptoms within the last two days. Exclusion criteria were: a) deny to consent; b) known infection by the human immunodeficiency virus (HIV); c) neutropenia defined as an absolute neutrophil count equal to or below 500 neutrophils per microliter (µl) of blood; d) oral intake of corticoids defined as more than 1 mg/kg of body weight of equivalent prednisone for more than one month. Enrolled patients underwent a detailed work-out comprising case-history and epidemiological history, thorough physical examination, chest X-ray if considered necessary, white blood cell count, and urine analysis for detection of leukocytes and nitrate and detection of antigens of *Streptococcus pneumoniae* and of *Legionella pneumophila*. If the above work-out failed to disclose the presence of bacterial infection, a pharyngeal smear was collected with a swab. Infection by the H1N1 virus was diagnosed by the real-time reverse transcriptase polymerase chain reaction detecting the presence of H1N1 RNA in the infected cells (rRT-PCR) [@pone.0008393-Petric1]. A patient was considered to present with a lower respiratory tract infection (LRTI) due H1N1 virus when a local infiltrate was diagnosed in chest X-ray and H1N1 RNA was detected in the pharyngeal swab, as defined elsewhere [@pone.0008393-PerezPadilla1], [@pone.0008393-Chowell1]. Patients were followed-up until complete resolution of symptoms. In parallel with the collection of the pharyngeal smear, 25 milliliters of whole blood were collected after venipuncture of one forearm antecubital vein under sterile conditions. Twenty ml blood were collected into two EDTA-coated tubes (Vacutainer, Becton Dickinson, Cockeysville Md) and another 5 were collected into sterile and pyrogen-free tubes (Vacutainer) and transferred immediately to the laboratory for further analysis. Blood was also sampled from 10 healthy volunteers. For patients diagnosed with H1N1 infection, 5 ml of blood was also sampled after 48 hours as described above. Laboratory Procedure {#s4c} -------------------- In whole blood collected into the first tube, red blood cells were lyzed with ammonium chloride 1.0 mM. White blood cells were washed three times with phosphate buffered saline (PBS, pH 7.2) (Merck, Darmstadt, Germany) and subsequently incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD3, anti-CD4, anti-CD14 and anti-CD19 and the protein ANNEXIN-V at the flurochrome fluorescein isothiocyanate (FITC, emission 525 nm, Immunotech, Marseille, France); with the monoclonal antibodies anti-CD4, anti-CD8, anti-CD14, anti-CD(16+56), anti-CD25 and anti-HLA-DR at the fluorochrome phycoerythrin (PE, emission 575 nm, Immunotech); with the monoclonal antibody anti-CD3 and anti-CD127 at the fluorochrome ECD (emission 613 nm, Immunotech); with the monoclonal antibody anti-CD45 at the fluorochrome PC5 (emission 650 nm, Immunotech); and with 7-AAD at the fluorocolour PC7 (emission 670 nm, Immunotech). Fluorospheres (Immunotech) were used for the determination of absolute counts. The following combinations were studied: anti-CD3/anti-CD4/anti-CD45 for CD4-lymphocytes; anti-CD3/anti-CD8/anti-CD45 for CD8-lymphocytes; anti-CD3/anti-CD(16+56)/anti-CD45 for Natural Killer (NK)-cells and for NKT-cells; anti-CD4-/anti-CD25/anti-CD127/anti-CD45 for T-regulatory cells (Tregs); anti-CD19/anti-CD45 for B-lymphocytes; anti-CD14/anti-HLA-DR/anti-CD45 for immunoparalysis of monocytes; ANNEXIN-V/anti-CD4/anti-CD3/7-AAD for apoptosis of CD4-lymphocytes; ANNEXIN-V/anti-CD8/anti-CD3 for apoptosis of CD8-lymphocytes; ANNEXIN-V/antiCD14/7-AAD for apoptosis of monocytes; ANNEXIN-V/anti-CD19/7-AAD for apoptosis of B-lymphocytes. Cells were analyzed after running through the EPICS XL/MSL flow cytometer (Beckman Coulter Co, Miami, Florida) with gating for lymphocytes or monocytes based on their characteristic FS/SS scattering. Cells staining negative for CD3 and positive for CD(16+56) were considered NK-cells; those staining positive for both CD3 and CD(16+56) were considered NKT- cells. Cells staining positive for both CD4 and CD25 and negative for CD127 were considered Tregs, according to other authors [@pone.0008393-2], [@pone.0008393-3]. In all analyzed cell subtypes, apoptotic cells stained positive for ANNEXIN-V and negative for 7-AAD. Isotypic IgG controls were used for each patient. To verify the appropriateness to discriminate cells staining positive for both CD4 and CD25 and negative for CD127 as Tregs, blood sampling was repeated for seven newly enrolled patients; two healthy volunteers, two patients with flu-like syndrome and three patients with H1N1 infection. Absolute counts of Tregs were estimated both as described above and with the PE anti-human Foxp3 staining set (eBioscience Inc., San Diego, USA). Median difference of the two assays was 5.88%. Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected in the second tube after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany) for 20 minutes at 1400 g. After three washings in ice-cold PBS pH 7.2, PBMCs were counted in a Neubauer plate with trypan blue exclusion of dead cells. They were then diluted in RPMI 1640 enriched with 2 mM of L-glutamine, 500 µg/ml of gentamicin, 100 U/ml of penicillin G and 10 mM of pyruvate (Biochrom) and suspended in wells of a 96-well plate. The final volume per well was 200 µl with a density of 2×10^6^ cells/ml. PBMCs were exposed in duplicate to the following stimuli for 24 hours at 37°C in 5% CO~2~: 10 ng/ml of *Escherichia coli* O55:B5 lipopolysaccharide (LPS, Sigma, St. Louis, USA); 5 µg/ml of phytohemagglutin (PHA, EMC microcolecctions, Tübingen, Germany); and 2×10^5^ colony forming units/ml of each of the following heat-killed isolates: *Candida albicans*; *Pseudomonas aeruginosa*; methicillin-resistant *Staphylococcus aureus* (MRSA) producing Panton-Valentine leukocidin (PVL); and penicillin-susceptible *S.pneumoniae* (PSSP). All the above were clinical isolates derived from the blood of different patients with documented infections. Resistance of *S.aureus* to methicillin and production of PVL were documented by the detection of the *mec*A gene and the *luc*S-PV gene respectively by PCR [@pone.0008393-4], [@pone.0008393-Witte1]. PSSP has been isolated from a patient with LRTI and bacteremia and it has already been described in a previous study [@pone.0008393-GiamarellosBourboulis1]. Concentrations of tumour necrosis factor-alpha (TNFα) and of interleukin-1beta (IL-1β) were estimated in cell supernatants in duplicate by an enzyme immunoassay (R&D, Minneapolis, USA). Those of interferon-gamma (IFNγ) were estimated only in supernatants of PHA-stimulated cells and of PSSP-stimulated cells (Diaclone, Paris, France). Those of IL-6, of IL-18 and of IFNα were estimated only in supernatants of PSSP-stimulated cells (Diaclone, Paris, France and MBL, Nagoya, Japan). The lowest detections limits were: for TNFα 80 pg/ml; for IL-1β 20 pg/ml; for IL-6 60 pg/ml; for IL-18 130 pg/ml; for IFNα 40 pg/ml; and for 100 IFNγ pg/ml. Blood collected into sterile and pyrogen-free tubes was centrifuged and serum was stored at −70°C until assayed. Concentrations of TNFα, IL-1β and IL-6 were estimated as described above. In blood samples collected 48 hours after the baseline, absolute counts of monocytes and of B-lymphocytes were determined as described above. Statistical Analysis {#s4d} -------------------- Subjects were divided into healthy volunteers; into those presenting with flu-like syndrome; and into those infected by the H1N1 virus. Results were expressed as means ± SE. Comparisons between groups were compared by one-way analysis of variance (ANOVA) with post-hoc Bonferroni analysis for the avoidance of random correlations. Comparisons between H1N1-infected patients without and with pneumonia were done by Student\'s "t-test". Comparisons between consecutive samplings were done by paired "t-test". Any value of p below 0.05 was considered significant. **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**This study was funded by kind donations of the following pharmaceutical industries in alphabetical order: Unipharma SA, Athens, Greece; Vianex SA, Athens, Greece; Wyeth Hellas SA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: EJGB HG. Performed the experiments: EJGB AS TK MG AP NP. Analyzed the data: EJGB MR AA FB PK TT SA LG EG Dk FK. Wrote the paper: EJGB. Data collection: MR, AA, FB, PK, DK, FK, GP, VS, PP, AP, KK, HG. Drafting of the manuscript: MR, AA, FB, PK, AS, TK, MG, AP, TT, NP, SA, LG, EG, DK, FK, MM, GP, VS, PP, AP, KK, HG.

See also: [G Kustatscher *et al*](https://doi.org/10.15252/msb.20177548) (August 2017) Since the earliest explorations in post‐genomics, one truth seems universal---genes in proximity in a genome tend to be co‐expressed (Hurst *et al*, [2004](#msb177961-bib-0003){ref-type="ref"}), at least when considering mRNA levels. Why is this? An appealing explanation is that functionally related genes whose protein products are needed at the same time, evolve to be close in the genome enabling coordinated regulation. A recent paper by Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}) puts a final nail in the coffin for this hypothesis, at least as an explanation for most mRNA‐level co‐expression in humans. They show only for a few rare gene pairs is co‐expression at the mRNA level translated into protein‐level co‐expression. For most genes, RNA‐level co‐expression is dampened at the protein level (Fig [1](#msb177961-fig-0001){ref-type="fig"}). This, however, they suggest, does not mean that the close gene coupling and co‐expression is not without utility or consequence. {ref-type="ref"}).](MSB-13-943-g001){#msb177961-fig-0001} Genes in a bidirectional orientation and in very close proximity (\< 1 kb) account for an unexpectedly high proportion (\~10%) of gene pairs in our genome (Trinklein *et al*, [2004](#msb177961-bib-0009){ref-type="ref"}). Such genes tend to be co‐expressed at the RNA level and seen together as a pair in the mouse genome (Trinklein *et al*, [2004](#msb177961-bib-0009){ref-type="ref"}). It could be supposed that the co‐expression of the two genes in such a pair enables protein‐level coordination. To test this idea, Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}) examined housekeeping genes expressed in human lymphoblastoid cell lines and, unusually, examined both RNA‐ and protein‐level co‐expression. Looking at 167 such very close bidirectional gene pairs, they found 31 showing strong co‐expression at the RNA level. Of these, only three gene pairs showed sustained co‐expression at the protein level. Generally, those buffered at the protein level do not share common functions, while those retaining co‐expression do (Kustatscher *et al*, [2017](#msb177961-bib-0004){ref-type="ref"}). The same RNA‐level co‐expression and protein‐level attenuation are seen for genes in close proximity not in bidirectional orientation. These results suggest that the protein‐level functional co‐operation model for gene order evolution explains some, but not very much, of the observed genome organization, concurring what prior evidence in yeast. Then why are so many genes so tightly coupled in bidirectional orientation (Trinklein *et al*, [2004](#msb177961-bib-0009){ref-type="ref"}) and why are so many neighbouring genes co‐expressed to some degree? An attractive null model to explain neighbour co‐expression proposes that open chromatin affects blocks of genes and enables their expression (Raj *et al*, [2006](#msb177961-bib-0007){ref-type="ref"}). Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}) add to this null model showing that genes that are co‐expressed at the mRNA level tend to reside in the same or interacting genome "compartments", identified in Hi‐C data as showing long‐range interactions. Importantly, this is true for neighbour genes and genes not in the immediate 1D vicinity but that nonetheless are interacting in 3D space. While this co‐expression does not translate to the protein level, the authors sensibly suggest that there is no reason to suspect any active buffering. Post‐transcriptional effects such as different miRNA regulation, different ribosome occupancy and different protein half‐lives are probably enough to explain why an initial signal of mRNA‐level co‐expression disappears at the protein level. Quantitative modelling of the expected collapse of co‐expression, from RNA into protein level, for random gene pairs would help clarify whether the attenuation is any more than would be expected. One enigma is why genes that interact in 3D are functionally more similar than expected by chance (Thevenin *et al*, [2014](#msb177961-bib-0008){ref-type="ref"}) but apparently not co‐expressed at the protein level. While this RNA co‐expression of co‐compartment genes does not translate to the protein level (Fig [1](#msb177961-fig-0001){ref-type="fig"}), Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}) also report that genes whose protein products are co‐expressed do not correlate with spatial organization of the DNA. For these functionally related proteins, the authors suggest that post‐transcriptional adaptations may well be required to preserve co‐expression (e.g. selection for similar protein half‐life). It is indeed striking that unlinked genes whose proteins are part of the same protein complex have stronger correlations at the protein level than at the RNA level (Kustatscher *et al*, [2017](#msb177961-bib-0004){ref-type="ref"}). So perhaps co‐expression is mostly just a product of chromatin dynamics and, with very few exceptions, there is nothing to write home about. Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}), however, argue that there is most likely more to it than this. Indeed, if gene expression parameters are modulated at a chromatin level or by local interactions, gene order might well evolve in response to such effects, assuming selection to be strong enough. In this context, a key further property of genomes is the observed clustering of housekeeping/essential genes (Lercher *et al*, [2002](#msb177961-bib-0005){ref-type="ref"}; Pal & Hurst, [2003](#msb177961-bib-0006){ref-type="ref"}). Co‐expression of essential genes is not so important but since essential genes are, by definition, dose‐sensitive, it is important to ensure that their dose never sinks to low levels by chance. Indeed, as expected, essential genes have low noise in expression and so rarely risk the calamitous fate of accidentally being depleted (Batada & Hurst, [2007](#msb177961-bib-0001){ref-type="ref"}). In this context, it was suggested that genomic organization on both a cluster (Batada & Hurst, [2007](#msb177961-bib-0001){ref-type="ref"}) and gene‐pair (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}) level might be part of the solution to the problem of noise control for dose‐sensitive genes. Keep chromatin permanently open and transcription is always possible. Allow genes to maintain each others' open chromatin status and expression level fluctuations can be smoothed. The effect can be most profound for genes in bidirectional orientation and genomically very close: one gene primes the neighbour (and vice versa) and in so doing stabilizes expression levels (low noise) (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}). Toy models of this mutual interdependence predict that RNA co‐expression is an epiphenomenon of gene pair dynamics of low noise genes (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}). Preliminary data supported this showing that essential gene clusters are indeed low noise clusters (Batada & Hurst, [2007](#msb177961-bib-0001){ref-type="ref"}), that transgenes inserted into such low noise clusters adopt a low noise level (Chen & Zhang, [2016](#msb177961-bib-0002){ref-type="ref"}), that genes in bidirectional orientation have especially low noise (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}) and that essential genes are especially likely to be found in bidirectional orientation (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}). Kustatscher *et al* ([2017](#msb177961-bib-0004){ref-type="ref"}) importantly show that, indeed, for highly gene dense regions gene clustering is associated with reduced noise at both RNA and protein levels, as this model predicts. Gene density is here potentially important as gene--gene non‐independence is to a first approximation dependent on between‐gene distance profile (Wang *et al*, [2011](#msb177961-bib-0010){ref-type="ref"}). Thus, there seem to be three viable models to account for pervasive co‐expression of neighbouring genes at the mRNA level. First, for a very limited number of neighbouring gene pairs co‐expression *per se* is an important part of their biology, these pairs being especially highly co‐expressed, functionally related and, at least in yeasts, preserved as a pair over evolutionary time. Second, many other signals of co‐expression are better considered a happenstance of chromatin dynamics. But, third, some co‐expression appears to viably be explained as an epiphenomenon of selection for low noise of dose‐sensitive genes, by adoption of the dynamics of DNA that enable co‐expression. What is unclear is how common this adoption is and whether, for example, it can explain why gene density varies around a genome. Are housekeeping clusters high‐density clusters to enable this sort of noise control by enabling non‐independence in expression? Or might we instead be looking at a mutation bias? For reasons still unclear, essential gene clusters in yeast have unusually low recombination rates (Pal & Hurst, [2003](#msb177961-bib-0006){ref-type="ref"}). Could a relationship between recombination and sequence gain/loss dynamics explain such a trend?

Introduction {#s1} ============ Biological invasions are problematic in terrestrial, marine, and freshwater ecosystems. Native communities are often severely affected by ecological effects of invaders, anthropogenic habitat alterations, and their combinations [@pone.0077597-Vitousek1], [@pone.0077597-Mack1], [@pone.0077597-Pyek1], [@pone.0077597-Strayer1]. In freshwaters, non-indigenous invasive crayfish species (NICS) belong to a group of invaders with substantial effects on autochthonous biota and habitats throughout the world [@pone.0077597-Holdich1]. They negatively influence indigenous crayfish populations, along with other aquatic organisms, through competition, disease transfer, and predation, and can alter habitat conditions through extensive burrowing, reducing available food, and resource depletion [@pone.0077597-Holdich2]. In addition, NICS often establish populations in areas of deteriorated environmental conditions and prevent restoration of native stocks even after improvement in water and habitat quality [@pone.0077597-SoutyGrosset1]. Non-indigenous invasive crayfish use a wide range of ecological features to invade inhabited sites, compete with native species, and spread to new localities. Once established, they may eradicate indigenous crayfish species (ICS) and reduce the abundance of forage species such as gastropods, algae, and macrophytes, thereby executing a trophic cascade effect on the ecosystem [@pone.0077597-Nystrm1], [@pone.0077597-Klocker1]. Other factors facilitating NICS establishment and spread are crayfish plague infection (*Aphanomyces astaci*, Schikora 1906) [@pone.0077597-Kozubkov1], climate fluctuations [@pone.0077597-DiguezUribeondo1], and human activity such as deliberate or accidental stocking and watercourse regulation [@pone.0077597-SoutyGrosset1]. In general, a complex system of mechanisms supports invasions [@pone.0077597-Strayer1]. The most important factors in establishing and spreading new invasive species involve anthropogenic activity [@pone.0077597-Pyek1], [@pone.0077597-Perdikaris1] and, in crayfish, the presence of the crayfish plague pathogen, fatal to European ICS [@pone.0077597-Vogt1]. Non-indigenous crayfish exhibit a number of competitive advantages enabling them to establish dense and expanding populations. In general, invasive NICS can be considered r-selected species compared to their K-selected ICS antagonists. This difference is demonstrated by earlier maturation, higher fecundity, faster growth, and a higher level of activity and aggressiveness in NICS [@pone.0077597-Lindqvist1]. With respect to invasive crayfish of the Cambaridae, reproductive characteristics play a significant role. Obligate parthenogenesis in marbled crayfish (*Procambarus fallax* f. *virginalis*) [@pone.0077597-Martin1], [@pone.0077597-Martin2] and facultative parthenogenesis in spiny-cheek crayfish (*Orconectes limosus*) [@pone.0077597-Bui1] enables the recovery of small populations and the establishment of a new viable population from few specimens. Apart from the marbled crayfish, which reproduces by apomictic parthenogenesis only, and the facultative parthenogenetic spiny-cheek crayfish, the only decapod species for which a potential for asexual reproduction has been suggested is the red swamp crayfish (*Procambarus clarkii*) [@pone.0077597-Yue1], [@pone.0077597-Yue2]. This characteristic undoubtedly plays an important part in the success of NICS invasion. However, other specifics of the reproduction and life cycle are still not completely understood. With respect to *O. limosus*, which colonized at least 20 European countries from 90 introduced specimens, without an apparent bottleneck effect [@pone.0077597-Filipov1], the role of environmental, behavioral, and chemical cues in the switch from sexual to asexual reproduction need to be investigated. The behavior may be associated with their prolonged mating period. Autumn and spring mating seasons extend from 7 to 8 months (∼ from September to April). Females store sperm in a ventral body cavity, the *annulus ventralis*, until spawning in April/May [@pone.0077597-Andrews1], [@pone.0077597-Hamr1], [@pone.0077597-Kozk1]. The hypothetical basis for this strategy are numerous, with three key provisions: a) multiple paternity, to achieve as many mates as possible to increase the genetic diversity of progeny; b) mate selection, the search for a male with a preferred traits, and c) increasing chance of successful mating in a low population. Probably only after an unsuccessful mating season does asexual reproduction come into play [@pone.0077597-Bui1]. The aim of this study was to increase understanding of reproductive mechanisms in an important aquatic invasive species by evaluating differences in reproductive output among crayfish groups under four mating regimes. In accordance to above described hypothetical basis we can indicate a number of potential differences among the studied mating regimes. Females with the opportunity to mate in both mating seasons should have the highest possibility of successful selecting of the best mates or collecting spermatophores from the maximum number of males without additional effort. On the other hand, females mating only in spring would have less time to seek a suitable mate and additionally, oogenesis could be hypothetically negatively influenced due to lack of chemical stimulation by mature males during the autumn period. Autumn only mating provides females a shorter time for seeking a suitable mate and necessitates the long term storage of the spermatophores until the spring spawning season. Such mechanism could also affect reproductive success of females. The fourth regime excludes male participation entirely. It implies the use of special mechanisms to reproduce in such conditions (switching to asexual reproduction). There were therefore assumed hypothetical conspicuous differences in reproduction success between above described mating regimes. There could be hypothetically assumed conspicuous differences in reproduction success between above described mating regimes. Materials and Methods {#s2} ===================== No specific permissions were required for sampling crayfish (capture, manipulation, transport) for presented study. The locality where capture was made is not included in any protected area with requirement of any permission. The field studies did not involve endangered or protected species. Crayfish used in experimental work is dangerous invasive species and there was not necessary any permission to capture them. Animals {#s2a} ------- Spiny-cheek crayfish were captured (n  =  1157; carapace length, CL  =  24.1±7.8 mm) in the Černovický Brook (South Bohemia, Czech Republic; 49°15'51"N, 14°43'02"E) in August 2007. Captured animals were separated according to sex and held under laboratory conditions for 30 days acclimation. Adult individuals (n  =  596) were selected according to glair gland development in females and form I gonopods presence in males [@pone.0077597-Bui2], [@pone.0077597-Bui3]. Of these, 150 females and 75 males were randomly selected for the trial. Experimental conditions {#s2b} ----------------------- Animals were maintained in circular tanks supplied with ∼ 3 shelters per crayfish. Photoperiod and water temperature were natural ambient, provided by natural daylight and a flow-through water supply. During the experiment, dissolved oxygen (Oxi 315i, WTW GmbH, Weilheim, Germany), and pH (pH 315i, WTW GmbH, Weilheim, Germany) were measured daily. Tanks were cleaned regularly. Crayfish were fed frozen chironomid larvae, carrots, and fish pellets to satiation. Experimental design {#s2c} ------------------- Selected crayfish were divided at random into five tanks. Mean carapace length (CL) and weight (w) did not differ among groups (ANOVA, F*~CL~*  =  0.55, P*~CL~*  =  0.650, F*~w~*  =  0.68, P*~w~*  =  0.565) for either females or males (ANOVA, F*~CL~*  =  1.27, P*~CL~*  =  0.287, F*~w~*  =  1.53, P*~w~*  =  0.223) ([Table 1](#pone-0077597-t001){ref-type="table"}). Three groups, thirty females and fifteen males per each tank, were allowed to move freely about the tanks, so that visual, chemical, and tactile contact, including mating behavior, was possible. Group 4 consisted of thirty free-ranging females and fifteen males placed in separate cages of ∼ 3 mm plastic mesh suspended approximately 0.3 m above the tank bottom. Therefore chemical communication between the sexes was possible, but physical contact was prevented. The groups described above differed only in the duration of male presence as follows: Group 1 - males available only during the autumn mating season (from October to January); Group 2 - males available only during the spring mating season (from January to May); Groups 3 - females and males maintained together throughout the mating season (from October to May); and Group 4 - without physical contact of sexes ([Table 1](#pone-0077597-t001){ref-type="table"}). 10.1371/journal.pone.0077597.t001 ###### Experimental groups with number of specimens, mean carapace length (CL), and the duration of male exposure. {#pone-0077597-t001-1} Group No. of females Females CL (mm) No. of males Males CL (mm) The exposure time of males ----------------------------- ---------------- ----------------- -------------- --------------- --------------------------------------------------------- 1--Autumn mating only 30 31.2±2.92^a^ 15 32.9±3.95^a^ 7.10.2007 -- 17.1.2008 2--Spring mating only 30 30.8±2.74^a^ 15 35.1±2.63^a^ 17.1.2008 -- 12.5.2008 3--Autumn and spring mating 60 31.7±3.20^a^ 30 33.9±3.57^a^ 7.10.2007 -- 12.5.2008 4--Without mating 30 31.2±2.88^a^ 15 33.1±2.55^a^ 7.10.2007 -- 12.5.2008[\*](#nt102){ref-type="table-fn"} Different alphabetic superscripts in the same column indicate significant differences at α  =  0.05 (ANOVA, Tukey post hoc test). Placed in cage 0.3 m above the tank bottom. Physical contact with females was prevented. When spawning was completed in all groups, males were removed and females were kept separately until the hatched juveniles reached the 2^nd^ developmental stage [@pone.0077597-Andrews1]. During incubation, egg clutches were examined weekly to assess egg loss. Juveniles were removed from the successfully reproducing females and counted. Seven microsatellite loci of the females and their offspring (randomly selected 40 specimens from each female) from Group 4 were analyzed, according to Buřič *et al*. [@pone.0077597-Bui1] and Hulák *et al*. [@pone.0077597-Hulk1], to confirm the asexual reproduction in non-mated females. In addition, females and their offspring from "mated" groups were analyzed in the same way to confirm paternal alleles and thus sexual reproduction. Data analysis {#s2d} ------------- The data were analyzed with Statistica 9.0 (StatSoft, Inc.). All values were examined for normal distribution (Kolmogorov-Smirnov test) and homoscedaticity (Levene test). The one way ANOVA with Tukey's post hoc test was used for comparing crayfish size, weight, and fecundity at the 2^nd^ developmental stage. The chi-square test was used for comparing crayfish mortality, egg loss, spawning and hatching success. The null hypothesis was rejected at α  =  0.05. Data are presented as means ± standard deviation. Results {#s3} ======= The lowest mortality of spiny-cheek crayfish females was observed in Group 4 (Chi-square  =  11.39, P  =  0.010). Female mortality in Groups 1, 2, and 3 occurred during the period when males were stocked with the females. Females in all experimental groups successfully produced eggs ([Table 2](#pone-0077597-t002){ref-type="table"}) without differences between experimental groups (Chi-square  =  0.36, P  =  0.948). Spawning took place from May 15 to 27. Significant, including total, eggs losses occurred during incubation for several females, mainly in Groups 3 and 4 ([Table 2](#pone-0077597-t002){ref-type="table"}, Chi-square  =  30.46, P \< 10^−5^). Hatching of viable offspring was observed in all groups without significant differences between groups (Chi-square  =  1.33, P  =  0.721) and took place between June 10 and 17. The number of juveniles per female at the 2^nd^ developmental stage, immediately prior to leaving the mother, was high in all groups ([Table 2](#pone-0077597-t002){ref-type="table"}). The number of offspring in the non-mated Group 4 was significantly lower (more than 40% fewer) than in the other four groups (ANOVA, F  =  5.45, P  =  0.002), which did not differ from one another. Analysis of seven microsatellite loci showed that females from Group 4 produced genetically homogeneous offspring identical to the mother [@pone.0077597-Bui1] ([Table 3](#pone-0077597-t003){ref-type="table"}). In mated groups were found paternal alleles, which implying sexual reproduction ([Table 4](#pone-0077597-t004){ref-type="table"}). 10.1371/journal.pone.0077597.t002 ###### The number (n) of females and percent (%) mortality, and spawning in each group, the number and percent of crayfish females in which \> 50% or total egg losses were observed, the number and percent of females hatching eggs, and the number of juveniles in 2^nd^ developmental stage (mean ± standard deviation) in each group. {#pone-0077597-t002-2} Experimental group Initial stock Mortality Successful spawning Loss of\> 50% eggs Total lossof eggs Successful hatching Number of juveniles ----------------------------- --------------- ----------- --------------------- -------------------- ------------------- --------------------- --------------------- --- --------- ---- ---------- ----------------- 1--Autumn mating only 30 4 13.3^a^ 25 96.2^a^ 1 4.0^b^ 0 0.0^b^ 25 100.0^a^ 142.96±55.38^a^ 2--Spring mating only 30 6 20.0^a^ 22 91.7^a^ 1 4.6^b^ 0 0.0^b^ 22 100.0^a^ 133.73±43.95^a^ 3--Autumn and spring mating 60 8 13.3^a^ 52 100.0^a^ 10 19.2^a^ 7 13.5^a^ 45 86.5^a^ 138.00±44.19^a^ 4--Without mating 30 1 3.3^b^ 28 96.6^a^ 8 28.6^a^ 2 7.1^a^ 26 92.9^a^ 96.46±46.82^b^ Different alphabetic superscripts in the same column indicate significant differences at α  =  0.05 (Chi-square test for mortality, spawning, egg loss and hatching; ANOVA, Tukey post hoc test for number of juveniles). excluding dead females, \*\* in successfully spawned females. 10.1371/journal.pone.0077597.t003 ###### Example of multilocus genotypes of 5 spiny-cheek crayfish females reproduced by apomictic parthenogenesis (Group 4), and their offspring. {#pone-0077597-t003-3} Allele sizes (bp) for seven microsatellite loci --------------- ------------------------------------------------- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- Female 1 297 301 278 285 171 195 145 158 151 153 208 222 297 301 juvenile 1.1 297 301 278 285 171 195 145 158 151 153 208 222 297 301 juvenile 1.2 297 301 278 285 171 195 145 158 151 153 208 222 297 301 juvenile 1.3. 297 301 278 285 171 195 145 158 151 153 208 222 297 301 Female 2 294 302 274 285 195 195 158 158 147 147 222 227 297 297 juvenile 2.1 294 302 274 285 195 195 158 158 147 147 222 227 297 297 juvenile 2.2 294 302 274 285 195 195 158 158 147 147 222 227 297 297 juvenile 2.3 294 302 274 285 195 195 158 158 147 147 222 227 297 297 Female 3 297 306 281 283 195 225 158 158 147 161 224 229 293 297 juvenile 3.1 297 306 281 283 195 225 158 158 147 161 224 229 293 297 juvenile 3.2 297 306 281 283 195 225 158 158 147 161 224 229 293 297 juvenile 3.3 297 306 281 283 195 225 158 158 147 161 224 229 293 297 Female 4 301 301 278 278 195 225 158 158 147 147 208 229 295 301 juvenile 4.1 301 301 278 278 195 225 158 158 147 147 208 229 295 301 juvenile 4.2 301 301 278 278 195 225 158 158 147 147 208 229 295 301 juvenile 4.3 301 301 278 278 195 225 158 158 147 147 208 229 295 301 Female 5 292 298 274 274 171 225 158 158 147 161 222 229 301 301 juvenile 5.1 292 298 274 274 171 225 158 158 147 161 222 229 301 301 juvenile 5.2 292 298 274 274 171 225 158 158 147 161 222 229 301 301 juvenile 5.3 292 298 274 274 171 225 158 158 147 161 222 229 301 301 Alleles are given as fragment sizes in base pairs. All analyzed juveniles of these females had multilocus genotypes identical to their mothers, so only three juvenile genotypes are shown. 10.1371/journal.pone.0077597.t004 ###### An example of allelic inheritance after sexual reproduction in spiny-cheek crayfish. {#pone-0077597-t004-4} Allele sizes (bp) for seven microsatellite loci --------------- ------------------------------------------------- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- ----- Female 1 297 297 278 283 195 225 158 158 147 161 227 227 291 295 juvenile 1.1 297 297 283 285 195 195 145 158 147 161 227 227 295 295 juvenile 1.2 297 297 283 285 195 195 145 158 147 161 227 227 295 295 juvenile 1.3. 297 297 281 283 195 195 145 158 147 161 227 227 291 301 Female 2 297 297 278 283 195 229 158 158 147 161 227 227 291 295 juvenile 2.1 297 301 274 278 225 229 145 158 147 161 227 227 291 295 juvenile 2.2 297 301 274 278 225 229 145 158 147 161 227 227 291 295 juvenile 2.3 297 301 274 278 225 229 145 158 147 161 227 227 291 295 Female 3 294 294 274 274 225 225 158 158 147 161 208 208 295 295 juvenile 3.1 294 304 274 274 225 237 158 158 147 163 208 208 295 295 juvenile 3.2 294 304 274 274 225 237 158 158 147 163 208 208 295 295 juvenile 3.3 294 304 274 274 225 237 158 158 147 163 208 208 295 295 Female 4 294 294 283 283 225 225 145 145 147 147 227 227 301 301 juvenile 4.1 294 306 283 283 225 232 145 145 147 147 219 227 301 301 juvenile 4.2 294 306 283 283 225 232 145 145 147 147 227 227 301 301 juvenile 4.3 294 306 283 283 225 232 145 145 147 147 219 227 301 301 Female 5 301 304 274 274 195 225 145 158 147 147 208 227 293 301 juvenile 5.1 294 304 274 278 195 225 145 158 147 147 208 227 295 301 juvenile 5.2 294 301 274 281 225 225 145 158 147 161 208 227 295 301 juvenile 5.3 294 304 274 278 225 229 145 158 147 161 208 208 293 295 Multilocus genotypes for 5 females and their offspring (juveniles) carrying paternal alleles are presented as the sizes (in base pairs) of alleles at seven microsatellite loci. Discussion {#s4} ========== Crayfish are key inhabitants of aquatic ecosystems [@pone.0077597-Momot1]. They often have significant impact, positive as well as negative, on benthic communities. In addition to their ecological role, crayfish have economic value [@pone.0077597-Holdich2]. For these reasons, crayfish have often been transferred from their natural range and, since the 19^th^ century, even across continents, mainly from North America to Europe. This allowed introduction of new pathogens including the oomycete *Aphanomyces astaci*, Schikora, 1903, a crayfish plague disease lethal to European crayfish, leading to massive losses of autochthonous crayfish species. The newly introduced crayfish vectors of the disease successfully established populations in abandoned habitats [@pone.0077597-SoutyGrosset1]. This resulted in the establishment of two groups of crayfish on the old continent: an indigenous species (ICS), characterized by usually a sharply demarcated occurrence, decreased populations, and localization in headwaters or separate backwaters, and a non-indigenous crayfish species (NICS), distinguishable by high population density, rapid spread and establishment in new localities, and wide tolerance to varying habitat conditions. One of the most important NICS is the spiny-cheek crayfish (*Orconectes limosus*), which has been found in European freshwaters for more than 120 years. Despite its wide distribution throughout the continent [@pone.0077597-SoutyGrosset1], the probable source of all *O. limosus* populations in Europe is suggested, according to haplotype variation [@pone.0077597-Filipov1], to be the introduction of 90 specimens into Poland at the end of 19^th^ century [@pone.0077597-McDonald1], [@pone.0077597-Kossakowski1]. The overwhelming success of further invasions of *O. limosus* into numerous European waters probably arose from a combination of factors. First, there was the effect of *O. limosus* resistance to crayfish plague and its transmission to indigenous species. This could have reduced competition with native crayfish populations [@pone.0077597-Kozubkov1], [@pone.0077597-Lindqvist1] and facilitated establishment in newly vacant habitats. Secondly, *O. limosus* exhibit characteristics favorable for competition with ICS, such as aggressive behavior [@pone.0077597-Lozan1], [@pone.0077597-Musil1], tolerance to poor environmental conditions [@pone.0077597-SoutyGrosset1], and the capability of rapid migration [@pone.0077597-Puky1], [@pone.0077597-Bui4]. However, the current distribution in Europe would not have been possible through natural dispersal alone, and was accomplished by a combination of natural spreading from the region of first introduction (via rivers and connecting canals between watersheds) and long distant transport by humans within or between watersheds [@pone.0077597-SoutyGrosset1], [@pone.0077597-Filipov1], [@pone.0077597-Holdich3]. Finally, the uncommon reproductive plasticity of *O. limosus* reinforces these other characteristics. These features, common in the majority of Cambaridae, but specifically *O. limosus* consist of early maturation enabling a short generation cycle [@pone.0077597-Hamr1], [@pone.0077597-Holdich3], [@pone.0077597-Schulz1]; high fecundity even of small females maximizing numbers of offspring [@pone.0077597-Kozk1], [@pone.0077597-Pieplow1], [@pone.0077597-Stucki1]; periodic occurrence of morphologically distinct sexually active (form I) and sexually inactive forms (form II) in males and females providing a mechanism to utilize resources effectively in the most important life stages [@pone.0077597-Bui2], [@pone.0077597-Bui3], [@pone.0077597-Smith1], [@pone.0077597-Hobbs1]; autumn and spring mating maximizing the probability of successful mating [@pone.0077597-Hamr1], [@pone.0077597-Bui4], [@pone.0077597-Stucki1], [@pone.0077597-VanDenBrink1]; the capability of sperm storage to circumvent extreme conditions [@pone.0077597-SoutyGrosset1], [@pone.0077597-Andrews2], [@pone.0077597-Albaugh1]; and parthenogenesis as a tool for alternative reproduction if males are not available [@pone.0077597-Bui1]. The goal of the present work was to clarify reproductive factors having substantial impact on successful establishment of populations at new sites and their subsequent spread. The reasons underlying the double mating season are not known, nor do we have information regarding the impact on reproductive success if a mating season is absent. The four mating regimes used in this trial, optimal as well as deficient, resulted in successful reproduction. The most appropriate situation was when males were present during both the autumn and spring mating seasons. The less adequate regime provided a single mating period either in autumn (expected long term spermatophores storage in *annulus ventralis*, or spring (extensive wait for mating). Under those regimes, a female:male ratio of 2:1 was chosen to provide a sufficient number of potential mates [@pone.0077597-Taugbl1], [@pone.0077597-Reynolds1], [@pone.0077597-Wickins1], while limiting effects of aggressive encounters with males on the fitness of females [@pone.0077597-Gherardi1]. Males were proportionally larger ([Table 1](#pone-0077597-t001){ref-type="table"}) than females, as is recommended for successful mating [@pone.0077597-Andrews2], [@pone.0077597-Wickins1]. The Group 4 regime was the most inadequate variant, as there was no possibility of mating. What are the hypothetical differences among the regimes? Females with the opportunity to mate in both mating seasons had the highest possibility of selecting the best mates or collecting spermatophores from the maximum number of males. Mechanisms of sexual selection in crayfish have been described [@pone.0077597-Villanelli1], [@pone.0077597-Gherardi2], [@pone.0077597-Galeotti1], but it is unclear whether females can strictly limit mating only to the best males. In addition, the number of males from which a female can store the spermatophores is not known. Studies reporting multiple paternity have detected offspring from 2--4 males for a single female [@pone.0077597-Walker1], [@pone.0077597-Yue3]. Moreover, males are known to damage or remove the sperm from prior matings [@pone.0077597-Villanelli1], [@pone.0077597-Berrill1], [@pone.0077597-Rubolini1]. The primary effect of the long mating season may be to maximize the probability of finding a suitable mate. Females mating only in spring would have less time to seek a suitable mate. In addition, oogenesis could hypothetically be negatively influenced due to lack of chemical stimulation by mature males during the autumn period [@pone.0077597-Butler1], [@pone.0077597-CluttonBrock1]. However, this has not been well-documented, in contrast to observations of stimulation of males by female pheromones [@pone.0077597-Stebbing1], [@pone.0077597-Belanger1] or the effect of chemical stimuli on sexual selection [@pone.0077597-Berry1], [@pone.0077597-Gherardi3]. Autumn only mating provides females a shorter time for seeking a suitable mate and necessitates the long term storage of the spermatophores (four months) until the spring spawning season. Long-term sperm storage has been confirmed in cambarid crayfish [@pone.0077597-SoutyGrosset1], [@pone.0077597-Albaugh1], [@pone.0077597-Butler1]. Despite these variables, suggesting differences in reproductive output among mating regimes, no significant difference was found among the experimental groups ([Table 2](#pone-0077597-t002){ref-type="table"}). Females in all mated groups produced eggs and hatched and carried viable offspring. The number of 2^nd^ developmental stage juveniles per female was high in all groups ([Table 2](#pone-0077597-t002){ref-type="table"}) and was comparable to the literature [@pone.0077597-SoutyGrosset1], [@pone.0077597-Filipov1], [@pone.0077597-Kozk1]. This suggests that omitted or truncated mating periods, long-term sperm storage, and absence of chemical stimulation by males at the start of a mating season did not reduce fecundity, and, conversely, the completion of both mating periods had no positive influence on reproductive output. The present study included another regime, in which females had no possibility of meeting males for 10 months. In this situation, reproductive success was observed in the vast majority of females, showing the facultative parthenogenesis in *O. limosus* [@pone.0077597-Bui1]. However, compared to the mated groups, the number of hatchlings was significantly lower (by \>40%) in asexually reproducing females ([Table 2](#pone-0077597-t002){ref-type="table"}). Butler & Stein [@pone.0077597-Butler1] argued that reproductive success would decline with fewer mates, nevertheless that is probably not relevant to the asexual reproduction observed in the present study. Secondary factors may affect reproduction, such as the ability to manage clutch size, and size of eggs relative to the quality of mate [@pone.0077597-Galeotti2]. Decreased reproductive success due to partial egg loss during incubation suggests a cost of asexuality, probably associated with alternating between sexual and asexual reproduction [@pone.0077597-Bui1]. The mechanisms underlying the switching between sexual and asexual reproduction are still unclear. Further research is necessary, as recent studies indicate that this reproductive feature could be more widespread [@pone.0077597-Yue1], [@pone.0077597-Yue2]. Other variables determining the success of reproduction include the percent of females that successfully spawned eggs and carried offspring, egg losses during incubation, and the mortality of females. Considerable differences would be expected, at least between mated groups and the non-mated group. However, minimal variations in these parameters were observed among groups. The timing of spawning and hatching was similar among the experimental groups. The timing can be influenced by environmental cues, mainly water temperature and photoperiod [@pone.0077597-Stucki1], [@pone.0077597-Dub1], or by the size (age) of females [@pone.0077597-Smith1], [@pone.0077597-Reynolds2]. Our experimental groups were kept in the same conditions, and females were of similar size, suggesting similar age. We can therefore conclude that differences in the reproductive regimes did not influence the time of spawning and the hatching of juveniles. The factors determining reproduction success are successful egg laying, incubation, and hatching of viable offspring. All experimental groups, regardless of the mating regime, showed high spawning success ([Table 2](#pone-0077597-t002){ref-type="table"}), with more than 90% of females producing eggs. In groups in which both mating seasons were completed, spawning success was 100%, but it is not clear whether other groups were negatively influenced by fewer matings [@pone.0077597-Butler1]. Contrary to spawning success, loss of full egg clutches was observed for several females in the non-mated group (7%) and 7 and 20% of females in the two groups with continuous mating ([Table 2](#pone-0077597-t002){ref-type="table"}). Such egg losses could be caused by failed fertilization, unsuccessful egg attachment, or lack of female fitness [@pone.0077597-Reynolds2]. Partial egg losses occurred in the non-mated group, with nearly 30% of females losing more than 50% of the egg clutch. This could be a further reason for decreased fecundity in this experimental group. However, a number of partial egg losses also occurred in the groups completing both mating seasons (11 and 28%). This could be the result of inadequate egg attachment to pleopods, poor egg quality, or removal of dead eggs by the female [@pone.0077597-Gherardi1], [@pone.0077597-Reynolds2]. The fecundity of only non-mated females was affected, which can be ascribed to a combination of the mentioned factors (cost of asexuality, secondary reproductive effort, poor egg attachment, environmental conditions, and possible reduced fitness of the parthenogenetic eggs) [@pone.0077597-Bui1], [@pone.0077597-Gherardi1], [@pone.0077597-Galeotti2], [@pone.0077597-Dub1], [@pone.0077597-Reynolds2]. On the other hand, asexual reproduction would have benefits in eliminating aggressive encounters with males, which probably caused the observed mortality (10 -- 20%) in the mated groups, since it only occurred when males were present in the experimental tanks. The omitted autumn or spring mating period, long-term sperm storage, and the absence of chemical stimulation by males at the start of mating season was not shown to affect fecundity, and conversely, the completion of both mating periods did not influence reproductive output. Even with no males present, fecundity was relatively high. Moreover, discussed mating scenarios have fundamental effect not only for female reproductive output, but also for male contribution on offspring. Males which failed suitable mate search in autumn, can still deposit their spermatophores to females during spring mating period and vice versa, males which participated in autumn period only, have still good probability of their contribution on offspring. On the other hand, females are able to store spermatophores from more males (evidently from both, autumn and spring mates), resulting in multiple paternity [@pone.0077597-Walker1], [@pone.0077597-Yue3]. They therefore can increase the variability within offspring. Such mechanism can hypothetically restore the lost diversity after potential previous introduction bottlenecks, and therefore can maintain population viability. However, there are much more unanswered questions waiting for future scientific work - including e.g. switching mechanism between sexual and asexual reproduction, male contribution on offspring according to their phenotypic quality [@pone.0077597-Galeotti2] or their time and order of copulation). The result of the complex mechanisms described in the present study is to achieve optimal production of offspring in the shortest possible time and under a wide spectrum of ecological conditions. The reproductive plasticity of *O. limosus* directly determines its status of a high risk invasive species [@pone.0077597-Gherardi4] and contributes to its successful and rapid spread into new localities. *Orconectes limosus* is a highly adaptive species, which can exert a strong direct and indirect influence on the environment. The present data also indirectly supports the results of Filipová *et al*. [@pone.0077597-Filipov1] that, based on haplotype variation of populations in North America and Europe, all European *O. limosus* populations are descendants of only 90 specimens initially introduced into one small pond. Moreover, the recently acquired knowledge of *O. limosus* reproduction may question the effectiveness of eradication techniques [@pone.0077597-Aquiloni1], [@pone.0077597-Gherardi5]. Currently, there is no known means of eliminating *O. limosus* spread. The reproductive plasticity of *O. limosus* implies a serious threat, not only for autochthonous European astacofauna, but for other aquatic organisms, as well as entire ecosystems [@pone.0077597-Holdich2], [@pone.0077597-Holdich3], [@pone.0077597-Gherardi4]. The combined effects of *O. limosus* invasive behavior, continued habitat destruction, pollution of waters, and human activity can accelerate an invasion and prevent future ICS restoration. In order to decelerate *O. limosus* invasion, steps must be taken to increase public awareness to prevent new crayfish transfers, and to continue NICS invasion monitoring and research. Thanks to the Lucidus Consultancy for language corrections. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: MB AK PK. Performed the experiments: MB AK. Analyzed the data: MB. Contributed reagents/materials/analysis tools: MB AK PK. Wrote the paper: MB AK.

*Funding information* No funding Presented to the 99th meeting of the Association of Surgeons of Great Britain and Ireland, Telford, UK, May 2019; winner of the Moynihan prize Introduction {#bjs550277-sec-0005} ============ In the West, most patients who are offered attempted curative therapy for oesophageal adenocarcinoma (OAC) will undergo a multimodal treatment involving either neoadjuvant chemotherapy (NAC) followed by surgery[1](#bjs550277-bib-0001){ref-type="ref"}, perioperative chemotherapy[2](#bjs550277-bib-0002){ref-type="ref"}, or neoadjuvant chemoradiotherapy[3](#bjs550277-bib-0003){ref-type="ref"}. Despite evidence of survival benefit, a meta‐analysis[4](#bjs550277-bib-0004){ref-type="ref"} comparing NAC with surgery alone in 2062 patients found only a 5·1 per cent absolute 2‐year survival advantage after NAC, because only a small minority experienced a significant pathological response. In a multicentre cohort study[5](#bjs550277-bib-0005){ref-type="ref"}, a clinically meaningful local response to NAC was restricted to the 14·8 per cent of patients with a tumour regression grade (TRG) of 1--2. Inflammation is now widely recognized as a feature of many cancers[6](#bjs550277-bib-0006){ref-type="ref"}. Among a variety of inflammatory markers, derivative biomarkers -- neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), neutrophil to platelet score, and the modified Glasgow Prognostic Score (mGPS) -- have been reported to be associated with poor survival[7](#bjs550277-bib-0007){ref-type="ref"}, [8](#bjs550277-bib-0008){ref-type="ref"}, [9](#bjs550277-bib-0009){ref-type="ref"}. The aim of this study was to determine whether clinically readily available serum markers of inflammation obtained from routinely performed patient screening blood profiles might predict response to potentially curative NAC in OAC, and whether there was a subsequent relationship with survival after potentially curative oesophagectomy. Methods {#bjs550277-sec-0006} ======= To test the proposed hypotheses, a single cohort of patients diagnosed with OAC between 1 January 2010 and 31 August 2018 was recruited, and included patients with radiological TNM stage I--III deemed amenable to treatment with curative intent. All patients were managed by a multidisciplinary specialist team (MDT) with an interest in oesophageal cancer, including clinical nurse specialists, gastroenterologists, surgeons, oncologists, radiologists, anaesthetists and pathologists[10](#bjs550277-bib-0010){ref-type="ref"}. Management plans were individually tailored according to both patient and disease factors. Staging was done by means of CT, endoscopic ultrasonography, CT--PET and staging laparoscopy as appropriate. The South‐East Wales MDT treatment algorithms for oesophageal carcinoma have been described previously[11](#bjs550277-bib-0011){ref-type="ref"}, [12](#bjs550277-bib-0012){ref-type="ref"}, [13](#bjs550277-bib-0013){ref-type="ref"}. The majority of patients received two cycles of cisplatin 80 mg/m^2^ and 5‐fluorouracil 1000 mg/m^2^ for 4 days. A minority received three cycles of epirubicin 50 mg/m^2^, cisplatin 60 mg/m^2^ and 5‐fluorouracil 200 mg/m^2^ or capecitabine 625 mg/m^2^ (ECF/X regimen). Definitive chemoradiotherapy was offered to patients with localized squamous cell carcinoma and those with adenocarcinoma deemed unsuitable for surgery because of disease extent and/or medical co‐morbidity[14](#bjs550277-bib-0014){ref-type="ref"}, [15](#bjs550277-bib-0015){ref-type="ref"}. The standard surgical approach was subtotal transthor‐acic oesophagectomy (TTO), as described by Lewis[16](#bjs550277-bib-0016){ref-type="ref"} and Tanner[17](#bjs550277-bib-0017){ref-type="ref"}. Transhiatal oesophagectomy (THO), as described by Orringer[18](#bjs550277-bib-0018){ref-type="ref"}, was used selectively in patients with adenocarcinoma of the lower third of the oesophagus who had significant cardiorespiratory co‐morbidity, cT1--3 N0 disease. A modified extended D2 lymphadenectomy (preserving the pancreas and spleen where possible) was performed. Ethical approval was sought, but the chair of the Cardiff and Vale University Health Board ethics committee confirmed that individual patient consent was not required to report clinical outcomes alone, and no formal approval was necessary. Clinicopathological characteristics {#bjs550277-sec-0007} ----------------------------------- Tumours were staged using the seventh edition of the AJCC/UICC TNM staging system. Pathological factors were recorded from pathology reports issued at the time of surgery, and included tumour differentiation, margin status and the number of lymph nodes with and without metastasis. The TRG was quantified using the Mandard system[19](#bjs550277-bib-0019){ref-type="ref"} by a histopathologist with a special interest in oesophagogastric cancer. Briefly, Mandard TRGs range from 1 to 5, based on the ratio of fibrosis to viable cancerous cells[5](#bjs550277-bib-0005){ref-type="ref"}, [19](#bjs550277-bib-0019){ref-type="ref"}. In keeping with the Oesophageal Cancer Clinical And Molecular Stratification (OCCAMS) reporting methodology, TRGs of 1 and 2 were considered to constitute good response, with TRGs of 3, 4 and 5 constituting poor response[5](#bjs550277-bib-0005){ref-type="ref"}. Routine laboratory measurements of haemoglobin, whole white cell count, neutrophil, lymphocyte and platelet counts at the time of diagnosis were recorded. Derivate measurements of systemic inflammation were constructed by calculating the NLR and PLR[7](#bjs550277-bib-0007){ref-type="ref"}, [20](#bjs550277-bib-0020){ref-type="ref"}. Patients were followed up at regular intervals of 3 months in the first year and 6 months thereafter. In the event that patients developed symptoms suggestive of recurrent disease, investigations were undertaken sooner. Follow‐up surveillance was conducted for 5 years or until death, whichever was sooner. Overall survival was calculated from time of diagnosis to the date of death or censoring. Disease‐free survival was measured from the date of surgery to the date of recurrence or censoring. The time of recurrence was taken as the date of the confirmatory investigation, on an intention‐to‐treat basis. Death certification was obtained from the Office for National Statistics via the Cancer Network Information System Cymru. Statistical analysis {#bjs550277-sec-0008} -------------------- Grouped data were expressed as median (i.q.r.) values, and non‐parametric methodology was used throughout. Receiver operating characteristic (ROC) curve analysis was employed to assess the predictive value of continuous variables with the primary outcome measure. ROC analysis was also used to determine dichotomization thresholds for poor Mandard TRGs, as described by Youden[21](#bjs550277-bib-0021){ref-type="ref"}. Univariable and multivariable logistical regression analysis was used to identify independent associations of categorical variables with poor Mandard TRGs. Variables with *P* \< 0·100 were included in the model using backward conditional methodology. Patient demographics were analysed between the treatment modalities by means of χ^2^ or non‐parametric tests, including the Mann--Whitney *U* test. These tests were also employed in the analysis of disease recurrence and time to recurrence for the treatment groups. Overall survival was measured from the date of diagnosis, and disease‐free survival from date of surgery. This approach was adopted in the randomized trials to allow for the variable interval to surgery after diagnosis, depending on whether NAC was prescribed[22](#bjs550277-bib-0022){ref-type="ref"}. As in the trials, events resulting in a failure to complete curative treatment, such as not proceeding to surgery, open and close laparotomy, palliative resection and in‐hospital mortality, were assumed to have occurred at this landmark time, in order to maintain the intention‐to‐treat analysis. Cumulative survival was calculated according to the Kaplan--Meier method, with differences between groups analysed using the log rank test. A univariable analysis examining factors influencing survival was performed initially by the life‐table method of Kaplan and Meier, and factors with associations found to be significant at the *P* \< 0·010 level were retained in a Cox proportional hazards model using backward conditional methodology to assess the prognostic value of individual variables. All statistical analysis was performed in SPSS® Statistics v25.0.0.0 (IBM, Armonk, New York, USA) with extension R. Results {#bjs550277-sec-0009} ======= A total of 136 patients with OAC were identified and underwent surgery after NAC; the operative approach was open in 120 patients with 16 patients undergoing laparoscopically assisted surgery. Details of patients\' clinicopathological factors can be found in *Table* [1](#bjs550277-tbl-0001){ref-type="table"}. Their median age was 68 (i.q.r. 63--73) years; 106 (77·9 per cent) were men and 30 (22·1 per cent) were women. Twenty‐three patients (16·9 per cent) had a good pathological response to NAC (TRG 1--2); the Mandard TRG groupings were: TRG 1, 18 (13·2 per cent); TRG 2, five (3·7 per cent); TRG 3, six (4·4 per cent); TRG 4, 56 (41·2 per cent); and TRG 5, 51 (37·5 per cent). During follow‐up, 36 patients (26·5 per cent) developed cancer recurrence and 69 (50·7 per cent) died. Median follow‐up of survivors was 27 (range 6--60) months. Around two‐thirds of the patients were followed up for at least 5 years or until death. ###### Clinicopathological patient factors No. of patients (*n* = 136) --------------------------------------- ----------------------------- **Age (years)** \< 65 46 (33·8) 66--75 68 (50·0) \> 75 22 (16·2) **Sex ratio (F** : **M)** 30 : 106 **Operative approach** *n* = 113 TTO 71 (62·8) THO 42 (37·2) **Neutrophil** : **lymphocyte ratio** \< 2·25 36 (26·5) ≥ 2·25 100 (73·5) **pT category** pT0 11 (8·1) pT1 10 (7·4) pT2 10 (7·4) pT3 69 (50·7) pT4 13 (9·6) No resection 23 (16·9) **pN category** pN0 35 (25·7) pN1 34 (25·0) pN2 26 (19·1) pN3 18 (13·2) No resection 23 (16·9) **Mandard TRG** Good 23 (16·9) Poor 113 (83·1) **Differentiation** Well or moderate 64 (47·1) Poor 72 (52·9) **CRM** Negative 58 (42·6) Positive 55 (40·4) No resection 23 (16·9) **Lymph node yield** *n* = 113 \< 15 43 (38·1) ≥ 15 70 (61·9) Values in parentheses are percentages. TTO, transthoracic oesophagectomy; THO, transhiatal oesophagectomy; TRG, tumour regression grade; CRM, circumferential resection margin. Baseline and area under the ROC curve (AUC) values for markers of the systemic inflammatory response (SIR) are shown in *Table* [2](#bjs550277-tbl-0002){ref-type="table"}. The median value for NLR was 3·00 (i.q.r. 2·15--3·89). NLR was strongly associated with a poorer Mandard TRG (AUC 0·71, 95 per cent c.i. 0·58 to 0·83; *P* = 0·002) (*Fig*. [1](#bjs550277-fig-0001){ref-type="fig"}). Using the Youden index, the optimal dichotomization threshold was 2·25, with 70·5 per cent considered to have a raised NLR. This gave sensitivity and specificity values of 80·5 and 60·9 per cent respectively. To adjust for potential confounding, a binary logistic regression model was developed to include the clinical factors available to the MDT at the point of commencing neoadjuvant therapy (*Table* [3](#bjs550277-tbl-0003){ref-type="table"}). ###### Association between pretreatment markers of systemic inflammatory response and poor Mandard tumour regression grade No. of patients with marker level[†](#bjs550277-note-0006){ref-type="fn"} ROC analysis -------------------------------- ------------------- --------------------------------------------------------------------------- -------------- ---- ------------------- ------- Haemoglobin (g/l) 138 (128--148) 33 103 0 0·47 (0·33, 0·62) 0·699 White cell count (× 10^9^/l) 7·6 (6·2--8·1) 2 124 10 0·59 (0·46, 0·72) 0·158 Neutrophil count (× 10^9^/l) 5·0 (3·9--6·1) 2 118 16 0·68 (0·56, 0·80) 0·008 Lymphocyte count (× 10^9^/l) 1·7 (1·3--2·1) 8 123 5 0·40 (0·25, 0·54) 0·115 Platelet count (× 10^9^/l) 281 (233--330) 3 123 10 0·52 (0·40, 0·65) 0·691 Neutrophil to lymphocyte ratio 3·00 (2·15--3·89) 0·71 (0·58, 0·83) 0·002 Platelet to lymphocyte ratio 160 (122--198) 0·66 (0·52, 0·79) 0·019 Values in parentheses are 95 per cent confidence intervals unless indicated otherwise; values are median (i.q.r.). Based on local thresholds. AUC, area under the receiver operating characteristic (ROC) curve. {#bjs550277-fig-0001} ###### Logistic regression analysis of preoperative factors associated with poor Mandard tumour regression grade Univariable analysis Multivariable analysis ---------------------------------------------------------- ---------------------- ------------------------ --------------------- ---------- Age (\< 65 *versus* 66--75 *versus* \> 75 years) 1·11 (0·58, 2·15)   0·753 Sex (F *versus* M) 1·31 (0·47, 3·68)   0·610 Differentiation (well/moderate *versus* poor) 17·09 (3·82, 76·55) \< 0·001 15·92 (3·42, 74·02) \< 0·001 cTNM (1 *versus* 2 *versus* 3 *versus* 4) 1·38 (0·76, 2·48)   0·289 Neutrophil to lymphocyte ratio (\< 2·25 *versus* ≥ 2·25) 6·43 (2·47, 16·77) \< 0·001 5·86 (2·03, 16·92)   0·001 Values in parentheses are 95 per cent confidence intervals. The relationship between clinicopathological factors and overall survival is shown in *Table* [4](#bjs550277-tbl-0004){ref-type="table"}, and that between clinicopathological factors and disease‐free survival in *Table* [5](#bjs550277-tbl-0005){ref-type="table"}. Five‐year overall survival rates for patients with a low and high NLR were 50 and 20 per cent, respectively. Five‐year disease‐free survival rates for low and high NLR were 80 and 40 per cent, respectively. ###### Cox proportional hazards analysis of factors associated with overall survival Univariable analysis Multivariable analysis ------------------------------------------------------------- ---------------------- ------------------------ -------------------- ------- Age (\< 65 *versus* 66--75 *versus* \> 75 years) 0·87 (0·58, 1·30)   0·494 Sex (F *versus* M) 1·13 (0·57, 2·21)   0·731 Operative approach (TTO *versus* THO) 1·25 (0·69, 2·24)   0·462 Neutrophil to lymphocyte ratio (\< 2·25 *versus* ≥ 2·25) 2·33 (1·16, 4·68)   0·017 2·26 (1·03, 4·93) 0·042 pT category (0 *versus* 1 *versus* 2 *versus* 3 *versus* 4) 2·14 (1·43, 3·21) \< 0·001 1·81 (1·06, 3·08) 0·029 pN category (0 *versus* 1 *versus* 2 *versus* 3) 1·70 (1·34, 2·15) \< 0·001 1·57 (1·14, 2·17) 0·006 Mandard TRG (good *versus* poor) 2·67 (1·22, 5·86)   0·014 4·28 (1·37, 13·34) 0·012 Differentiation (well/moderate *versus* poor) 2·26 (1·27, 4·02)   0·006 2·71 (1·39, 5·29) 0·004 CRM (negative *versus* positive) 2·46 (1·43, 4·22)   0·001 0·171 Lymph node yield (\< 15 *versus* ≥ 15) 0·75 (0·43, 1·31)   0·306 Values in parentheses are 95 per cent confidence intervals. TTO, transthoracic oesophagectomy; THO, transhiatal oesophagectomy; TRG, tumour regression grade; CRM, circumferential resection margin. ###### Cox proportional hazards analysis of factors associated with disease‐free survival Univariable analysis Multivariable analysis ------------------------------------------------------------- ---------------------- ------------------------ ------------------- ------- Age (\< 65 *versus* 66--75 *versus* \> 75 years) 0·95 (0·59, 1·53) 0·823 Sex (F *versus* M) 1·08 (0·49, 2·38) 0·847 Operative approach (TTO *versus* THO) 2·64 (1·36, 5·11) 0·004 3·10 (1·58, 6·12) 0·001 Neutrophil to lymphocyte ratio (\< 2·25 *versus* ≥ 2·25) 2·48 (1·08, 5·67) 0·032 0·288 pT category (0 *versus* 1 *versus* 2 *versus* 3 *versus* 4) 1·54 (1·09, 2·17) 0·014 1·72 (1·01, 2·93) 0·047 pN category (0 *versus* 1 *versus* 2 *versus* 3) 1·17 (0·98, 1·40) 0·081 0·091 Mandard TRG (good *versus* poor) 3·68 (1·12, 12·08) 0·032 0·936 Differentiation (well/moderate *versus* poor) 1·82 (0·94, 3·53) 0·078 0·372 CRM (negative *versus* positive) 1·38 (0·69, 2·74) 0·361 Lymph node yield (\< 15 *versus* ≥ 15) 0·71 (0·37, 1·36) 0·302 Values in parentheses are 95 per cent confidence intervals. TTO, transthoracic oesophagectomy; THO, transhiatal oesophagectomy; TRG, tumour regression grade; CRM, circumferential resection margin. {#bjs550277-fig-0002} Discussion {#bjs550277-sec-0010} ========== The principal finding of this study is that a single biomarker of SIR, a raised NLR, was a significant and independent prognostic indicator of response to NAC before potentially curative oesophagectomy for cancer. Based on a dichotomization threshold of 2·25, 100 of the 136 patients (73·5 per cent) had a raised NLR, and were nearly sixfold more likely to have a poor TRG response to NAC. Median overall survival in patients with a low NLR was, on average, 34 months longer than that in patients with a high NLR. Similarly, 5‐year disease‐free and overall survival rates in patients with a low NLR were around 80 and 50 per cent respectively, approximately double those of patients with a high NLR. The relationship between the SIR and TRG in oesophageal cancer has been described previously[23](#bjs550277-bib-0023){ref-type="ref"}, in a study in which no association between NLR and TRG was identified. Key differences in methodology from that in the present study may account for these different findings. The definition of good TRG response differed. In the earlier study[23](#bjs550277-bib-0023){ref-type="ref"}, patients with TRG 1--3 were considered good responders, whereas in the present study TRG 1--2 was considered to represent a good response. The OCCAMS research consortium currently favours the TRG 1--2 as indicative of good response[5](#bjs550277-bib-0005){ref-type="ref"}. The statistical methods also differed. The earlier study[23](#bjs550277-bib-0023){ref-type="ref"} looked only at differences in NLR measurements between responders and non‐responders (2·26 *versus* 2·73; *P* = 0·127), but did not examine the predictive value of NLR by ROC curve or logistic regression analysis. Statistical nuances may, of course, be overanalysed, but half of the TRG responders in the earlier study had a NLR below 2·26, which implicitly supports the critical threshold of 2·25 employed in the present study. At the very least, markers of SIR require further evaluation. The prognostic power of SIR in relation to neoadjuvant therapies has been reported previously, involving rectal[24](#bjs550277-bib-0024){ref-type="ref"}, [25](#bjs550277-bib-0025){ref-type="ref"}, ovarian[26](#bjs550277-bib-0026){ref-type="ref"}, lung[27](#bjs550277-bib-0027){ref-type="ref"} and breast[28](#bjs550277-bib-0028){ref-type="ref"} cancers. A high mGPS was associated with poor response to NAC in rectal cancer (odds ratio (OR) 0·18, *P* = 0·006), and low NLR was associated with a good pathological response (OR 0·27, *P* = 0·046), although this did not retain independent significance in multivariable analysis[24](#bjs550277-bib-0024){ref-type="ref"}. Although other studies have not specifically examined the role of SIR\'s association with pathological response to NAC, preoperative NLR has been reported to predict overall survival, with high NLR values being associated with poor survival[29](#bjs550277-bib-0029){ref-type="ref"}. Exactly why SIR should be associated with poor response to neoadjuvant chemotherapy in OAC is not understood, although *in vivo* and *in vitro* evidence suggests that activation of the JAK/STAT3 (Janus kinase/signal transducers and activators of transcription) pathway by interleukin 6 may play a role in chemoresistance[30](#bjs550277-bib-0030){ref-type="ref"}, [31](#bjs550277-bib-0031){ref-type="ref"}. There are a number of inherent limitations to all studies of this type, which have been reported previously[7](#bjs550277-bib-0007){ref-type="ref"}, [32](#bjs550277-bib-0032){ref-type="ref"}, [33](#bjs550277-bib-0033){ref-type="ref"}. Cohort size was modest, and stage‐by‐stage subanalysis was therefore impractical. The patients represented a selected cohort (most had undergone a potentially curative oesophagogastrectomy) and were consequently not representative of all patients diagnosed with oesophageal cancer; indeed, only about one‐quarter of all patients in South Wales with OAC undergo potentially curative surgery[34](#bjs550277-bib-0034){ref-type="ref"}. The strengths of the study, nevertheless, included robust follow‐up data with no patient lost to follow‐up, a reasonable duration of follow‐up, and accurate causes and dates of death. A National Health Service laboratory using standardized techniques performed the serum analyses and histopathological examinations, so reproducing these results should be easy. Patients were recruited from a consecutive series diagnosed with OAC, from a single UK geographical region, all treated by the same group of specialists, using a standardized staging algorithm and operative techniques, with internationally recognized and published key performance indicators[10](#bjs550277-bib-0010){ref-type="ref"}. Despite improvements in staging and surgical technique, approximately half of the patients who undergo potentially curative oesophagectomy for cancer will suffer disease recurrence[13](#bjs550277-bib-0013){ref-type="ref"}, [22](#bjs550277-bib-0022){ref-type="ref"}. Determination of the NLR, derived and calculated from absolute counts of serum lymphocytes and neutrophils, is performed routinely during preoperative blood profile work, and is readily available. The findings suggest that SIR offers a novel therapeutic target for patients susceptible to NAC resistance and cancer recurrence. Incorporation of the NLR into management pathways is presently limited by inconsistent dichotomization thresholds. Adequately powered studies comparing critical dichotomization or categorization thresholds are needed. Given the association between SIR and relative chemoresistance, the identification of the group with high NLR would suggest that these patients might benefit from alternatives to NAC at the outset. Collaborators {#bjs550277-sec-0011} ============= Members of the South‐East Wales Oesophagogastric Cancer Collaborative: G. Blackshaw, G. Clark, X. Escofet, A. Foliaki, T. Havard, M. Henwood, J. Witherspoon, W. G. Lewis. Disclosure {#bjs550277-sec-0012} ========== The authors declare no conflict of interest.

Introduction {#ccr3875-sec-0001} ============ Primary aldosteronism (PA) is a major cause of endocrine hypertension [1](#ccr3875-bib-0001){ref-type="ref"}. There are two major subtypes. One is unilateral PA, mainly due to aldosterone‐producing adenoma (APA) and the other is bilateral PA, mainly due to idiopathic hyperaldosteronism (IHA). It is important to determine the PA subtype because the recommended treatment for APA is an adrenalectomy and that for IHA is pharmacological therapy with a mineralocorticoid receptor antagonist [2](#ccr3875-bib-0002){ref-type="ref"}. Adrenal venous sampling (AVS) is recommended for determining the PA subtype [3](#ccr3875-bib-0003){ref-type="ref"}. Regardless of its diagnostic efficacy, AVS is a difficult procedure. The catheterization of the right adrenal vein is particularly difficult because of its small diameter [4](#ccr3875-bib-0004){ref-type="ref"}. By contrast, the left adrenal vein is catheterized in almost all patients because it enters the left renal vein, which can be used as a guide to the left adrenal vein. However, in the rare case of an anomalous left adrenal vein, it is difficult to cannulate the left adrenal vein [5](#ccr3875-bib-0005){ref-type="ref"}. In this report, we describe the successful left adrenal vein cannulation under contrast‐enhanced computed tomography (CT) guidance in a patient with PA and left inferior vena cava (IVC) whose left adrenal vein drained directly into the IVC. Case Report {#ccr3875-sec-0002} =========== A 43‐year‐old man presenting with a 6‐year medical history of hypertension was referred to our hospital for suspected PA. He was taking antihypertensive drug of amlodipine 5 mg. The laboratory data were as follows: creatinine, 0.67 mg/dL; plasma aldosterone concentration (PAC), 249 pg/mL; plasma renin activity (PRA), 0.7 ng/mL/h; and PAC/PRA ratio, 356. Captopril challenge test and saline infusion test confirmed the diagnosis of PA. Contrast‐enhanced CT showed no adrenal tumor. CT also revealed that the left inferior vena cava joined the left renal vein and crossed over the aorta to the right side (Fig. [1](#ccr3875-fig-0001){ref-type="fig"}). {#ccr3875-fig-0001} Adrenal venous sampling was performed using a sheath inserted in the right femoral vein. The right adrenal vein was cannulated using a catheter. The left adrenal vein was then explored through the left renal vein. However, venography showed no left adrenal vein joining the left renal vein. A coronal section of the CT indicated that the left adrenal vein directly drained into the left IVC at a point close to the center of the left renal vein (Fig. [2](#ccr3875-fig-0002){ref-type="fig"}). After examining the area, it was found that the left adrenal vein could be cannulated (Fig. [3](#ccr3875-fig-0003){ref-type="fig"}A). However, a blood sample could not be obtained because the catheter was wedged; therefore, a multipurpose 4.2 French catheter was used to obtain a blood sample (Fig. [3](#ccr3875-fig-0003){ref-type="fig"}B). AVS was performed after adrenocorticotropic hormone stimulation. The patient was diagnosed with bilateral adrenal hyperplasia (Table [1](#ccr3875-tbl-0001){ref-type="table-wrap"}) and treated with mineralocorticoid receptor antagonist. {#ccr3875-fig-0002} {#ccr3875-fig-0003} ###### Adrenal venous sampling results after adrenocorticotropic hormone stimulation Aldosterone (pg/mL) Cortisol (*μ*g/dL) Aldosterone cortisol ratio -------------------- --------------------- -------------------- ---------------------------- Right adrenal vein 34,189 1130 30 Left adrenal vein 24,379 1100 22 Inferior vena cava 212 20 11 John Wiley & Sons, Ltd Discussion {#ccr3875-sec-0003} ========== The IVC can present with a multitude of anatomical variations, such as double and left IVC, which are caused by complex embryonic developments. Based on the involvement of iliac and gonadal veins, several classifications have been proposed for IVC variations [6](#ccr3875-bib-0006){ref-type="ref"}, [7](#ccr3875-bib-0007){ref-type="ref"}, [8](#ccr3875-bib-0008){ref-type="ref"}. To perform a successful AVS, knowledge of possible anatomical variations related to adrenal vein drainage is crucial. For example, in patients with double IVC, the left adrenal vein may drain either directly into the IVC or into the left renal vein. In patients with left IVC, the left adrenal vein drains directly into the IVC [6](#ccr3875-bib-0006){ref-type="ref"}. In almost all individuals, the right adrenal vein drains directly into the IVC. Alper et al. reviewed the anatomy of adrenal veins [9](#ccr3875-bib-0009){ref-type="ref"}. Contrast‐enhanced CT is useful in planning for AVS because it reveals the positions of the adrenal veins [5](#ccr3875-bib-0005){ref-type="ref"}. In this case, a coronal section of CT was helpful in detecting the left adrenal vein. Stack et al. reported a case where the left adrenal vein drained directly into the IVC [10](#ccr3875-bib-0010){ref-type="ref"}. To our knowledge, our patient is the first reported case of AVS being performed in the left IVC wherein the left adrenal vein directly into the IVC. Conclusion {#ccr3875-sec-0004} ========== We report an unusual case wherein the left adrenal vein drained directly into the left IVC. This anomaly makes cannulation of the left adrenal vein for AVS difficult. Contrast‐enhanced CT should be routinely performed in all patients undergoing AVS to rule out or clearly left any unusual anatomical variations that might complicate the procedure. Authorship {#ccr3875-sec-0005} ========== YF and HT: drafted the article and conception of this study; YF and TU: performed the adrenal venous sampling; YU: performed the consultation and evaluation; YF: revised the article critically for important intellectual content. Conflict of Interest {#ccr3875-sec-0006} ==================== None declared.

The term *working memory* refers to the process or collection of processes responsible for the complex cognitive coordination necessary for everyday human thoughts and actions. Researchers generally agree about the importance of working memory for human cognition. There is also general agreement that it supports the ready availability of a small amount of information in support of current tasks, and has a key role in updating and processing that information moment to moment (e.g., [@c30]; [@c52]). However, there are multiple different definitions of working memory (see [@c30], for a discussion), and each definition gives rise to different theoretical assumptions and different experimental paradigms designed to test those assumptions. Contrasting results across labs might then reflect the specific experimental paradigms adopted, and theoretical debates may be based on differences that are more apparent than real ([@c49]). Rarely do researchers who assume different definitions of working memory adopt the exact same paradigm to directly test their contrasting predictions. We present four experiments that addressed the debate about what limits the capacity of working memory to undertake both memory maintenance and ongoing processing. Unlike most studies in this area, the experiments were carried out across different labs within an "adversarial collaboration" in which the coauthors agreed on a common experimental paradigm to test predictions from their contrasting, and well-established theoretical frameworks for working memory. The experiments described here are part of a larger project called Working Memory Across the Adult Life Span: An Adversarial Collaboration (WoMAAC; <https://womaac.psy.ed.ac.uk>). Specifically, these frameworks are referred to as the multiple-component model (MCM; [@c7]; [@c49], [@c50]), time-based resource sharing (TBRS; [@c11], [@c12]), and embedded processes (EP; [@c26], [@c27]). This approach allows a more direct test of the different predictions than is possible across different studies, with the aim of contributing new insights, both theoretically and empirically, to this important area of cognition. First, we give an overview of each of the three theoretical frameworks that motivated our experiments, and then go on to describe the expectations from each for the series of experiments that follow. All of the predictions from each theory, and the experimental methods, were preregistered on the Open Science Framework (OSF, see the project page at <https://bit.ly/2KTKMgb>). Multiple-Component Model (MCM) {#s2} ============================== The MCM assumes a coordinated system of specialized cognitive resources serving specific functions in online cognition. The model specifies separate components for storage and processing, with distinct stores based on modality-specific codes that need not match the modality of presentation. For example, words may be stored as visual codes or as phonological or semantic codes, regardless of whether they are presented visually or aurally, and nonverbal stimuli such as shapes and colors may be stored as visual codes or as phonological or semantic codes for the associated names. Originally ([@c2]; [@c5]), a central executive was proposed as a domain-general processing and control mechanism, but subsequently ([@c4]; [@c50]), a number of separate executive functions were proposed, such as inhibition, updating, task-switching ([@c60]), dual-tasking ([@c51]; [@c59]), and the manipulation of mental images ([@c15]; [@c79]). Executive functions have therefore been suggested to be emergent properties of the interaction between these multiple functions ([@c49], [@c50]). The phonological loop has been proposed as a temporary store for serial ordered phonological codes (e.g., [@c3]). Items stored within the phonological loop are said to be vulnerable to interference among themselves due to phonological similarity ([@c23]) and interference from asking participants to repeat aloud an irrelevant word (e.g., the--the--the) while encoding or retaining verbal sequences (a technique known as articulatory suppression \[AS\]), as well as from presentation of irrelevant speech ([@c71]). While the limited capacity store can maintain small list lengths without any attentional cost, the MCM also proposes a separate subvocal rehearsal mechanism that can "boost" performance. Maintenance of longer lists through subvocal rehearsal has been found to be affected by a number of temporal factors, such as the length of words in a sequence and individual reading and speech rates ([@c8]; [@c42]), although some recent studies have debated this issue ([@c41]; [@c44]; [@c65]). The links between memory performance and phonological characteristics of the to-be-remembered items are therefore argued as evidence for a specific verbal store. Additional evidence has come from studies of brain damaged individuals who appear to have very specific impairments of short-term retention of phonological sequences ([@c76]; [@c78]). The visual cache is said to store an array of visual items or a single visual item that may vary in complexity ([@c47], [@c48], [@c49]). The broader concept of visuospatial working memory is assumed to comprise separable resources and mechanisms dedicated to visual and spatial information ([@c49]; [@c56]; [@c57]). Evidence for separate visual and spatial components also comes from the finding that spatial and visual memory spans increase at different rates with age during childhood, and are poorly correlated within age groups ([@c57]). While separate stores for verbal and visuospatial material are assumed by the MCM, the theory also states that material is often recoded for storage in other formats. For example, evidence that verbal material is represented in memory in the form of the visual appearance of the letters comes from the presence of visual similarity effects in serial written recall for visually presented verbal materials ([@c54]; [@c58]; [@c70]), and other evidence has pointed to the use of verbal labels for abstract visual patterns ([@c16]). MCM also assumes that different participants may approach tasks in multiple different ways that may not include phonological or visuospatial rehearsal mechanisms, using strategies such as employing mnemonics for remembering lists of words ([@c53]). In sum, working memory is viewed as a set of mental tools that can be applied in different combinations to support task performance, and the same task may be performed in different ways depending on which combination of working memory components are deployed. The structure of working memory proposed by the MCM assumes a separation of processing and storage functions. In their seminal article, [@c5] investigated the effect of concurrent memory load on processing tasks (e.g., sentence verification/comprehension, logical statement verification), and found that dual-task costs to processing were only observed at longer list lengths, and that greater interference effects than those observed should be expected if both storage and processing relied on a single limited resource. This argument has been made in a number of subsequent studies citing small or null effects as evidence for separate resources for each type of task (e.g., [@c37]; [@c38], [@c39]). Evidence for the separation of memory and processing is further provided by reports of low correlations between measures of memory span and measures of processing span (e.g., [@c35]; [@c55]; [@c82]). Neuropsychological studies have also been used to argue for a dual-tasking ability based on coordination of multiple components; for example, [@c51]) and [@c59]) identified a specific dual-task deficit in Alzheimer's patients that was not present in younger and older healthy controls. A key feature of dual-tasking studies within the MCM framework is that the cognitive demand of each task is adjusted (titrated) to the ability each individual participant, and this measured single-task ability is used to set the demand level both when performing each task on its own and when performing the two tasks together. This is done to ensure that any dual-task effect can be attributed specifically to the dual-task condition, and not because the individual-tasks were simply set at too high a level for the participant (for a more detailed discussion, see [@c51]). Time-Based Resource Sharing (TBRS) Model {#s3} ======================================== The TBRS model assumes that both functions of working memory, processing and storage, rely in part on a shared, general-purpose, limited capacity attentional resource. Because a central bottleneck constrains cognitive operations to take place one at a time, when attention is occupied by processing it is no longer available for maintaining memory traces and so these traces suffer from temporal decay and interference. However, decayed memory traces may be restored through attentional refreshing when attention is available during pauses in processing. While temporary verbal memory can be bolstered by subvocal rehearsal in a phonological loop, performance is highly dependent on access to the focus of attention. The empirical basis for the theory is a number of observations of how the demand of a secondary processing task is inversely correlated with memory performance in a dual-task complex span paradigm (see [@c12], for a review). This attentional demand of a processing task is discussed in terms of its "cognitive load," which refers to the proportion of time the processing task captures attention and therefore diverts the focus away from maintenance of temporary memory traces. Crucially, the TBRS model differentiates itself from pure decay-based theories of short-term forgetting in stating that it is not the overall duration of the processing component that matters but rather how much time between processing items is available for maintaining the representations of the memoranda. TBRS research has demonstrated how cognitive load can be increased by increasing the number of retrievals from long-term memory (LTM; or the number of responses required by a secondary task), increasing the time taken to respond to each item of a distractor task, and decreasing the time of the processing period while keeping other factors constant (resulting in a smaller proportion of the time being available to refresh memory traces). These manipulations all result in higher cognitive load and thus poorer memory performance (e.g., [@c9]; [@c10]). Attentional refreshing, the specific process that is interrupted by high cognitive load tasks, is described as separate from the subvocal rehearsal that is assumed to take place in the phonological loop (for reviews, see [@c17]; [@c18]). Supporting evidence from brain imaging studies shows different activation patterns for each form of maintenance ([@c68]; [@c77]). The TBRS model states that refreshing can be actively or passively engaged depending on whether subvocal rehearsal is available or effective given task parameters or indeed whether participants are instructed to rehearse or refresh ([@c19]). In the same way as processing prevents refreshing, refreshing activities postpone processing, as [@c81] observed a slowing of processing task responses with increasing memory loads (see also [@c20]). It is important to note that this effect occurs only when the phonological loop is unavailable (e.g., under AS) or when its capacity is exceeded. Importantly, the same study by [@c81] provided evidence that, contrary to verbal information for which a domain-specific storage system exists (i.e., the phonological loop), visuospatial information is not maintained by any domain-specific storage system and so its maintenance relies entirely on attention ([@c61]; see also [@c62]). Embedded Processes (EP) {#s4} ======================= The EP model, in its iterations over the years, has been developed to account for a wide range of empirical findings within a single framework ([@c25], [@c26], [@c27], [@c28], [@c29]). According to the model, a subset of features from environmental stimuli and past events associated with present thoughts are temporarily activated within LTM. This embedded subset of information then enjoys a heightened state of activation while remaining vulnerable to time-based decay and similarity-based interference. A subset of the activated features can be made further salient and integrated into coherent objects and scenes when placed under the focus of attention, which allows a deeper semantic analysis of stimuli. The focus of attention is said to be limited to somewhere between three and five representational units ([@c31]), which may be single-featured items or "chunked" items with multiple features (e.g., shape, color, location, orientation; [@c27]). The EP model assumes a limited-capacity domain-general central attentional controller ([@c26]). Its role is to supervise covert processes that serve to maintain information over time by reactivating decaying memory representations via subvocal rehearsal, as well as × activation × way of the focus of attention. These activation procedures have been found to have an observable cost to processing tasks within a dual-task paradigm, such as drop in accuracy on nonverbal choice reaction time (RT) tasks with increasing concurrent verbal memory load ([@c20]). Temporary information in working memory is therefore represented within this hierarchical system. LTM representations are initially activated by incoming stimuli and information is then further activated within the focus of attention where it must be maintained. Once information leaves the focus of attention it begins to decay, and this decay can only be combated by reactivation within the focus of attention or through subvocal rehearsal. Although items represented within activated LTM memory are partially protected from decay, interference between items can occur based on overlapping features between individual items. Comparisons Between the Theoretical Views {#s5} ========================================= In the present work, the three theoretical views we have described were compared in terms of the effects of processing on storage and vice versa, in a dual-task setting in which a verbal recall task is combined with processing in a different domain. A conundrum that must be appreciated to understand our approach is that all three of the views are capable of predicting interference between tasks under some circumstances. In the MCM approach, if the capacity of verbal storage is reached, additional items can be saved by recoding the information in visuospatial terms (or semantic representations), at the expense of visuospatial or semantic aspects of processing. In the TBRS approach, any attention needed for processing conflicts with attention needed for refreshing of the items to be retained. Finally, in the EP approach, the limited capacity of the focus of attention must be shared between items to be remembered and the goals, procedures, and data for processing. Given this convergence between approaches, a comparison of the models depends on more specific predictions and suppositions related to the experimental tasks. The detailed predictions from the three theoretical frameworks will be presented after the task methods. Crucially, these methods incorporate key features that were intended to avoid some procedural differences across labs that might have given rise to contrasting results between testing sites. One aspect of working memory that is widely accepted is that its capacity varies from one individual to another, even if there are debates about how that individual variability should be measured. However, in many studies in which working memory load is manipulated, the task demands in different conditions are the same for all participants. This means that for someone with a high working memory capacity, an experimental manipulation intended to impose a high cognitive load, might, for them, actually be a low load relative to their capacity. Conversely, for someone with a low working memory capacity, what is deemed to be a low cognitive load in an experiment might, for this individual, effectively be a high cognitive load. By averaging the results across participants, in one lab that happens to recruit high capacity individuals, they might observe little or no effect of increasing the load of a single-task, or of requiring a processing task to be performed while retaining a memory load. In labs that happen to recruit lower capacity individuals, there will be very clear effects observed for cognitive load and of dual-task manipulations. We addressed this possible sampling error in two ways. One was to run each experiment in parallel in two independent labs that have previously reported contrasting results, and to use identical equipment and software to rule out subtle, but potentially important differences between labs. More importantly, in all experiments we measured the memory span and processing span for each participant. Then the memory load without and with a processing task was set at the span-level for each participant. Likewise, the processing load without and with a memory load was set at the level of the processing span for each participant. This process of adjusting, or titrating, cognitive demand according to the span of each participant is commonly used by labs that work within the MCM framework (e.g., [@c37]), but tends not to be adopted by other labs. A second important procedural detail is the extent to which trade-offs between memory and processing arise because of input and output conflicts when the two tasks are performed concurrently, or incompatibility between input modalities or output modalities, rather than because they require overlapping cognitive resources. Two tasks might mutually interfere because they both involve visual input, or both require an oral or keypress response. So, presenting verbal material visually and requiring an oral response, or presenting verbal material aurally and requiring a written response will require more cognitive operations than if the input and output modalities are more compatible, that is, aural input and oral response or visual input and written/typed response. We can avoid input and output conflicts by using a memory preload, with the processing task performed during the retention interval. Again, the extent to which these procedural details are considered varies across laboratories. Therefore in our experiments we avoid these potential artifacts by contrasting conditions in which there is aural presentation and oral recall of verbal memoranda with visual presentation and typed recall of these memoranda, without and with a visually presented processing task with a speeded single keypress response during a retention interval. This is illustrated in [Figure 1](#fig1){#fgc1-1 ref-type="fig"}.[](#fig1){ref-type="fig-anchor"} Finally, when comparing single- and dual-task conditions, in some experiments, the single-task conditions always come first, or the order of single- and dual-tasks is counterbalanced across participants. The former approach could lead to practice effects on the tasks that could reduce the potential impact of requiring dual-task performance. The latter approach could lead to half of the participants showing a dual-task trade-off, because of unfamiliarity with each task and with performing two tasks together when the dual-task condition comes first, and the other half showing no such trade-off. We avoided these potential problems by requiring single-task performance before and after the dual-task condition. Comparing before and after single-task allowed an assessment of whether practice effects were evident in the tasks being combined. Also, the procedure for assessing span on each task acted to familiarize participants with each task before assessing single- and dual-task performance, and this should help to reduce the impact of task practice. In all of the experiments reported here we observed either null or small practice effects between the first and second single-task blocks, but crucially these practice effects did not change the observed patterns of statistically significant dual-task effects. For this reason the results of these analyses of practice effects are reported in the [online supplementary materials](#supp). Overview of Experiments {#s6} ======================= In the current article, we present the results of four experiments with young adults. These experiments were designed to address differences among the assumptions and associated predictions from the three theoretical frameworks regarding whether or how the combination of processing and remembering affects performance of each relative to when they are each performed on their own. The theories also predict different effects of AS on visually or aurally presented verbal memory stimuli due to differences in the number of components or subsystems each framework contains. In all of the experiments reported here, the focus was on how processing during a memory retention interval affects, or is affected by, serial ordered recall of a verbal memory preload when both the memory load and the processing load are set at the measured span (titrated) for each individual. The memory task involved presentation of a random letter sequence, followed by a blank retention interval (single-task) or a processing task (dual-task), then serial ordered recall of the letter sequence. The processing task involved speeded verification of simple arithmetic. The materials for each task were chosen to be compatible with testing English-speaking (U.K.) participants and French-speaking (Swiss) participants. The tasks were performed without or with AS, for reasons given later in the predictions from each theoretical framework. In line with our earlier discussion about possible procedural artifacts, in Experiments 1 and 3, the memory list was presented visually and recall responses were typed on the computer keyboard. In Experiments 2 and 4, the memory list was presented aurally and participants recalled the list orally. In Experiments 1 and 2, titration of span was carried out without AS, while it was carried out under AS in Experiments 3 and 4. For each experiment we tested differential predictions from each of the three theoretical frameworks. Experiment 1 {#s7} ============ The starkest contrast between the theories is MCM's assumption that, with healthy adults, storage and processing can occur in parallel with little to no effect on performance in either task (e.g., [@c55]), particularly if tasks are titrated according to each participant's individual abilities (e.g., [@c37]; [@c51]), while both TBRS (e.g., [@c11]; [@c9], [@c10]) and EP (e.g., [@c20]; [@c32]) argue for interference effects due to a shared central resource. MCM also argues for a visual store to support memory for visually presented verbal material (see [@c47]; [@c54], [@c58]; [@c70]) and use of mnemonics (e.g., [@c53]; [@c66]) that can have a small effect on concurrent processing accuracy when rehearsal is prevented by AS, and so predicts more complex interaction effects than the additive main effects predicted by TBRS, and different patterns of interactions than the slot-based capacity of temporary memory argued by the EP theory. Experiment 1 aimed to investigate different predictions from each theory for the effects on a visually presented verbal memory task and a visually presented verbal processing task of performing both memory and processing together relative to performing each on its own, and also the effect of AS on the presence or magnitude of these effects. Method {#s8} ------ This experiment, and all subsequent experiments, were approved by the ethics committees for The University of Edinburgh, The University of Fribourg, and The University of Geneva. The general trial sequences for all experiments are shown in [Figure 1](#fig1){#fgc1-2 ref-type="fig"}. ### Participants {#s9} Participants were recruited from the student populations at the University of Edinburgh, United Kingdom, and the Universities of Fribourg and Geneva, Switzerland. They received different honoraria in each country due to concerns about differing motivation for cash rewards in each location. In the United Kingdom, participants were compensated for their time with an honorarium of £12. In Switzerland, participants were either offered cinema vouchers (equivalent to 16 CHF) or course credit. Sixty-four participants were recruited in total, 32 from each country (48 female and 16 male, *M*~age~ = 22.19, *SD* = 2.56). The sample size in each lab was selected to be comparable with previous research in the working memory literature, but to consist of a relatively large sample when compared to previous MCM, TBRS, and EP research. ### Apparatus {#s10} Because the experiment was conducted across laboratories, efforts were made to ensure that the same equipment was used in each location. Each lab was equipped with the same model of laptop running PsychoPy (Version 1.84.2; [@c67]), connected to the same models of external monitor, headphones, and button boxes. Due to differences in British English and Swiss French keyboard layouts, different models of keyboards were used at each site. PsychoPy settings and external monitors were set so that text stimuli were presented with an approximate vertical visual angle height of 1.3°. The same equipment and settings were used for all other experiments described in this article. The experimenter remained in the room during the experiment. ### Procedure {#s11} The session began with a recognition task, in which participants were shown letters on screen and immediately typed the presented letter. Data from the pretest served as a check that the memory stimuli were sufficiently distinguishable from each other, and are reported in the [online supplementary materials](#supp). The pretest was followed by the memory and processing titration conditions, which set the load levels for the single- and dual-task conditions for each participant. Participants completed the single- and dual-task conditions without and with AS, with half the participants completing the no-AS condition first and half starting with the AS condition. In each no-AS and AS block, participants started a single-task memory block and a single-task processing block consisting of 10 trials each (the order of the memory and processing blocks were also counterbalanced). This was followed by two blocks of 10 dual-task trials, followed again by two single-task blocks of memory and processing. Each participant therefore completed 40 single-task memory trials (20 without and 20 with AS), 40 single-task processing trials (20 without and 20 with AS), and 40 dual-task trials (20 without and 20 with AS). #### Memory and processing titration procedure {#s12} Before the experimental conditions, both memory and processing loads were titrated to each participant's individual abilities. The titration conditions followed a "staircase" procedure, in which the demand of a task was increased or decreased depending on a participant's performance. Sixteen trials were presented in total, in pairs of two set at each level of demand, starting at five items for both tasks. If accuracy across a pair of trials was ≥80%, the demand of the task was increased for the next two trials; if accuracy was below 80% the demand was decreased. If a participant passed the final two trials (i.e., the eighth pair, Trials 15 and 16), and these two trials were the highest "level" they had reached up until that point, then additional pairs of trials were administered until failure to reach the 80% correct criterion. Participants' memory and processing spans were recorded as the highest level at which they achieved 80% accuracy or above. Three practice trials were given at the start of each titration, with demand set to four items. Memory and processing titration were completed without AS in this experiment. #### Single-task memory {#s13} The same set of letters was used for both English and French stimulus sets, which contained all the letters of the alphabet except vowels (to reduce pronounceability of memory sequences), and multisyllable letters from either language (*w*, *y*). The letter *z* was also excluded due to the desire to maintain parity with the stimulus sets for WoMAAC aging studies conducted across U.K. and U.S. laboratories, as *z* is pronounced differently in British and American English. Lists were randomly generated for each trial, without replacement. Participants initiated each trial with a button press, which was followed by a 2-s interval. Letters were then presented in the center of the screen sequentially for 250 ms each, with a 750-ms interstimulus interval (ISI). Therefore, the study phase lasted *n* × 1,000 ms. The onset of the last letter was followed by a two second interval, followed by a 10-s retention interval that consisted of five circles flashed on the monitor at a rate of one every 2 s, with a 250-ms ISI. Following the retention interval a 400-Hz tone sounded to prompt recall. Participant recalled items using the keyboard, and were able to pass on a letter by pressing the *0* key. The AS conditions proceeded in much the same way, except that 1 s before the presentation of the first letter, a 400-Hz tone sounded to prompt participants to begin repeating "ba" at a rate of two per second (see [Figure 1](#fig1){#fgc1-3 ref-type="fig"}). Before each AS condition participants were presented with a tone playing twice every second to demonstrate the speed they should be repeating "ba." Participants were instructed to cease AS when they heard the second tone (after the 10-s interval), and recall the memory items by typing them on the keyboard. To be clear, AS commenced prior to the start of the presentation of the memory sequence, and continued until after the filled or unfilled retention interval. This procedure was important for the MCM, which assumes that AS disrupts the use of phonological encoding and subvocal rehearsal of the visually presented letter sequence. #### Single-task processing (arithmetic verification) {#s14} The processing task required participants to verify simple equations (e.g., "3 + 5 = 8, correct/incorrect?"). These equations were randomly generated for each trial, with each equation having a 50% probability of being presented with a correct solution. Participants initiated trials with a button press, after which they heard five 250-ms-long, 300-Hz "placeholder" beeps played once every second. Two seconds after the onset of the final beep, the first equation appeared for (10,000/*n*) -- 250 ms (where *n* is the number of items to be presented), followed by a 250-ms ISI, then the next equation. Following the presentation of the final equation, a 400-Hz tone played to signify the end of the trial. Participants pressed a button marked with a "tick" (or "check") for correct equations, and a button marked with a "cross" for incorrect equations (as they appeared on the screen). The task progressed whether the participant responded within the presentation time or ISI or not, that is, the sums remained on screen during their entire presentation window, and the ISI always occurred in full, regardless of the RT of the participant. In the AS condition, a 400-Hz beep preceded the first 300-Hz placeholder beep to prompt participants to begin repeating "ba--ba--ba." They were instructed to cease AS once they heard the second 400-Hz beep. #### Dual-task {#s15} The single-task memory and processing procedures were designed to match the timing of the dual-task condition with the use of placeholder beeps or circles. Dual-task trials therefore proceeded in a similar fashion to the single-task memory condition, both without and with AS, except that instead of the placeholder circles appearing during the 10-s retention interval the arithmetic verification task appeared. Participants were instructed to complete both tasks, with no importance being placed on one task or the other by the instructions or by the experimenter. Participants were given three practice trials before the first 10 experimental dual-task trials were presented. The demand for the dual-task practice trials was set at one item below each participant's span. ### Predictions {#s16} Although each of the theoretical frameworks incorporates different assumptions, and therefore makes different predictions, none is a formal computational model and therefore the predictions are qualitative. The predictions refer to whether or not an effect is expected to be present, and whether any such effect will be small, medium, or large. Because the models cannot make specific predictions for the size of effects, particular emphasis was placed on predicting the size of effects in relation to other factors within the experiment (e.g., the size of the dual-task effect compared to the AS effect), and in later experiments predicting effect sizes in relation to previous experiments. The hierarchical models we describe in the upcoming analysis section estimate a random participant effect standard deviation, therefore summarizing the average difference between participants in the dependent variable (i.e., accuracy, or more specifically the log odds of a correct response). It is therefore possible to specify the size of effects arising from experimental designs by placing them on a scale of differences due to individual differences. WoMAAC partners were asked to generate their predictions with this scale in mind. Predictions were specified in terms of small, medium, and large effects. Translating these into a common scale we used conventional criteria to refer to effects on the scale of expected individual differences ([@c22]). Consequently, 0.2 of the average difference between individuals represents a small effect, 0.5 a medium effect, and 0.8 a large effect. These values were chosen as reasonable for effect sizes in research on memory ([@c63]).[^1^](#fn1){ref-type="fn"} To supplement the description of each account's predictions simulated data conforming to the described expectations were generated and plotted and can be found on the OSF. Although each framework was required to generate predictions on the full set of variables, some predictions were speculative and not central to a particular theory. For example, the TBRS model has in the past largely focused on costs on memory, so predicted effects of dual-tasking on processing were generated from what the model would ideally expect when attention is split between tasks. Predictions were also generated in each theory's proponents own chosen format: MCM and TBRS predictions focused on previous findings in the working memory literature, while EP generated predictions based on a simple capacity model created specifically for this experimental paradigm. The mathematical model generated by EP is available to view on the OSF, while a written summary of it is reported here for easy comparison with the predictions from the other theories. [Table 1](#tbl1){ref-type="table"} summarizes the predictions made by each of the theories, and the full descriptions of these predictions are described in the next sections.[](#tbl1){ref-type="table-anchor"} ### Multiple components {#s17} In the MCM, serial-ordered recall with visual presentation of a letter sequence is assumed to reflect (a) translation of the visually presented items into a phonological code; (b) the involvement of the phonological loop, comprising a passive phonological store and subvocal articulatory rehearsal to retain both item and serial order information as phonological codes; (c) visual encoding of the letters in a visual cache or temporary visual memory that can support item and order information; and (d) activation of representations of the visual and phonological information (of item, but not order) about the letters in LTM. All elements are thought to contribute to the observed span score. However, phonological encoding will dominate span performance when subvocal articulatory rehearsal is available. For memory above the span levels that are typical of healthy adults, there is thought to be an additional contribution from a range of mnemonic strategies such as chunking or semantic associations. Visually presented items for arithmetic verification are assumed to involve activation of arithmetic knowledge in LTM and a decision process together with initiation of a manual response. None of these aspects of the task is thought to require use of the phonological loop, and so no effect of AS on processing is predicted by the MCM. Visually presented memory items may be disrupted by the arithmetic verification task during the retention interval due to the concurrent activation in LTM of arithmetic knowledge and of letter representations. In addition to these disruptive effects, there may be an additional small disruption to memory because of the visually presented arithmetic disrupting the contents of the visual cache. The overall disruption will be seen as a small effect size because the operation of the phonological store and articulatory rehearsal will be unaffected by visually presented arithmetic verification. This prediction is derived from previous studies that have shown no, or small dual-task costs when combining an at-span verbal memory preload with a processing task (e.g., [@c21]; [@c51]), and evidence showing low correlations between processing and memory performance (e.g., [@c35]; [@c55]; [@c82]). MCM assumes that AS during the encoding and retention phases will prevent phonological encoding and articulatory rehearsal of the memory items, and encourage the use of visual codes (e.g., [@c54], [@c58]; [@c70]). Memory for visually presented letters will be impaired, because of a lack of phonological encoding and articulatory rehearsal, but will only be a medium effect size and will remain well above floor through a combination of passive storage within the visual cache, and activation of letter representations in LTM. For dual-task with AS, memory for visually presented items will be impaired with a medium effect size because of the use of visual codes to support memory even when there is a lack of phonological encoding and articulatory rehearsal. This means there will be a Dual-Task × AS interaction, with a larger dual-task effect under AS. The support from visual codes may be less effective than for memory alone plus suppression because of interference from the visual presentation and manual response for arithmetic verification. Under AS, there will be a small dual-task effect on verification because of participants attempting to use mnemonic strategies for retaining the letters to try to compensate for the lack of articulatory rehearsal. Therefore, for processing, a small interaction is also predicted such that there is a dual-task effect only under AS. ### Time-based resource sharing {#s18} Verbal memory span reflects the involvement of both the phonological loop and the executive loop in the TBRS model (see [@c18] for a review). At span (single-task, no AS), participants should recruit all the resources at their disposal, that is, because the phonological loop is limited to about four letters, the executive loop is used to "boost" performance beyond this limit. Thus, performing a processing task that involves attention (i.e., addition verification task) should disrupt the maintenance of verbal information through the executive loop and lead to a poorer memory performance than in the single-task condition. The addition of concurrent articulation will impair the use of the phonological loop, resulting in poorer recall performance. Previous experiments showed that such a reduction is stronger than the reduction produced by a concurrent attentional-demanding task (e.g., [@c17]). Thus, TBRS predicts a medium main effect of task and a large main effect of suppression. Finally, the joint impairment of the phonological and executive loops by a concurrent articulation and the addition verification task, respectively, should lead to additive effects, and to a minimum recall performance. This should constitute a residual memory performance that remains when working memory maintenance mechanisms are blocked. For the processing task, performing the addition verification task involves the executive loop. Because maintaining letters at span also involves the executive loop, a medium detrimental effect on processing should be observed in the dual-task condition compared with the single-task condition. AS should not have any effect on addition verification, except if AS induces a small attentional capture. In such a case, the addition of AS should result in a small reduction in processing performance. Therefore, two additive main effects are predicted, with the possibility of a small interaction to the extent that the addition task requires phonological processes. ### Embedded processes {#s19} The EP model assumes that task relevant information from LTM is held in a heightened state of activation subject to decay and interference from other items with similar features. A subset of that activated information can be held in the focus of attention, which helps to overcome decay and interference. Additionally, a way to prolong and improve the maintenance of some verbal information with very little contribution of attention is through subvocal rehearsal. To coordinate a verbal memory and verbal processing, dual-task participants must share the capacity of the focus of attention between these tasks. Compared to single-task performance, dual-task accuracy on memory and processing is predicted to be lower due to the need to split attention between these two tasks. Both tasks are assumed to benefit from subvocal rehearsal, and so an effect of AS on both tasks is predicted. However, memory performance also benefits from both rehearsal during encoding (as there is no AS during encoding for visually presented memory items) and visual sensory memory (due to memory items being presented visually). While rehearsal prevents time-based decay, visual sensory memory supports performance by providing additional storage while also freeing up the focus of attention for storage of other memory items. Likewise, the arithmetic task is assumed to rely on some mechanisms that are not relevant to the memory task (likely well-learned mathematical rules that can be recalled from LTM). This task also benefits from visual sensory memory, as the use of this separate storage frees up the focus of attention for processing. These different factors contributing to single- and dual-task performance for each task lead to a set of predictions based on the overlap in shared mechanisms for each tasks. To make these predictions, some assumptions need to be made regarding the behavior of participants: (a) that participants are motivated to use all available resources to complete tasks and (b) that the attentional costs of the processing task can be expressed in terms of the number of items held in the focus of attention, as it is with the memory task. Although the theory does not specify the allocation of attention between tasks, when encouraged to make a guess at the allocation, the protagonists of this theory simply guessed that participants would split attention and other shared resources equally between the memory and processing tasks. In sum, based on the assumptions made by the model as to the separate and shared mechanisms utilized for the memory and processing tasks, EP predicts large dual-task and AS costs to both memory and processing tasks. The model also predicts a smaller dual-task cost under AS (i.e., a medium interaction effect), as the shared subvocal resource is no longer split between the two tasks in single- and dual-task conditions, so the dual-task costs are reduced compared to the no AS condition. Results {#s20} ------- ### Analysis method {#s21} To avoid the potential pitfalls of conventional methods (e.g., ANOVA and other normal models can lead to spurious results, particularly in the interpretation of interaction effects [@c36]; [@c43]), data were analyzed using generalized linear mixed effects models ([@c14]). This method allowed modeling of non-normal response variables (via a logit link function) while also acknowledging that observations are nested within individuals (i.e., repeated measures). The analyses were conducted using the lme4 package (Version 1.1--17, [@c13]), and the full analysis scripts for the experiments reported in this article are available on the OSF. List of memory items and sequences of sums were analyzed on a by item basis: that is, if a participant remembered/responded correctly to three out of four items in a list/sequence, then the log odds would be modeled on this performance. Although participants were able to answer pass for the memory task, these responses were simply coded as incorrect for the purposes of analysis. As detailed in the previous section, WoMAAC partners provided effect size predictions, but the first step of our analyses involved reducing the complexity of models to effects of interest. Initially full models, with all main effects and interactions plus a random intercept for each participant, were fitted to the memory and processing data. For both memory and processing data the main effects were task (single vs. dual), AS (without vs. with), and site (Switzerland vs. United Kingdom), and all interactions, including the three-way Task × AS × Site interaction, were included. The first model comparison involved removing the highest order interaction (the three-way interaction), and comparing it with the reduced candidate model. Model comparison was based on Bayesian information criterion (BIC) values ([@c75]): If these values were lower for the candidate model, this was evidence for the removal of the effect and to use the new simpler model for future comparisons. Two-way interactions, and then main effects, were then considered in turn. Each two-way interaction and main effect was considered separately with a model containing all other effects (apart from already removed higher order effects). If model comparison favored the inclusion of an interaction, lower order interactions or main effects contained within that interaction were not considered for removal later in the chain. Summaries of the best-fitting statistical models from each experiment are reported in this article, but the full analysis script showing each step is available on the OSF. ### Analyses {#s22} Mean memory span was 6.34 (*SD* = 1.28), and mean processing span was 8.00 (*SD* = 2.0). The best fitting memory and processing statistical models are summarized in [Table 2](#tbl2){ref-type="table"}. Because model comparison was conducted via BIC comparison, it is possible to calculate a Bayes factor comparing the winning statistical models to the next best candidate model. The Bayes factor in favor of the best fitting statistical model for memory was 31.34 (BIC for best fitting statistical model = 21,696.57; BIC for next best candidate model = 21,703.46), and for processing the Bayes factor in favor of the best fitting statistical model was 6,734.51 (BIC = 16,022.03; BIC for next best candidate model = 16,039.67). For memory, there were statistically significant main effects of dual-task (*ES*~scaled~ = −0.73) and of AS (−2.96). Although the effect of site was not statistically significant, the model comparison method described earlier resulted in the retention of Condition × Site and AS × Site interactions, both of which were statistically significant in the model (*ES*~scaled~ = −0.30 and 0.39, respectively). These interactions reflect a larger dual-task effect at the U.K. lab, and a smaller AS effect in the U.K. lab compared to the Swiss lab (note that the former interaction effect runs counter to the pattern that would be expected due to testing site bias). [Figure 2](#fig2){#fgc2-1 ref-type="fig"} summarizes dual-task and AS effects split across labs and clearly demonstrates the source of the interactions is the larger single-task AS effect in the Swiss lab reducing the dual-task effect in the same lab. Contrary to the memory task, processing performance was not affected by either dual-task or AS manipulations (see [Figure 3](#fig3){#fgc3-1 ref-type="fig"} for plotted data).[](#tbl2){ref-type="table-anchor"}[](#fig2){ref-type="fig-anchor"}[](#fig3){ref-type="fig-anchor"} Discussion: Summary of Experiment 1 {#s23} ----------------------------------- All three theories made clear predictions for the outcome of Experiment 1, ranging from null effects (MCM), to additive effects of dual-task and AS (TBRS), to interactions between these two effects (MCM/EP). While each of the models predicted some of the observed effects, no account predicted the complete pattern of results. A large dual-task effect was observed for memory performance. This does not fit with the predictions from the MCM of a small disruptive effect of processing on memory accuracy. Both TBRS and EP predicted the dual-task effect, yet both models predicted medium effect sizes where a very large effect size was observed. All three models predicted an effect of AS, though both MCM and EP predicted a medium effect size where a large effect as predicted by TBRS was in fact observed. It is important to note that constrained effect sizes were used for predictions of small, medium, and large effect sizes (0.2, 0.5, and 0.8, respectively), so it may be considered more informative to compare each model's predicted magnitude of dual-task and AS effects. Thus, TBRS correctly predicted that the dual-task effect would be smaller than the AS effect. MCM also predicted this pattern, but only because such a small effect of dual-task was predicted and the predicted size of the AS effect was still smaller than that observed. When forced to make a prediction of the relative effect sizes for dual-task and AS, EP assumed equal contribution of attention to rehearsal and processing and so predicted that these effects would be equal, which the data do not support. MCM and EP both predicted Dual-Task × AS interactions with memory, though each predicted different patterns. Neither of these interactions was present in the data. Contrary to the MCM prediction, the effect of dual-task was present without and with AS, and the introduction of AS did not reduce the size of the dual-task effect as predicted by EP. That is, it appeared that the effects of dual-task and of AS were independent and additive. TBRS predicted a medium dual-task effect and a small AS effect on processing with no interaction, while the EP model predicted the same Dual-Task × AS interaction as it did for memory where a smaller dual-task effect was observed under AS. Neither of these patterns was observed in the data. The MCM prediction of no dual-task effect on processing when there was no concurrent AS was accurate, yet the Dual-Task × AS interaction prediction was not confirmed, as AS did not introduce a statistically significant effect of task. Finally, although large effects of AS and dual-task were found for memory performance, performance levels were still well above chance even when both dual-task and AS were required. This highlights a difference in emphasis between the three theoretical approaches, with MCM studies typically pointing to the size of the residual performance levels, even under high cognitive load, whereas TBRS and EP typically note the reduction in performance relative to baseline levels. In summary, while each model predicted some trends no account provided a satisfactory approximation of all the observed data patterns. Where some models succeeded, for example TBRS and EP in predicting dual-task effects on memory, those same models failed to predict patterns in the processing task. The opposite pattern was partially true for MCM, where small dual-task effects on processing were predicted while the dual-task effects on memory were not. Considering that the models all specify some interplay between memory and processing in working memory, accurate or semiaccurate predictions of one half of the data are not sufficient to identify a "winning" framework. Experiment 2 {#s24} ============ Experiment 1 investigated the effect of the dual-task and AS on memory and processing, and found large effects of both on memory but no effects on processing. Experiment 1 featured visual presentation of memory items, which, according to the MCM, meant that these items were verbally recoded when there was no concurrent AS but that suppression prevented recoding leading to a dual-task effect. It occurred that there was a dual-task effect in both no-AS and AS conditions, but such a recoding hypothesis was only presented by the MCM and so may be of use when differentiating between the models. Experiment 2, therefore, replaced the visually presented memory task and typed recall with an aurally presented task and oral recall. In Experiment 2, we aimed to investigate whether the presentation format changed the pattern of statistically significant effects or increased/decreased the magnitude of these effects, as only the MCM would make strong predictions regarding differences in performance due to presentation format. Method {#s25} ------ ### Participants {#s26} As mentioned previously, data collection for Experiments 1 and 2 ran concurrently, and so participants were recruited in the same way as described in Experiment 1, resulting in a sample of 64 participants, 32 from the United Kingdom and 32 from Switzerland (46 female and 18 male, *M*~age~ = 20.96, *SD* = 2.46). The samples for Experiments 1 and 2 were independent. ### Procedure {#s27} The procedure for Experiment 2 proceeded in the same way as in Experiment 1, except for the substitution of an aurally presented task in place of the visually presented memory task, and participants responded orally rather than typing their responses. ### Aurally presented verbal memory task {#s28} Memory task stimuli were generated using the built-in Apple OS × 10.11.4 voice. The American English voice "Allison" was used in the U.K. lab, and the French voice "Audrey" was used in the Swiss lab. The same list of letters from Experiment 1 was used in Experiment 2, and lists were again randomly generated for each trial without replacement. The auditorily presented memory task proceeded with the same timing as the visual presentation memory task in Experiment 1. Memory item onsets were separated by 1,000 ms, so that the study phase (as with Experiment 1) was *n* × 1,000 ms. Following the blank retention interval, or the retention interval filled with the processing phase, a 400-Hz tone prompted participants to orally recall the letters, saying, "pass" for any letter they could not remember. The experimenter typed the participants' responses on a separate keyboard and monitor. Both the experimenter's keyboard and monitor were out of view of participants. In the AS conditions, the 400-Hz tone signaling the beginning of the AS component of the task was played 1,000 ms after the onset of the last memory item, rather than before the onset of the memory items as it had in Experiment 1. In Experiment 1 the AS during encoding was to maximize the use of nonphonological memory processes (i.e., to avoid phonological storage through recoding of the memory items); the encoding phase in the AS condition for Experiment 2 was presented in silence to maximize the likelihood of phonological storage of memory items---an important procedural consideration for the MCM. ### Predictions {#s29} Data for Experiments 1 and 2 were collected concurrently, so the predictions for Experiment 2 do not take into account the findings from Experiment 1. The predictions for Experiment 2 are summarized in [Table 1](#tbl1){ref-type="table"}. ### Multiple components {#s30} In the MCM, serial-ordered memory span with aural presentation of letters is assumed to reflect (a) a passive phonological store, (b) articulatory rehearsal, and (c) activation of representation of the letters in LTM for items, but not order. All three elements are thought to contribute to the observed span score. For memory above span levels that are typical for healthy adults, there is thought to be a contribution from a range of mnemonic strategies such as chunking or semantic associations. When arithmetic verification is performed during a retention interval for an aural letter sequence, it is expected that the concurrent activation in LTM of arithmetic knowledge and of letter representations may result in some disruption of letter memory, because of a small contribution of LTM activation to item memory in auditory, serial order letter span. However, this disruption will not be statistically reliable because the operation of the phonological store and articulatory rehearsal will be unaffected by visually presented arithmetic verification. Thus, no dual-task cost is predicted. It is expected that there will be no effect on arithmetic verification of a memory preload of an at-span aurally presented letter sequence. AS was added during a blank retention interval, but not during encoding. This is important because it allows for initial phonological encoding and rehearsal during presentation of the at-span letter sequence, but prevents articulatory rehearsal to retain the sequence during the retention interval. Memory for aurally presented letters will be impaired, showing a large effect of AS. Memory performance will remain above floor through a combination of passive storage within the phonological store and activation of letter representations in LTM. When AS is added to visually presented arithmetic verification, it is anticipated that there will be no effect on verification performance. When AS is added to arithmetic verification after presentation (without suppression during encoding) of an aural preload of an at-span letter sequence, memory for the letter sequence will be impaired for the same reasons as for suppression during memory retention without arithmetic verification. The extent of the disruption will show as a large effect on memory. Thus there is no interaction predicted between suppression and task (single- vs. dual). There will be a small dual-task effect on verification under AS because of participants attempting to use mnemonic strategies for retaining the letters in an attempt to compensate for the lack of articulatory rehearsal. Therefore, for processing a small interaction is predicted such that performance should be below span (\<80%) in the dual-task with AS condition. ### Time-based resource sharing {#s31} The TBRS predictions for Experiment 2 are unchanged from Experiment 1, with medium effect of dual-task, a large effect of suppression on memory, and a small dual-task effect on processing. ### Embedded processes {#s32} EP predictions for Experiment 2 closely match those from Experiment 1, and follow a similar set of assumptions. While in Experiment 1 letter memory was assumed to be supported by visual sensory memory, in this experiment memory performance is assumed to be supported by auditory sensory memory. Auditory sensory memory is assumed to be more efficient than visual sensory memory for verbal materials, providing an additional source of memory that does not have to be divided between storage and processing, and so medium dual-task and AS costs are predicted in contrast to the large effects predicted in Experiment 1. As in the previous experiment, EP predicts a medium interaction between dual-task and AS in which the dual-task cost under AS is smaller due to the fact that subvocal mechanisms are no longer utilized and therefore shared between memory and processing tasks. Results {#s33} ------- Data from Experiment 2 were analyzed using the same methods as Experiment 1. Mean memory span was 6.52 (*SD* = 1.04), and mean processing span was 8.61 (*SD* = 2.00). The best fitting statistical model for memory is summarized in [Table 3](#tbl3){ref-type="table"}, which displays coefficient estimates for each effect. The Bayes factor in support of this model over the more complicated candidate model (calculated using BIC values, winning model = 21,293.38, more complicated candidate = 21,309.80) was 3,677.54, and over 1 million for the simpler candidate model (BIC for simpler model = 22,739.29). There were statistically significant dual-task and AS effects. Scaling the dual-task effect in terms of average differences between participants, the effect of going from single- to dual-task results in an effect size of −1.21. The scaled AS effect size was −2.00.[](#tbl3){ref-type="table-anchor"} There was also a large effect of site (0.68), with U.K. participants performing better on the memory task than Swiss participants. As with Experiment 1, and contrary to what would be expected by site bias, there was also a slightly larger dual-task effect in U.K. participants (Condition × Site interaction, −0.34). Interpreting this main effect of site and interaction is straightforward when splitting participants' performance across site (see [Figure 4](#fig4){#fgc4-1 ref-type="fig"}): The higher single-task performance in U.K. participants explains the larger dual-task effect. It is difficult to explain why Swiss participants did not perform at the 80% titration level, but because the interaction effect is small (and does not include the AS effect) it does not complicate interpretation of the overall data pattern.[](#fig4){ref-type="fig-anchor"} The best fitting statistical model for processing is also summarized in [Table 3](#tbl3){ref-type="table"}. Unlike memory performance, processing performance was only affected by the introduction of a dual-task (*ES*~scaled~ = −0.43). Note that this dual-task effect was not present in Experiment 1. Processing data are summarized in [Figure 5](#fig5){#fgc5-1 ref-type="fig"}. The Bayes factor in support of the best fitting statistical model was 4103.13 (BIC for best fitting model = 15,853.39, next best candidate model BIC = 15,870.03).[](#fig5){ref-type="fig-anchor"} Discussion {#s34} ---------- ### Comparison of Experiments 1 and 2 {#s35} Memory and processing performance in Experiments 1 and 2 were compared using the same analysis method utilized for the separate analyses, except with the addition of a format between-subjects factor. The model comparison followed the same procedure of removing effects from the model and comparing BIC values, and the winning models for each task are summarized in [Table 4](#tbl4){ref-type="table"}. The Bayes factors supporting best fitting statistical models for memory and processing were 40.20 (BIC for winning model = 42,986.90, next best candidate model = 42,994.29) and 3,344.26 (winning model = 31,876.44, next candidate = 31,892.66), respectively.[](#tbl4){ref-type="table-anchor"} For memory, aside from the clear effects of dual-task and AS (*ES*~scaled~ = −1.65 and −2.89), the best fitting statistical model also contained format interactions (though the main effect of format was not statistically significant). The Dual-Task × Format interaction reflects a larger dual-task effect for the auditory/oral task in Experiment 2 compared to the visual/typed task of Experiment 1 (*ES* = 0.57). However the AS effect was smaller for auditory/oral compared to visual/typed (*ES* = −1.38). There was also a Format × Site interaction as U.K. participants' auditory/oral performance was higher than Swiss participants' (this effect was also detected in the memory analysis of Experiment 1). For processing, there was an overall statistically significant dual-task effect (*ES* = −0.61) which was driven by the effect observed in the auditory/oral condition (Experiment 2) as evidenced by the Dual-Task × Format interaction (0.46). ### Summary of Experiment 2 {#s36} As with Experiment 1, a large dual-task effect on memory was observed with aural presentation of stimuli. MCM did not predict an effect of dual-task (either with or without AS), while TBRS and EP both predicted medium dual-task effects. The AS effect was predicted by all three theories, but only TBRS correctly predicted that this effect would be larger than the dual-task effect. For processing, a medium dual-task effect was observed. TBRS predicted a small effect, and EP predicted a medium effect. MCM, however, predicted that the dual-task effect would only be present under AS (the same prediction as for Experiment 1), but this was not the case as no interaction between dual-task and AS was observed. The between-experiment comparison revealed that the dual-task effect on memory was larger than that observed in Experiment 1. For processing, the between-experiments comparison confirmed the different patterns of data in Experiments 1 and 2 where a dual-task effect was only observed in the auditory/oral format condition. However, it is important to note the methodological differences between Experiments 1 and 2 relating to the onset of AS: For Experiment 1 (visual presentation), AS was carried out during the encoding phase, whereas in Experiment 2 the AS onset was after the presentation of the last memory item and before the processing phase/retention interval. This difference was important theoretically, as discussed in the introduction to Experiment 2. However, it may be that the differences in dual-task effect sizes were due to this difference in procedure, as AS may have interfered with encoding in Experiment 1 while having a start up cost that interfered with processing in Experiment 2. MCM was the only model to propose different patterns of memory performance between Experiments 1 and 2, predicting a small dual-task effect with visual presentation and no effect for aural presentation. However, the opposite pattern was observed with a larger effect of dual-task on memory being observed in Experiment 2 compared to Experiment 1. While EP stated that different supporting memory processes were involved in visual and aural presentation tasks (i.e., visual and auditory sensory memory), the model did not predict that these differences would have an observable outcome on behavior. TBRS specifically predicted no difference between experiments, but differences were observed with a larger dual-task effect of memory in Experiment 2 than in Experiment 1, and a dual-task impact on processing in Experiment 2 that was not observed in Experiment 1. So, none of the three theoretical frameworks correctly predicted the full pattern of results observed across the two experiments. ### Titration under AS {#s37} Experiments 1 and 2 revealed large dual-task effects on memory with both visual and auditory presentation formats, and null/small dual-task effects on processing. The three models had mixed success in predicting the patterns of results, though all three missed large trends in the data. Because Experiment 1 (visual/typed) most closely conformed to TBRS/EP for memory data, and to MCM for processing data, Experiment 3 adapted this procedure to investigate further the different assumptions regarding maintenance and processing and how maintenance and processing are affected by AS. Each of the models makes some assumptions regarding the involvement of phonological/verbal rehearsal of memory items, and that these processes are affected by the addition of concurrent AS to the dual-task conditions. The goal of the titration procedure was to ensure that all participants were performing tasks set at appropriate levels of demand, but also to provide a reliable single-task measure of memory and processing performance. Titration of memory and processing tasks were completed without concurrent AS suppression, meaning that the memory task demand was adjusted to a level where memory was being supported by rehearsal. Whereas all three models agreed that memory was supported by some form of subvocal rehearsal, only the MCM states that a small number of verbal memory items can be maintained with no requirement to rehearse or refresh (i.e., no attentional requirement). In MCM, subvocal rehearsal is said to "boost" memory performance beyond the capacity of this store. In Experiments 1 and 2 this means that, according to MCM, single-task memory performance is a product of not only attention-free storage but also rehearsal methods that are also affected by concurrent AS ([@c6]; [@c64]). Experiments 3 and 4 aimed to test the MCM's proposal of an attention-free verbal store by titrating memory under AS for both visual and auditory presentation formats in an attempt to more accurately measure the capacity of memory for verbal items when subvocal rehearsal is not available. Experiment 3 {#s38} ============ Method {#s39} ------ ### Participants {#s40} Participants were recruited in the same way as in previous experiments, half in the United Kingdom and half in Switzerland. The total sample consisted of 32 participants who had not taken part in either of the previous experiments (24 female and eight male, *M*~age~ = 21.72, *SD* = 2.25). ### Procedure {#s41} The procedure for Experiment 3 closely resembled that of Experiment 1, with visual presentation and typed recall of memory items. The primary way in which the procedure deviated was that titration of memory and processing tasks was completed under AS. The trial procedures for memory and processing trials in the titration conditions followed the same timings as the AS conditions from Experiment 1. Single- and dual-task conditions were then completed in the same order as in previous experiments, however only data for performance under AS were collected. ### Predictions {#s42} Predictions are summarized in [Table 1](#tbl1){ref-type="table"}. ### Multiple components {#s43} The MCM predicted that there would be no subvocal rehearsal for the memory items because this would be prevented by the AS. There may be both phonological and visual encoding, with retention in passive, domain-specific temporary memory systems. Without suppression in previous experiments, rehearsal is assumed to be a strategy to boost temporary memory performance, and so span without suppression overestimates temporary memory capacity. Because rehearsal cannot be used under AS, the titrated spans will provide a more accurate measure of the capacity of the temporary memory systems. However, there might be attempts by some participants to use mnemonic strategies instead of rehearsal, and this would use a small amount of processing resource. Thus, MCM predicts that there will be at most a small dual-task effect, but possibly no effect on memory performance (contrary to Experiment 1), and no dual-task effect on processing performance (as was found in Experiment 1). ### Time-based resource sharing {#s44} Under AS, memory span reflects the involvement of the executive loop in the TBRS model. Thus, performing a processing task that involves attention (i.e., the addition verification task) should disrupt the maintenance of verbal information through the executive loop and lead to poorer memory performance than in the single-task condition. The model therefore predicts a medium dual-task effect on memory. For processing, performing the addition verification task involves the executive loop. Because maintaining letters at span also involves the executive loop, a detrimental effect on processing should be observed in the dual-task condition compared to the single-task condition. The TBRS model predicts a large dual-task effect on processing. ### Embedded processes {#s45} In Experiments 1 and 2 participants were able to make use of subvocal rehearsal to reach a high span level during the titration procedure. The data from these previous experiments have led us to revise our account such that we no longer assume that rehearsal makes a contribution to processing. Thus, the manipulation of suppression and single- versus dual-task are assumed to be independent. Therefore, we predict a large effect of single- versus dual-task on memory in the present experiment where participants are titrated under suppression. Further, we predict that the dual-task cost on memory will be larger in this experiment relative to that found in Experiments 1 and 2. This is because we assume that the processing task consumes a constant "number of items" worth of attention and consequently it will have a greater cost in terms of proportion correct items recalled in position on the smaller list lengths obtained via titration under suppression. For processing, there is a clear asymmetry in the data from Experiments 1 and 2. According to the EP account this is due to the preferential allocation of attention to the processing items as they appear at the expense of maintaining items in memory. Therefore, we predict no effect of single- versus dual-task on processing performance. Results {#s46} ------- Data from Experiment 3 were analyzed using the same methods as previous experiments, yet because all the conditions were performed with suppression the process was simplified because there were only two main effects to consider: dual-task and site. Mean memory span under AS was 5.00 (*SD* = 1.00), and mean processing span under AS was 8.56 (*SD* = 2.00). The best fitting statistical model for memory is summarized in [Table 5](#tbl5){ref-type="table"} and contained a significant main effect of dual-task (*ES*~scaled~ = −1.64) and a Dual-Task × Site interaction (−0.49). The model comparison procedure produced a Bayes factor of 1.06 against the removal of the Dual-Task × Site interaction (BIC full model = 4,498.70, BIC for model without interaction = 4,498.81). As stated in the preregistered materials, we treated BIC as a binary choice in our model comparison procedure despite the inconclusive Bayes factor. The interaction reflects a larger dual-task cost in U.K. participants. There were no effects of dual-task or site on processing, with a Bayes factor of 361.41 supporting the removal of both of these factors (BIC for best fitting statistical model = 3,813.78, BIC for next best candidate model = 3,825.56). Memory and processing data are summarized in [Figures 6](#fig6){#fgc6-1 ref-type="fig"} and [7](#fig7){#fgc7-1 ref-type="fig"}.[](#tbl5){ref-type="table-anchor"}[](#fig6){ref-type="fig-anchor"}[](#fig7){ref-type="fig-anchor"} Discussion: Summary of Experiment 3 {#s47} ----------------------------------- MCM predicted a small or null effect of dual-task on memory due to titration under AS resulting in a more accurate measure of the verbal memory store. Conversely, TBRS and EP predicted medium and large effects respectively. Contrary to MCM predictions, and in line with TBRS and EP, a large dual-task effect on memory was observed in Experiment 3. Both EP and MCM predicted no effect of processing (as was observed in Experiment 1 with visual presentation and typed recall), though for different reasons. MCM predicted no effect due to separation of processing resources from memory, while EP predicted no effect on processing due to preferred allocation of attention to this more immediate task. TBRS predicted a dual-task effect on processing due to the involvement of the executive loop in maintaining memory items when subvocal rehearsal is prevented by AS. The results from Experiment 3 revealed no dual-task effect on processing---the same as was observed in Experiment 1. Experiment 4 {#s48} ============ Experiments 3 and 4 were run consecutively (unlike Experiments 1 and 2), and so some predictions for the latter experiment were influenced by the results from the former. Method {#s49} ------ ### Participants {#s50} Thirty-two participants took part in Experiment 4, split evenly between the two labs as with previous experiments (23 female and nine male, *M*~age~ = 21.66, *SD* = 2.39). None of the participants had taken part in previous experiments. ### Procedure {#s51} The procedure for Experiment 4 followed that of Experiment 3, with titration under suppression. However, Experiment 4 utilized the aural presentation and oral recall memory task from Experiment 2. ### Predictions {#s52} Predictions are summarized in [Table 1](#tbl1){ref-type="table"}. ### Multiple components {#s53} MCM assumes that AS will prevent rehearsal of memory items but will not prevent temporary phonological storage. Participants may attempt to use mnemonic strategies instead of rehearsal, which would use a small amount of processing resources leading to (at most) a small dual-task effect on memory and processing. So, while a large dual-task effect on memory was observed for the visual/typed experiment with titration under AS (Experiment 3), a small or zero effect is predicted by MCM with auditory presentation because aurally presented memory items will have direct access to the phonological store. A small or zero dual-task effect is also predicted for processing, with any effect due to the aforementioned potential use of mnemonics. ### Time-based resource sharing {#s54} The TBRS model predicts the same pattern of results as observed in Experiment 3. The TBRS model does not make specific predictions about differences in effect sizes, but states that titration with AS will result in participants relying to different degrees on the phonological and executive loops. The extent to which participants will rely on one mechanism or the other is not precisely predictable, but the switch from a visual/typed memory task to auditory/oral is not predicted to make a difference for the effect size, so TBRS predicts that the observed dual-task effect size for memory will be at least as large as the effect observed in Experiment 3 (−1.64). TBRS amends their processing task predictions to state only that a dual-task effect will be present (without specifying an effect size) because the theory does not specify working memory mechanisms or resources uniquely related to arithmetic verification, but that it induces an attentional cost that will disrupt refreshing via the executive loop. ### Embedded processes {#s55} As with Experiment 1 and 3, EP again predicts that the dual-task cost will be *larger* in this experiment compared to that observed in Experiment 2, because processing task has greater cost in terms of the number of items in smaller lists. The full analysis of Experiments 1 and 2 revealed a two-way interaction between format (auditory/oral, visual/typed) and task (single, dual). Given that this comparison was, in part, made between subjects, this interaction is not expected to replicate. Consequently, with regards to comparison to the follow-up study with visual presentation and typed response titrated under AS (Experiment 3), EP predicts that the dual-task cost for memory in this auditory/oral experiment will be at least as large if not larger. For processing, EP predicts no effect of dual-task because of the preferential allocation of attention to the processing items in the retention interval. While Experiment 2 revealed a small dual-task cost for processing, EP does not predict a replication of this pattern in this follow-up experiment. A replication of a dual-task processing cost with auditory/oral presentation of memory items when we have not observed this with visual/typed (Experiments 1 and 3) would require further theoretical changes to the EP model. Results {#s56} ------- Mean memory span under AS was 5.20 (*SD* = 0.94), and mean processing span under AS was 7.66 (*SD* = 2.00). The best fitting statistical models for the memory and processing are summarized in [Table 6](#tbl6){ref-type="table"}, and data are summarized in [Figures 8](#fig8){#fgc8-1 ref-type="fig"} and [9](#fig9){#fgc9-1 ref-type="fig"}. Statistically significant dual-task effects were found for both memory (*ES*~scaled~ = −1.32) and processing (−0.42). For memory, a Bayes factor of 30.67 was found in support of the best fitting statistical model (BIC = 4,432.40) over the next best candidate model (BIC = 4,439.25). For processing the best fitting statistical model was supported by a Bayes factor of 33.78 (BIC = 3,648.41) over the next best candidate model (BIC = 3,655.45). As with previous experiments, no one theoretical framework correctly predicted the full pattern of results.[](#tbl6){ref-type="table-anchor"}[](#fig8){ref-type="fig-anchor"}[](#fig9){ref-type="fig-anchor"} Discussion {#s57} ---------- ### Full comparison of Experiments 1--4 {#s58} Following completion of the fourth experiment, we found it pertinent to compare it with all previous experiments (and EP specifically made predictions regarding effect sizes between experiments). The analysis method followed the same procedure as for individual experiments, and the best fitting statistical models for memory and processing are summarized in [Table 7](#tbl7){ref-type="table"}. For memory, the Bayes factor in support of the full model was over a million (BIC = 56,563.51) compared to the next simplest candidate model (BIC = 56,614.56), and for processing the winning model was preferred by a Bayes factor of 106.17 (BIC = 39,313.58) over the next more complex candidate model (BIC = 39,322.91).[](#tbl7){ref-type="table-anchor"} For memory, a number of statistically significant effects were found. The dual-task and format effects and the Dual-Task × Format interaction were observed in previous analyses. The titration effect and the Format × Titration reveal performance was *higher* with titration under AS. However, these effects are artifacts due to the differences in experimental designs of Experiments 1 and 2 versus 3 and 4: Mean performance was lower in the former two experiments because AS was added after titration levels were set. This means that in Experiments 1 and 2, on average, performance was lower as the mean was "pulled down" by the AS conditions. In Experiments 3 and 4, task demands were titrated under AS to 80% performance levels, and no additional load was added apart from dual-task. Of interest is the Dual-Task × Titration Type interaction for memory, which reveals that the dual-task cost to memory was larger when titration was performed under AS (Experiments 3 and 4 vs. 1 and 2). Also, the three-way Dual-Task × Format × Titration Type interaction reveals a larger dual-task effect in Experiment 3 compared to other experiments. ### Summary of Experiments 3 and 4 {#s59} For both Experiments 3 and 4, MCM predicted a small or null effect of dual-task due to the memory task being titrated under AS, which was assumed to result in a more accurate measure of the verbal memory store by removing the "boost" to memory performance from rehearsal. However, a large effect of dual-task on memory was observed in both experiments (TBRS predicted a medium effect, while EP predicted a large effect). The between-experiment comparison revealed that this effect was in fact larger than the memory dual-task effects in Experiments 1 and 2, in which memory (and processing) were titrated without concurrent AS. This larger effect was predicted by the EP model, and was attributed to the fact that the attentional cost of the secondary task will result in a larger proportion of the shorter list lengths being forgotten (the shorter lists being a result of titrating under AS). Experiments 3 and 4 also replicated the finding in Experiment 1 and 2, where a dual-task cost to processing was only observed when the memory stimuli were presented aurally. However, as discussed previously, it is difficult to ascertain whether this effect on processing is related to the presentation format of the memory task or due to the differences in AS onset. Specifically, the EP model predictions stated that this pattern might not be replicated in Experiments 3 and 4. MCM predicted no effect on processing in either Experiment 3 or 4, while TBRS predicted a large effect in Experiment 3 and a measurable effect (with an unspecified magnitude) in Experiment 4. As noted earlier, none of the theoretical frameworks predicted the pattern of observed results. General Discussion {#s60} ================== Theories of working memory attempt to both explain existing behavioral data and to predict performance on tasks based on an assumed structure and functional organization of working memory. One of the starkest differences between working memory theories, and the focus of the present study, is the effects of dual-tasking on memory and processing performance; specifically whether or not retention of memoranda relies on continued or repeated access to an attentional resource, and the performance cost of this access to a concurrent processing task. The three theories investigated in this article provided predictions ranging from no effect of dual-task on memory or processing (MCM), to a linear trade-off between the two tasks (TBRS), and to an interactive pattern of effects due to the allocation of attention to different mechanisms supporting maintenance of memory items and verifying equations (EP). No one set of predictions matched the results obtained. One of the possible explanations for differences between studies that found null/small dual-task effects in younger adults (e.g., [@c51]) and studies that found large trade-offs between processing and storage (see review [@c12]) is that they could be due to a lack of titration in the latter body of research which instead focused on the maximum memory span achievable under dual-task rather than performance at span. For this reason, a titration procedure was utilized to ensure demand was set at appropriate levels for individual participants, therefore (according to the MCM) maximizing the likelihood that they would rely on specialized verbal stores rather than resorting to potentially attention-demanding strategies to cope with high task demand. The titration under suppression procedure in Experiments 3 and 4 aimed to further increase the use of a dedicated verbal store by removing participants' ability to subvocally rehearse. Despite setting memory and processing demand according to each participant's individually measured spans, clear dual-task costs were observed in memory performance in all four experiments. This finding differed from previous MCM research with titrated demand that found little or no effect on memory ([@c21]; [@c37]; [@c51]), and were more consistent with dual-task costs observed in previous EP and TBRS studies. In contrast, dual-task costs on processing were either not present or very small which was consistent with previous MCM studies on younger and older adults but not consistent with EP and TBRS. Predictions from each framework were based on supporting evidence from the literature associated with each theoretical framework. The MCM predicted no dual-task effects based on previous findings (e.g., [@c37]) and based on the assumption of a dedicated verbal store. As discussed previously, the assumption of a dedicated store dates back to the findings of [@c5] in which dual-task costs were only observed at longer list lengths (hence the use of a titration procedure here to ensure list lengths, and processing task speed, were appropriate for individual participant's abilities). In Experiments 1 and 2 (for memory), only the prediction by MCM for the effect of AS for memory was supported by the data as a large effect of single- versus dual-task was observed in both experiments. TBRS predicted an additive effect of dual-task and AS on memory accuracy in Experiments 1 and 2, as was found. As summarized previously, the TBRS theory assumes that both storage and processing share, on a temporal basis, a common limited attentional resource through the alternating occupation of an executive loop while, for verbal maintenance, a domain-specific phonological loop can store some additional items to supplement the executive loop ([@c12]). The predicted pattern of additive effects of dual-task and AS predicted by TBRS and borne out in the data from Experiments 1 and 2 is argued by TBRS to result from independent effects of diverting attention away from refreshing and preventing subvocal rehearsal. TBRS also predicted the relative magnitude of dual-task and AS effects, with AS having a greater impact on memory accuracy presumably due to greater reliance on subvocal rehearsal mechanisms when they are available, with the comparatively lower reliance on attention-based resources remaining great enough to evoke a substantial dual-task cost. EP also correctly predicted dual-task (and AS) effects on memory in Experiments 1 and 2, yet attributed the cause to different mechanisms. The EP and TBRS approaches are consistent in many ways, most notably the use of attention to assist memory maintenance. It is therefore difficult to distinguish between the TBRS view in which the speed of attention-based refreshing explains capacity, and the EP view in which capacity may determine the speed of refreshing, with multiple items up to the capacity limit refreshed in parallel (for simulations of these models, see [@c46]). EP also predicted an interaction between dual-task and AS, where a smaller dual-task cost under AS was expected. The fact that these interactions were not observed is relatively inconsequential for the framework as they were predicted based on arbitrary parameter values; there was no attempt to tweak the model or optimize it to get the best fit, as is often done in a model-fitting approach. Unlike TBRS, EP does not view the lack of interaction between dual-task and AS factors as evidence for separate systems, as it is not clear whether they would benefit performance in an additive or subadditive manner. The MCM interpretation of the interim memory data from Experiments 1 and 2 was that allowing participants full use of subvocal rehearsal and some attention-demanding maintenance mechanism during the memory titration (i.e., titration being conducted in silence) resulted in spans representing input from additional resources (e.g., a visual store, mnemonics) rather than only the specialized short-term verbal memory store. This interpretation is supported by [@c37] in which dual-task costs to processing were observed with no cost to memory spans, argued to be due to the fact that domain- or task-general attention-based sources could support memory performance (at a cost to the processing task) but that memory could not support processing due to the specialized nature of short term verbal storage resources. However, in Experiments 1 and 2 dual-task effects on processing were null and small respectively (Experiments 3 and 4 replicated the same pattern),[^2^](#fn2){ref-type="fn"} suggesting no drop in performance to support memory. This contrast with the findings from [@c37] merits exploration in future studies. It is notable that the lack of dual-task cost for processing is consistent with other previous MCM studies ([@c51]). To further investigate the possible additional support from attention-demanding maintenance mechanisms, Experiments 3 and 4 aimed to reduce spans to be more representative of the capacity of the verbal store argued by the MCM. Titrating under AS, MCM presumed, would remove or reduce the ability of the participants to subvocally rehearse verbal memory items, and so performance would rely solely on the number of items they could store in verbal memory without rehearsal (auditory presentation), or on the support afforded by both a verbal and a visual store (visual presentation). For Experiments 3 and 4 (visual and auditory presentation respectively) MCM therefore predicted at most small effects of dual-task on verbal memory due to reliance on the verbal store and support from the visual store, with a small cost to memory performance potentially arising from the use of mnemonics being impaired by the processing task. However the MCM memory prediction was not supported by the data, as dual-task effects were larger than those observed in Experiments 1 and 2 were observed. The MCM interpretation of the observed effects speculates that, in the absence of rehearsal, people try to use mnemonic techniques to support performance, and this involves repeated access to LTM that is also required for the arithmetic verification task. It is notable that, in the original [@c5] experiments, a memory load of 3 items resulted in no impact on a reasoning or language comprehension task performed during a retention interval. A memory load of six items did affect performance on the interpolated processing task, but only on response time, not on accuracy. It is possible that titrated span scoring generates an overestimate of the capacity of the phonological store, and as with the six-item memory list used by [@c5], our titrated memory span exceeded that capacity. Conversely, TBRS and EP both correctly predicted that the dual-task effects on memory in Experiments 3 and 4 would be larger than those observed in the previous experiments. According to TBRS, the larger dual-task effect on memory in Experiments 3 and 4 is interpreted as demonstrating the cost of diverting attention once tasks have been titrated to a level relying solely on this mechanism due to the prevention of subvocal rehearsal by AS. Forcing participants to rely on attentional refreshing results in span levels indicative of the lower capacity of this mechanism for maintenance of verbal memoranda compared to subvocal rehearsal. According to TBRS, the larger dual-task effect was observed in Experiments 3 and 4 because of greater reliance on refreshing throughout. Conversely, EP interpreted the larger dual-task effect to be due to the fact that the processing task costs memory a certain fixed number of items by taking attention, and that number of items results in a larger proportional loss when span has been reduced by eliminating the contribution of subvocal rehearsal. While both interpretations are similar the key difference is that TBRS specifies that the loss of memoranda during dual-task is due to participants reduced ability to attentionally refresh memoranda, while EP attributes forgetting to displacement of items from attention by the processing task. The null/small dual-task effects on processing in Experiments 1--4 most closely match MCM predictions, as both TBRS and EP predicted medium/large effects. However, EP revised their predictions for Experiments 3 and 4, removing the assumption of an involvement of AS and interpreting the asymmetry in dual-task effects as being due to preferential allocation of attention to the processing task at the expense of memory performance. TBRS had assumed that because attention must be shared between memory and processing that participants would share "perfectly" between these two tasks and so the framework predicted the same dual-task cost would be observed in both. However, typical TBRS methodology has always placed a high priority on ensuring that participants are performing the processing task at a reliable level of accuracy (typically 80%) to ensure that the task reliably diverts attention away from refreshing memoranda. This emphasis typically leads to the removal of participants who perform below the accuracy criterion, though the majority of the sample is retained (e.g., [@c17], between ∼ 1% and 5% of participants removed; [@c80], between ∼6% and 8%). It appears, therefore, that although TBRS predicted dual-task costs in both tasks, the asymmetry in which the dual-task costs are present only in memory is not inconsistent with previous TBRS findings in which there are often large dual-task effects on memory, yet the majority of participants are able to maintain processing performance \>80% accuracy. EP had predicted dual-task costs to processing based on other situations in which a processing task has, in fact, been affected by a memory task. For example, [@c20] presented a three-choice task, in which participants had to press one of three buttons corresponding to a light on screen, with the task speed adjusted to produce errors. When this processing task occurred between digits to be recalled, the increasing memory load had a strong impact on three-choice performance. The results of [@c81], in which increasing memory loads affected processing task RTs, also influenced EP predictions on the speeded choice RT task used in this set of experiments. One difference between these findings is that the arithmetic verification task is more demanding ([@c81] featured relatively simple spatial and parity judgment tasks), and so EP speculates that it may not be possible for participants to divert attention during any one processing episode to engage in mnemonic restoration. There was mixed success by each framework in predicting trends in the data, but all missed large trends in the data. Each theory requires some reconsideration of its core assumptions, or at least under what circumstances expected effects should be observed. For example, MCM consistently predicted no dual-task effects on memory accuracy, and incorrectly predicted that the titration under suppression manipulation would remove the unexpected dual-task effect on memory observed in Experiments 1 and 2. MCM, however, was the only theory to predict small/null dual-task effects on processing, though the framework also predicted small Dual-Task × AS interactions that were not observed. These interactions were predicted as evidence for a trade-off from the processing resource to support memory when subvocal rehearsal was prevented/reduced by AS (small dual-task effects were tentatively predicted by the MCM in Experiments 3 and 4 for the same reason). Small yet statistically significant dual-task effects were only observed in auditory/oral experiments, in which the MCM would assume that aurally presented verbal memoranda had more immediate access to a phonological store and so performance would rely less on recruitment of additional resources or the use of mnemonics and so should predict smaller effects of dual-tasking on processing than when material is presented visually. In sum, the MCM did not predict the large dual-task effects on memory accuracy, even when the experimental procedure was altered with the goal of maximizing the use of a dedicated verbal store. The MCM processing predictions were a close approximation of the processing data and the lack of small predicted interactions is not crucial for the framework which assumes separate resources for memory and processing. The between-experiment interactions cannot be easily explained by the framework or serve as clear cut evidence of the trade-offs in performance the theory assumes. By virtue of predicting small dual-task effects on memory, the MCM did expect the large residual performance in memory performance that was observed. MCM proposes that this residual memory performance is evidence for the involvement of multiple supportive mechanisms for memory, because if only subvocal rehearsal or attention supported verbal memory performance then the introduction of both these costs should have very substantially reduced performance to a larger absolute degree than observed. Although the effects on memory were medium or large relative to the intersubject variability, even the statistically large effects were small compared with the overall performance. For example, from [Figure 2](#fig2){#fgc2-2 ref-type="fig"} (Experiment 1), the dual-task condition showed a ∼10% drop in mean proportion correct relative to single-task both with and without suppression. In [Figure 4](#fig4){#fgc4-2 ref-type="fig"} (Experiment 2), the drop is around 15% in mean proportion correct. These drops in accuracy are comparable with previous dual-task studies in the MCM framework (e.g., [@c2]; [@c39]), although previous research analyzed data using ANOVA models, whereas here we analyzed data using more appropriate methods for accuracy data. While these effects may typically be labeled as "small" in terms of changes in proportion correct, predictions on proportion correct are only appropriate when dealing with computational models, and so scaling effects in the way described in this article provide information regarding the size of the dual-task cost in relation to a reliable metric, that is, between participant variability. To qualify predictions expressed in terms of proportion correct one solution might be for MCM to develop a computational model, or to adapt the existing qualitative model to predict effects scaled to between-participants variability. Although the MCM expected large residual performance, it should be noted that neither TBRS and EP accounts predicted a performance drop to zero; TBRS would require both AS and a cognitive load of 1, that is, complete attentional capture, to predict floor performance. In fact, the residual memory performance observed in these experiments closely resembles that observed under extreme conditions of cognitive load (e.g., [@c9]). Likewise, EP posits that participants are able to split attention between tasks while also benefiting from activations in LTM, and so would not expect floor performance with the dual-task procedure utilized in the reported experiments. While neither EP nor TBRS makes predictions about the size of the residual performance, even if they have implicit assumptions that allow a plausible explanation for the residual that was observed, MCM is more explicit in predicting a large residual. This illustrates a difference in emphasis between the theoretical frameworks, with the former two focusing on the dual-task costs, while the latter focuses on the substantial residual memory performance relative to modest dual-task effect costs to proportion correct. Also, the MCM assumption of separate storage and processing stores was based on previous findings where low correlations between memory and processing spans were observed (e.g., [@c35]; [@c55]; [@c82]), and a post hoc analysis of the data from the current experiments reveals no statistically significant correlations between memory and processing spans (for Experiments 1, 2, 3, and 4, Pearson's *r* coefficients were .24, .23, .2, and .01, respectively, all *p* \> .05). The low level of shared variance between memory and processing spans, to the MCM, indicates evidence for separate components contributing to performance on each task and could explain the large residual performance observed in even the most demanding experimental conditions reported here. Again, the MCM focus on what performance remains and how separate working memory components could account for this performance further demonstrates differences in approaches between the theoretical frameworks and warrants further investigation. The TBRS model successfully predicted both the presence of dual-task effects on memory, their relative magnitude to AS effects, and that the dual-task effect size would increase when span was measured under suppression. TBRS failed to predict the small/null dual-task effects, and the lack of AS effects, on processing. It remains unclear whether this theoretical framework requires modification to accommodate these findings. As already discussed, the asymmetric dual-task costs between memory and processing is not inconsistent with previous TBRS research. However, the lack of an effect is somewhat inconsistent with the findings of [@c81], where memory load was observed to affect processing RTs. Because processing titration relied on increasing the speed of the arithmetic verification task until participants' accuracy dropped below 80%, it is logical to assume that any RT cost to processing performance should be reflected in accuracy. A post hoc analysis of RT revealed a small dual-task cost (see [online supplementary materials](#supp) to this article). This RT cost was either too small to impact speeded-response accuracy, or participants may be engaging in some speed/accuracy trade-off that preserves performance on the task enough to prevent a measurable drop in accuracy. According to the TBRS model, a possible explanation for the lack of dual-task effects on processing (one that does not require the separation of memory and processing resources, or speculation of some representation-based interference based on Presentation/Recall × Processing Dual-Task interactions[^3^](#fn3){ref-type="fn"}), is that participants prioritized the addition verification task over the memory task. Studies on dual-tasking have established that interference between tasks can be modulated by priorities ([@c74]) and external cues play a role in the way participants select their goals ([@c1]; [@c45]). It is possible that the successive presentation of additions on screen and the requirement to produce immediate responses led participants to prioritize the verification task over the maintenance of letter lists. [@c81] detected dual-task effects on processing only after trials with imperfectly recalled lists were removed from the analysis: it may be the case that the effects resulting from resource sharing mainly appear when tasks are explicitly or implicitly given priority by participants (e.g., due to their immediacy) or by researchers (e.g., by designing paradigms that emphasize perfect or high performance on one or the other task within a dual-task paradigm). Accounting for prioritization phenomena within the TBRS model would require specifying the mechanisms by which attention is devoted either to maintenance or processing activities and what are the mechanisms that lead the executive loop to switch from one activity to the other, something that the current version of the TBRS model does not. For example, it might be imagined that remembering memory items is participants' initial main goal in working memory tasks, and that the occurrence of a to-be-processed distractor on screen would trigger the reinstantiation of the task set associated with the concurrent task, thus leading attention to switch from maintenance to processing. Beyond this preliminary suggestion, what is needed is a temporally fine-grained description of the cognitive processes that successively take place during dual-task completion as well as the internal (volitional, strategic) and external cues that trigger them. The EP framework ([@c25], [@c26]) has evolved since it was first proposed. [@c25] left open the issue of how much semantic information is automatically analyzed and retained without attention, but the answer has to date appeared to be "little if any" (e.g., [@c24]). Also, assumptions about attention and information storage have changed; for example, dual-modality memory task results of [@c72] suggested that when participants cannot rehearse to-be-recalled items, memory is limited to three or four items. A psychometrically more thorough examination by [@c33] suggests that instead, participants first widen attention to take in three to four items in a set but then can quickly offload information to the activated portion of LTM. Cowan has long realized that the EP is a modeling framework to be filled in, not a complete model; an approach made clear by the revision of assumptions and predictions between Experiments 1 and 2 and Experiments 3 and 4 in this article. Although the EP framework correctly predicted effects of processing on storage, and its magnification under AS, the aspect of the results most surprising for the framework is the absence of effects of concurrent storage on processing. A post hoc interpretation would concern the nature of the processing task, which might require attention but in a manner that is obligatory rather than optional. Previous studies suggest that simple arithmetic can involve direct retrieval from LTM as a preferred route of performance (e.g., [@c40]), and other work suggests that this LTM retrieval is obligatory; people may not have the ability to modulate this use of attention to share with other tasks while the retrieval is ongoing ([@c34]; [@c73]). This assumption can be implemented without a change in the modeling framework but with an additional clarity in predictions, so that we would now predict that attention costs would accrue to processing as well as storage provided that the processing task was changed to one not requiring LTM retrieval (for a similar approach see [@c69]). The outcome of such research examining different processing tasks in a dual-task design might not only explain the results reported here but may also inform future iterations of the EP framework, and/or help distinguish between MCM, TBRS, and EP accounts. Conclusion {#s61} ========== The present work aimed to contrast predictions from MCM, TBRS, and EP theories of working memory by collaboratively designing a set of experiments for which (to the greatest extent possible) disparate predictions could be generated by each theory. We focused on the absence/presence/magnitude of dual-task effects on a pairing of verbal memory and verbal processing tasks, and on how AS modulated these effects. This research represents, to our knowledge, the first attempt at an adversarial collaboration to contrast working memory theories directly with the same experimental paradigm. Its main strength is the a priori design considerations made for each of the theories, resulting in outcomes that challenge the assumptions of all three models. The experiments also highlight two novel challenges for adversarial collaborations. First, despite our initial assumptions based on the high level of debate in the working memory literature, it is difficult to design experimental procedures that result in clearly contrasting predictions from all three theories. The main difference between theories, at least for dual-task effects, is in how effects are interpreted. This is most evident in how EP and TBRS each explain the increased dual-task cost between Experiments 1 and 2 and Experiments 3 and 4. By challenging the three theoretical frameworks with the observed data patterns, the current experiments have highlighted the strengths and limitations of those frameworks, while providing new insights into how working memory functions under dual-task demands. However, to fully disentangle the subtle differences in interpretation will require future effort for new experimental designs. The differences between the theoretical frameworks are also highlighted by the tendency for MCM to focus on the substantial residual performance that remains even under very demanding dual-task conditions, whereas EP and TBRS focus on the presence of a drop in performance relative to single-task or low cognitive load demands, suggesting that the differences may not be as substantial as they appear. However, each of the three approaches would require modification to develop a more integrated account for the current set of data, for previous data sets generated within each framework and to generate more accurate predictions for future experiments. Second, while the collaborative design process aimed to reduce post hoc interpretations of effects, such explanations are unavoidable. We do not, however, view this as a negative. Because the experiments were designed to take into consideration assumptions from each theoretical framework the scale of post hoc explanation is considerably reduced compared to what one might expect between competing theories researching and publishing work independently. Instead, the adversarial collaboration approach has resulted in a set of interpretations which rely on additional assumptions not directly tested here. These interpretations present a clear roadmap for future research; for example, whether task priority plays a role in the distribution of dual-task costs, if/how the input from additional resources supporting memory can be increased or reduced, and how the distribution of dual-task costs and/or the input from other mechanisms accounts for the residual performance in memory accuracy. Our findings support statistically large dual-task costs to memory accuracy that favor a shared resource structure of working memory such as that proposed by TBRS and EP accounts, but with residual memory performance that may indicate input from other resources or mechanisms argued by the MCM. While this residual performance in and of itself is insufficient to distinguish a "winning" framework, both it and the asymmetry between memory and processing dual-task costs pose questions as to whether working memory can ever be explained by any one of these three frameworks, or whether some integrated combination of the three accounts will be needed to provide a comprehensive explanation of these and both previously published and future behavioral data. Supplementary Material ====================== 10.1037/xlm0000668.supp Upon analysis of the data, effects far larger than 0.8 were in fact observed. Because predictions of large effect sizes were based on this smaller value, the magnitude of predicted effects were unavoidably underestimated. However, because each framework made predictions based on this same scale, it was still possible to evaluate contrasting predictions when data were analyzed. The differences in dual-task effects on processing across different memory presentation/recall formats were not predicted or easily explained by any of the three frameworks. A follow-up experiment had mixed success in replicating the pattern (i.e., dual-task effect on processing only in the auditory/oral memory condition), but this replication only occurred at the U.K. site. The effect remained small, and so we concluded that these small dual-task effects on processing are unreliable and possibly due to sampling effects. In any regard, these dual-task effects were always considerably smaller than for memory. The experiment is reported in the [online supplementary materials](#supp) to this article. See the [online supplementary materials](#supp) to this article for the between-subjects follow-up investigation of these interactions. ###### Summary of the Predictions From Each of the Three Models for Experiments 1--4 {#tbl1a} Effect MCM TBRS EP Observed ------------------ ------------ ----------------------- --------------------------------- ---------------------- Experiment 1  DT (mem.) Small Medium Large *ES*~scaled~ = −.73  DT (proc.) Null Medium Large *n.s*.  AS (mem.) Medium Large Large *ES*~scaled~ = −2.96  AS (proc.) Null Small Large *n.s*.  DT × AS (mem.) Small Null Medium *n.s.*  DT × AS (proc.) Small Null Medium *n.s.* Experiment 2  DT (mem.) Null Medium Medium *ES*~scaled~ = −1.21  DT (proc.) Null Medium Medium *ES*~scaled~ = −.43  AS (mem.) Large Large Medium *ES*~scaled~ = −2.00  AS (proc.) Null Small Medium *n.s*.  DT ×AS (mem.) Null Null Medium *n.s.*  DT × AS (proc.) Small Null Medium *n.s.* Experiment 3  DT (mem.) Null/Small Medium Larger than Experiments 1 and 2 *ES*~scaled~ = −1.64  DT (proc.) Null Large Null *n.s.* Experiment 4  DT (mem.) Null/Small Equal to Experiment 3 Larger than Experiment 2 *ES*~scaled~ = −1.32  DT (proc.) Null/Small Effect predicted^a^ Null *ES*~scaled~ = −.42 ###### Memory and Processing Analyses From Experiment 1, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl2a} Parameter Task ------------------ --------------------- -------------------- Intercept 1.190\*\*\* (.091) 1.410\*\*\* (.048) Dual-task −.356\*\*\* (.034) AS −1.436\*\*\* (.034) Site (Swiss/UK) .010 (.129) Dual-Task × Site −.143\*\*\* (.048) AS × Site .191\*\*\* (.049) ###### Memory and Processing Analyses From Experiment 2, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl3a} Parameter Task ------------------ -------------------- -------------------- Intercept 1.051\*\*\* (.083) 1.540\*\*\* (.054) Dual-task −.537\*\*\* (.033) −.175\*\*\* (.024) AS −.890\*\*\* (.024) Site (Swiss/UK) .304\*\*\* (.116) Dual-Task × Site −.152\*\*\* (.047) ###### Mixed-Factorial Analyses Comparing Memory and Processing Performance Between Experiments 1 and 2, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl4a} Parameter Task -------------------- -------------------- -------------------- Intercept 1.080\*\*\* (.087) 1.539\*\*\* (.052) Dual-task −.539\*\*\* (.029) −.175\*\*\* (.024) AS −.941\*\*\* (.029) Format (AO/VT) .086 (.122) −.107 (.073) Site (Swiss/UK) .246\*\* (.122) Dual-Task × Format .185\*\*\* (.034) .133\*\*\* (.035) AS × Format −.452\*\*\* (.034) Dual-Task × Site −.147\*\*\* (.034) AS × Site .104\*\*\* (.034) Format × Site −.186 (.168) ###### Memory and Processing Analyses From Experiment 3, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl5a} Parameter Task ------------------ --------------------- -------------------- Intercept 1.422\*\*\* (.178) 1.582\*\*\* (.064) Dual-task −1.076\*\*\* (.087) Site (Swiss/UK) .078 (.250) Dual-Task × Site −.321\*\*\* (.119) ###### Memory and Processing Analyses From Experiment 4, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl6a} Parameter Task ----------- -------------------- -------------------- Intercept 1.428\*\*\* (.111) 1.696\*\*\* (.086) Dual-task −.759\*\*\* (.057) −.182\*\*\* (.053) ###### Mixed-Factorial Analyses Comparing Memory and Processing Performance in Experiments 1 Through 4, Displaying Coefficient Estimates and Standard Errors From the Winning Models for Each Task {#tbl7a} Parameter Task -------------------------------- -------------------- -------------------- Intercept .725\*\*\* (.064) 1.536\*\*\* (.047) Dual-task −.585\*\*\* (.023) −.176\*\*\* (.022) Format (AO/VT) −.251\*\*\* (.091) −.097 (.061) Titration (no AS/AS) .696\*\*\* (.117) .163\*\*\* (.063) Dual-Task × Format .201\*\*\* (.033) .129\*\*\* (.031) Dual-Task × Titration −.172\*\*\* (.061) Format × Titration .286\* (.167) Dual-Task × Format × Titration −.687\*\*\* (.088) {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} {#fig7} {#fig8} {#fig9}

Background {#Sec1} ========== A terrorist attack may cause serious mental and physical health problems, and reliable estimates of subsequent morbidity and treatment needs are essential to develop an effective public health response \[[@CR1]\]. However, the unpredictable and chaotic circumstances of a terrorist attack make it difficult to conduct methodologically solid research. Previous post-disaster studies have often lacked a clear definition of the study population and not reported a participation rate \[[@CR2], [@CR3]\]. In addition, longitudinal studies have commonly failed to describe the characteristics of attrition \[[@CR4]\]. Therefore, little is known regarding research participation among individuals who survive terrorism. Typically, research respondents have a higher socioeconomic status and better health than non-respondents do \[[@CR5], [@CR6]\]. Terror-related experiences may also influence the willingness to participate in research \[[@CR4], [@CR7]\]. On the one hand, the most severely affected survivors might be less likely to participate because of impaired function or fear of being reminded of the attack. On the other hand, such survivors might be more motivated to participate to increase our understanding of the adverse effects of terrorism. More knowledge is required regarding research participation after acts of terrorism to interpret the results properly and strengthen future study methods. This article covers participation in two waves of an open cohort study of survivors of the Utøya attack and their parents. On July 22, 2011, a solitary perpetrator executed two terrorist attacks in Norway. After detonating a bomb in the Oslo Government Quarter, he committed a shooting massacre at the summer camp of the Norwegian Labour Party's youth organisation on the Utøya islet. Overall, 564 persons were isolated on the islet during a 1.5-hour-long shooting; 69 were killed, and many were injured or risked drowning trying to escape by swimming. The shooting is considered a severe trauma because the victims were young, designated targets, and many were killed or injured or lost close ones \[[@CR8], [@CR9]\]. The aim of this study was to gain insight into research participation among survivors of terrorism and thereby advance disaster research methodology. Our specific objectives were to (a) identify the factors associated with participation in an open cohort study on survivors of the Utøya attack and (b) assess the characteristics of the survivors based on parental participation in a complementary study on the survivors' parents. Methods {#Sec2} ======= Overall, 495 survivors who had been on the Utøya islet during the shooting were identified from police records. The survivors resided in rural and urban municipalities across all Norwegian counties. The recruitment consisted of three stages: (1) a postal invitation, (2) a telephone call, and (3) an interview with those who received the call and agreed to participate. Postal study invitations were sent to 490 survivors. In the invitation letter, the survivors received information about the aims of the study, the study procedures, and an overview of the content and duration of the interview. Furthermore, they were informed that they could withdraw from the study at any time, and the letter provided contact information in case they had questions regarding the study or did not want to be contacted by phone. Four survivors aged \<13 years and one survivor who lived abroad were excluded. The four survivors under 13 years of age were excluded because of their young age and the fact that they were mainly children accompanying leaders/guards. Moreover, the interview questionnaires were designed for adolescents and young adults. The study had an open cohort design in which all of the eligible survivors were invited to both waves. Semi-structured face-to-face interviews were performed at 4--5 months (wave 1) and 14--15 months (wave 2) after the shooting. The interviews included open-ended questions on several themes; such as their experiences during the shooting, the police interrogation, the trial, the return to school, and their use of social media, in addition to a wide range of topics assessed with closed-ended questions \[[@CR10]\]. The respondents could choose between being interviewed at home or being interviewed at a location arranged by the interviewer. When the interview was completed, the respondents completed a questionnaire. If a respondent was unable to respond to the interview in Norwegian or English, it was proposed to perform the interview using an interpreter. The parents of 482 survivors aged 13--32 years were eligible for participation in a parallel study focusing on the reactions and experiences of the survivors' parents. The current study assessed survivor reports only. A separate postal invitation addressed to parents/guardians of the survivor was sent to the addresses of the survivors. Next, the survivors were asked for the contact information of their parents during a telephone call. Parents of survivors born 1992 or later were eligible to participate by interview. Due to limited resources, parents of survivors born before 1992 were invited to participate by postal questionnaire. Reminders were sent to parents who did not answer the questionnaire on the first request. Variables {#Sec3} --------- Our sociodemographic data included age, gender, country of origin, and whether the survivors lived with their parents, had divorced parents, were financially disadvantaged or resided in a peripheral municipality. Age was measured at the time of the attack as a continuous variable in years with one decimal. Non-Norwegian origin was defined as having both parents born abroad. Survivors were asked how they perceived their parents' (those who lived with parents) or their own (those who did not live with parents) financial well-being compared with that of others. There were five response alternatives, which were dichotomized into financially disadvantaged (i.e., much or somewhat poorer) or not (i.e., similar, somewhat better, and much better). Peripheral residence described the location of the survivor's home municipality at wave 1 in relation to communities of a certain size, in accordance with Statistics Norway's centrality classification \[[@CR11]\]. Municipalities located more than 45 min travelling time from communities with at least 15,000 inhabitants were classified as peripheral. We obtained information for all survivors on age, gender and place of residence from police records and on admittance to somatic hospitals directly after the attack from hospital records. The terror exposure assessment was explicitly designed to cover 13 potentially traumatic events experienced during the attack and has been demonstrated to be independently associated with mental health problems \[[@CR12]\]. In the analyses, the mean sum score was applied. Posttraumatic stress reactions in the past month were assessed using the University of California at Los Angeles Post-traumatic Stress Disorder (PTSD) Reaction Index (UCLA PTSD-RI) \[[@CR13]\]. The total score includes 17 items that conform to the 17 DSM--IV symptoms of PTSD rated on a 5-point Likert scale that ranges from 0 (never) to 4 (most of the time) \[[@CR14]\]. Three items have two alternative wordings that are valued by the item with the highest score. The mean scores of the 17 items were used in the analyses. Cronbach's alpha was 0.89 (wave 1 and 2). Symptoms of anxiety and depression were measured with the Hopkins Symptom Checklist-8 (SCL-8), which is a short version of the SCL-25 \[[@CR15]\]. It measures symptoms of depression/anxiety that occurred during the preceding 2 weeks using eight items scored on a scale from 1 (not bothered) to 4 (very much bothered). The mean score was applied in the analyses, and Cronbach's alphas were 0.86 (wave 1) and 0.90 (wave 2). The short versions of the Hopkins Symptom Checklist have displayed high psychometric qualities in population-based studies \[[@CR16]\]. Somatic symptoms that occurred during the preceding 2 weeks were assessed using a short version of the Children's Somatic Symptoms Inventory (CSSI-8) \[[@CR17]\]. The eight items assessed pain in the stomach, head, lower back, and arms/legs; faintness/dizziness; rapid heartbeat; nausea/stomach problems; and weakness. Each item was scored on a scale from 1 (not bothered) to 4 (very much bothered). The mean score was used in the analyses. Cronbach's alphas were 0.77 (wave 1) and 0.78 (wave 2). Self-perceived social support was appraised using seven items from the Duke University of North Carolina Functional Social Support Questionnaire (FSSQ-7) and scored on a scale from 1 (much less than I would like) to 5 (as much as I would like) \[[@CR18]\]. Mental health service (MHS) utilization was measured with a question on contact with specialized mental health services (yes/no). Wave 1 covered MHS utilization since the attack until wave 1 (ca. 0--5 months post-disaster). Wave 2 covered utilization from January 1, 2012, until wave 2 (ca. 5--15 months post-disaster). Ethics {#Sec4} ------ Participants aged 16 years or older provided written informed consent. Written parental consent was required before survivors younger than 16 years old could participate in the study, as stipulated by Norwegian law. The interviewers were health practitioners who had received training in conducting research interviews of traumatised persons at a one-day seminar. The interviewers were instructed to offer help with contacting suitable services if they identified unmet needs. They worked in teams of two, and after each survey wave, they were invited to a one-day meeting to share experiences. In addition, a telephone line was provided for the interviewers where they could discuss the challenges that they encountered during the interviews and receive support. Most interviews were conducted at the homes of the respondents. If not, all travel expenses were covered. The participants did not otherwise receive financial compensation for study participation. After each survey wave, a brief summary of the initial results was sent to the respondents before the findings were published or reported to the media. The study was commissioned by the Norwegian Directorate of Health, and performed by the Norwegian Centre for Violence and Traumatic Stress Studies (NKVTS). The study was approved by the Regional Committees for Medical and Health Research Ethics South East and North in Norway, and the Norwegian police granted permission to access a list of the survivors' names. Statistics {#Sec5} ---------- Pearson's Chi squared tests (categorical variables) and independent t tests (continuous variables) were used to compare survivors by participation as follows. The exact test was used if the expected count was less than five for categorical variables. Survivors who participated in ≥1 waves were compared with non-participants (Table [1](#Tab1){ref-type="table"}). Survivors who participated in two waves were compared with participants in one wave only (Table [2](#Tab2){ref-type="table"}). Survivors with parental participation were compared with survivors without parental participation (Table [3](#Tab3){ref-type="table"}). The reported percentages were based on the total number of answers for each item. We applied a two-sided statistical significance level of 0.05. The analyses were conducted using IBM SPSS version 20.0.Table 1Characteristics of survivors according to participation in ≥1 study waves (n = 490)Characteristics of survivorsNon-participants (n = 135)Participants (n = 355)p valuen/mean(%/sd)n/mean(%/sd)Mean age in years19.0(3.9)19.3(4.6)0.473Male gender83(61.5)184(51.8)0.055Residing in peripheral municipality24(17.8)50(14.3)0.338Hospitalized8(5.9)28(7.9)0.457Any parental participation27(20.0)304(85.6)\<0.001Maternal participation22(16.3)289(81.4)\<0.001Paternal participation15(11.1)228(64.2)\<0.001Table 2Characteristics of survivors according to participation in one or both survey wavesCharacteristicsWave 1Wave 2Participated in wave 1 and 2 (n = 255)Participated in wave 1 only (n = 70)p valueParticipated in wave 1 and 2 (n = 255)Participated in wave 2 only (n = 30)p valuen/mean(%/sd)n/mean(%/sd)n/mean(%/sd)n/mean(%/sd)Mean age at attack (years)19.4(4.3)19.3(5.7)0.88719.4(4.3)18.8(4.1)0.444Male gender139(54.5)33(47.1)0.274139(54.5)12(40.0)0.132Non-norwegian origin25(9.9)14(20.3)0.01925(9.9)2(7.4)0.757Financially disadvantaged51(20.5)17(25.4)0.38751(20.5)6(21.4)0.906Living with parent(s)154(61.1)48(70.6)0.151118(47.0)13(44.8)0.823Divorced parents96(38.9)30(44.8)0.38196(38.7)13(44.8)0.523Youth Labour Party member234(94.4)61(87.1)0.040234(94.4)27(96.4)0.723Sibling(s) in the study27(10.6)5(7.1)0.39127(10.6)2(6.7)0.561Hospitalized19(7.5)5(7.1)0.93019(7.5)4(13.3)0.281Terror exposure (mean 0--13)8.28(2.24)9.32(1.91)\<0.0018.28(2.24)9.05(2.00)0.103Posttraumatic stress (mean PTSD-RI)1.53(0.72)1.67(0.68)0.1641.21(0.67)1.60(0.77)0.003Anxiety/depression symptoms (mean SCL-8)2.05(0.66)2.16(0.64)0.2041.77(0.62)2.11(0.86)0.043Somatic symptoms (mean CSSI-8)1.72(0.54)1.73(0.53)0.9441.62(0.48)1.90(0.67)0.033Social support (mean FSSQ-7)4.56(0.57)4.56(0.59)0.9514.56(0.60)4.53(0.61)0.753Mental health service utilization180(72.0)54(77.1)0.391169(67.3)23(79.3)0.188Wave 1 (n = 325) was performed 4--5 months after the attack; wave 2 (n = 285) was performed 14--15 months after the attackTable 3Survivor characteristics by maternal and paternal participation among survivors aged 13--32 years who participated in wave 1 or 2 (n = 348)Survivor characteristicsPaternal participationMaternal participationAny parental participationYes (n = 228)No (n = 120)p valueYes (n = 289)No (n = 59)p valueYes (n = 304)No (n = 44)p valuen/mean(%/sd)n/mean(%/sd)n/mean(%/sd)n/mean(%/sd)n/mean(%/sd)n/mean(%/sd)Mean age in years18.72(2.74)19.17(3.85)0.26318.62(2.84)20.15(4.24)0.01018.59(2.82)20.82(4.54)0.003Male gender118(51.8)63(52.5)0.895148(51.2)33(55.9)0.508154(50.7)27(61.4)0.184Non-norwegian origin14(6.1)25(21.7)\<0.00119(6.6)20(37.0)\<0.00123(7.6)16(41.0)\<0.001Financially disadvantaged42(19.2)29(24.6)0.24656(20.0)15(26.3)0.28659(20.1)12(27.9)0.239Divorced parents (wave 1)72(34.0)53(54.6)0.001112(42.4)13(28.9)0.087116(41.9)9(28.1)0.133Living with ≥ 1 parents (wave 1)138(65.7)64(61.5)0.467179(67.3)23(47.9)0.010188(67.4)14(40.0)0.001Sibling(s) in the study29(12.7)5(4.2)0.01131(10.7)3(5.1)0.18334(11.2)0(0.0)0.025Hospitalized17(7.5)11(9.2)0.57725(8.7)3(5.1)0.44227(8.9)1(2.3)0.152Terror exposure (mean 0--13)8.53(2.22)8.62(2.21)0.7248.52(2.16)8.74(2.50)0.5108.53(2.17)8.74(2.54)0.580Mental health service utilizationWave 1152(71.7)76(74.5)0.601190(71.7)38(77.6)0.399200(71.9)28(77.8)0.460Wave 2119(65.0)67(73.6)0.151155(66.5)31(75.6)0.251162(66.9)24(75.0)0.359Posttraumatic stress reactions (mean PTSD-RI)Wave 11.53(0.68)1.64(0.78)0.2021.51(0.70)1.86(0.71)0.0011.53(0.71)1.82(0.71)0.023Wave 21.21(0.66)1.33(0.74)0.1741.18(0.67)1.60(0.71)\<0.0011.20(0.67)1.60(0.74)0.002Anxiety/depression symptoms (mean SCL-8)Wave 12.04(0.63)2.13(0.71)0.2592.03(0.66)2.28(0.62)0.0152.04(0.66)2.26(0.63)0.067Wave 21.79(0.64)1.83(0.70)0.6091.74(0.63)2.12(0.71)\<0.0011.77(0.65)2.06(0.69)0.015Somatic symptoms (mean CSSI-8)Wave 11.67(0.51)1.82(0.58)0.0161.69(0.54)1.89(0.54)0.0171.70(0.54)1.87(0.53)0.075Wave 21.60(0.50)1.72(0.53)0.0691.61(0.51)1.81(0.51)0.0151.62(0.51)1.80(0.52)0.050Social support (mean FSSQ-7)Wave 14.65(0.45)4.37(0.75)0.0014.63(0.52)4.18(0.73)\<0.0014.61(0.53)4.16(0.76)0.001Wave 24.62(0.49)4.43(0.76)0.0354.60(0.55)4.33(0.80)0.0404.59(0.55)4.34(0.87)0.123 Results {#Sec6} ======= Altogether, 355 of 490 (72.4 %) survivors participated in the study, including 325 (66.3 %) in wave 1 and 285 (58.2 %) in wave 2. In wave 1, three survivors opted out when they received the invitation letter by sending a text message to the research team. In addition, 29 survivors could not be reached by telephone. In wave 2, seven survivors opted out before they were called, and 43 could not be reached by telephone. In wave 1, survivors were interviewed from 2 November 2011 to 5 March 2012, including \>95 % in November and December. In wave 2, the survivors were interviewed from 13 September 2012 to 5 February 2013, including 90 % during the first 2 months. Figure [1](#Fig1){ref-type="fig"} illustrates the survivor participation and parental participation by study wave. Overall, 255 (52.0 %) survivors participated in both waves: 70 (14.3 %) in wave 1 only and 30 (6.1 %) in wave 2 only. In total, 331 of 482 (68.7 %) survivors who were eligible for parental inclusion had ≥1 parents in the parental study, including 304 participating and 27 non-participating survivors. Therefore, we obtained data from either the survivor or the parents for 382 (78.0 %) survivors. There were 223 survivors with both maternal and paternal participation, 88 with only maternal participation, and 20 with only paternal participation. Altogether, 531 caregivers participated in ≥1 waves: 299 female (291 mothers, six stepmothers/foster mothers and two other female relatives) and 232 male caregivers (216 fathers and 16 stepfathers/foster fathers). Five survivors were represented by two female caregivers, i.e., a mother and a stepmother (n = 3) or other female relative (n = 2), and four survivors were represented by two male caregivers, i.e., a father and a stepfather. Because we examined parental participation, we excluded the two other female relatives.Fig. 1Flow chart of the study participation among survivors of the Utøya shooting (on the *left*) and survivors with parental participation (on the *right*). In total, 355 of the 490 (72 %) invited survivors participated in one or both study waves, and 331 of the 482 (69 %) survivors aged \<33 years had ≥1 parent(s) who participated in the study Non-participating survivors did not significantly differ from those who participated in ≥1 waves with respect to age, gender, hospitalization or geographical region of residence, although a nearly significantly (p = 0.055) larger proportion of participants were female (Table [1](#Tab1){ref-type="table"}). Compared with survivors who participated in both waves, those who only participated in wave 1 were more likely to be non-Norwegian, non-members of the youth Labour Party and to report higher levels of terror exposure. Those who only participated in wave 2 reported more posttraumatic stress reactions, anxiety/depression symptoms, and somatic symptoms (Table [2](#Tab2){ref-type="table"}). Table [3](#Tab3){ref-type="table"} presents factors associated with parental participation among the participating survivors. Altogether, 304 of 348 (87.4 %) participating survivors who were eligible for parental inclusion had ≥1 parents in the study: 289 with maternal and 228 with paternal participation. They represented 286 families: 270 families with one, 14 families with two, and two families with three participating survivors (i.e., siblings). Parental non-participation was associated with the following survivor characteristics: higher age; non-Norwegian origin; not living with parents; not having siblings in the study; and more posttraumatic stress reactions, anxiety/depression symptoms, somatic symptoms, and less social support. The same associations applied for maternal non-participation, except for not having siblings in the study. Paternal non-participation was associated with non-Norwegian origin, divorced parents, not having siblings in the study, somatic symptoms and less social support. Additionally, survivors with paternal non-participation in wave 1 were more likely to have higher levels of posttraumatic stress and anxiety/depression symptoms in wave 2. Moreover, survivors without maternal or paternal participation in wave 1 were more likely to use mental health services in wave 2 (Additional file [1](#MOESM1){ref-type="media"}: Appendix 1, Additional file [2](#MOESM2){ref-type="media"}: Appendix 2). A supplementary analysis of only survivors without siblings in the study (n = 314) yielded a similar pattern of associations. No maternal participation in wave 1 was associated with a loss of follow-up: 40 (16.0 %) of the survivors who participated in both waves had no maternal participation in the first wave versus 19 (27.5 %) of those who only participated in wave 1. No such associations were found with respect to paternal or any parental participation. Discussion {#Sec7} ========== The Utøya attack was a severe trauma that resulted in high levels of posttraumatic distress and extensive health-care utilization among survivors \[[@CR12], [@CR19]\]. After the attack, the survivors were geographically dispersed, and many soon relocated to start their studies. Despite these potentially unfavourable research conditions, nearly three of every four survivors participated in at least one survey wave. Therefore, satisfactory response rates can be obtained in studies launched after terrorism incidents. Longitudinal studies are essential to increase our understanding of how terrorism affects survivor health over time. However, our findings indicate that survivors who participate longitudinally differ from those who only participate once. Compared with survivors who participated in both survey waves, those who were lost to follow-up after wave 1 were more likely to be non-Norwegian and non-members of the political youth organization and to report higher levels of exposure. In contrast, the survivors who entered the study at wave 2 reported more posttraumatic stress reactions, symptoms of anxiety/depression and somatic symptoms. Therefore, the exclusion of survivors who do not participate longitudinally might increase the likelihood of selection bias. This would most likely lead to an underestimation of the impact of the disaster, since the levels of exposure and symptoms were higher among survivors who did not participate in both waves. This possibility can be counteracted by collecting longitudinal register-based data on, e.g., socioeconomics and health-care utilization for all participants. If possible, one should therefore consider requesting consent for data linkage when participants join the study. Another approach to account for attrition is the use of statistical methods, such as multiple imputation \[[@CR20]\]. Former longitudinal post-disaster studies have generally consisted of closed cohorts, in which only subjects who participated in the first assessment were invited to participate in ensuing assessments. The open cohort design of our study enabled survivors to join the study at wave 2 despite initial non-participation. This approach might have improved the response rate among survivors who were unable to participate directly after the event. The elevated symptom levels of survivors who entered our study at wave 2 might indicate that an open cohort design facilitates the inclusion of survivors who may have been unable to participate initially because of adverse health consequences (Table [2](#Tab2){ref-type="table"}). Therefore, an open cohort might yield a more representative sample. However, survivors who participated in wave 1 may also have been more likely to receive care than non-participants if the interviewers identified unmet needs, which may have contributed to lower symptom levels in the survivors who participated in wave 1. Our study supports previous findings that indicate lower response rates among immigrant survivors \[[@CR4], [@CR5]\]. These survivors were more likely to be lost to follow-up and less likely to have parents who participated in the study. These results suggest that additional attention should be paid to the recruitment of immigrants. A longitudinal study on survivors of a fireworks disaster in the Netherlands also found that the overall response rate was lower among immigrants \[[@CR4]\]. Additionally, the response patterns differed between immigrants and native Dutch survivors. Whereas health problems were associated with higher response among immigrant survivors, health problems were associated with lower response among non-immigrants. Therefore, the risk of selection bias might be higher for findings related to immigrant status and should be considered in the interpretation of findings. Although the results vary between studies, research respondents typically have higher socioeconomic status and better health than non-respondents \[[@CR5], [@CR21], [@CR22]\]. We did not find significant differences between participants and non-participants with respect to age, gender, centrality of residence, or hospitalization (Table [1](#Tab1){ref-type="table"}). However, for non-participants, we lacked information regarding the factors that differed between survivors who participated in both waves and one wave only (Table [2](#Tab2){ref-type="table"}). Therefore, it remains uncertain whether non-participants differed from participants with respect to ethnicity, terror exposure and health characteristics. Findings on factors associated with research participation among disaster survivors are conflicting. In certain post-disaster studies, non-participation has been associated with sociodemographic factors (such as being male, unmarried, and having low income) and health-related factors (such as symptoms of PTSD and depression) \[[@CR23], [@CR24]\]. Other studies have not found such associations \[[@CR25], [@CR26]\]. The results on exposure are also inconsistent. One study found that exposure was related to attrition \[[@CR27]\], whereas another found that a threat to life was associated with follow-up participation \[[@CR28]\]. Factors associated with research participation might also differ by groups of survivors and study method. Because the survivors in our study were confined to an islet during the attack, they may have been easier to identify than survivors of disasters without distinct geographical boundaries. Additionally, most of the survivors were young members of the same political youth party. This shared affiliation might have facilitated the dissemination of study information and motivated the survivors to participate. In fact, members were more likely to participate in both waves than non-members (Table [2](#Tab2){ref-type="table"}). It is also possible that the survivors of the Utøya attack, who were mostly politically active youth, were socioeconomically more homogenous than a random population-based sample, which might have contributed to the absence of sociodemographic differences with respect to participation except ethnicity. The interview experience of the participants during wave 1 might also have influenced subsequent participation. We lacked information regarding how the respondents experienced the interview. However, it has been demonstrated that a negative experience of an interview is associated with attrition \[[@CR21]\]. Our study was based on in-depth interviews performed across the entire country and was consequently highly demanding of resources. In addition to the research data that were collected, the health practitioners received up-to-date information on treating trauma, and the researchers gained insight into the challenges that health practitioners face when they met survivors. This integration of research and clinical practice might strengthen the understanding of trauma among clinicians and researchers, and be valuable in future research. Although our study may not be generalizable to low-resource settings, the integration of research and trauma education could be particularly important in low-income countries, where the risk of disasters is highest and there is often a lack of trained personnel \[[@CR29]\]. Population-based health studies on adolescents and young adults evince an overrepresentation of youth raised by two parents and from families with high income and education \[[@CR30]\]. We did not find significant differences between the survivors who participated in both waves and those who participated in only one wave with respect to divorced parents or self-perceived financial status. However, we lacked such information for non-participants. Regarding parental participation, findings from family studies indicate that fewer fathers than mothers participate in research \[[@CR31]\]. This finding agrees with our results. However, in our study, the difference was less pronounced, which might reflect that Norway is a country with relatively high gender equality. Thus, fathers might be more involved in the upbringing of their children compared with countries with less gender equality. Alternative explanations might be that fathers are more inclined to participate after catastrophic events than in everyday settings or that our study method was more successful in reaching fathers, e.g., flexibility with respect to the interview time and location. Parental non-participation was associated with non-Norwegian ethnicity, somatic symptoms and less social support. Otherwise, factors associated with maternal and paternal participation diverged (Table [3](#Tab3){ref-type="table"}). Paternal non-participation was associated with having divorced parents and not having siblings in the study, whereas maternal non-participation was associated with higher age, not living with parents, and higher levels of posttraumatic stress reactions and anxiety/depressions symptoms. Therefore, it is important to consider potential differential selection of mothers and father in the interpretation of parental data. For instance, a divorce might influence the health of both youth and their parents. Survivors were less likely to have participating fathers if the parents were divorced, whereas a nearly significantly larger proportion of survivors with participating mothers had divorced parents. Prior research that involved parents also indicated that well-functioning fathers with high socioeconomic status tend to be overrepresented \[[@CR31]\]. We obtained parent contact information from the survivors. Therefore, whether a parent was invited to participate in the study may have depended on the survivor. Thus, non-response among parents could reflect a weaker parent-offspring relationship. Non-participation does not necessarily result in bias. However, the estimated associations may be biased if non-participation is related to the severity of the outcome and/or the exposure under study \[[@CR32]\]. Therefore, non-response and attrition should be comprehensively assessed because they might threaten the validity of a study. Strengths and limitations {#Sec8} ------------------------- This study provides new data with respect to non-participation that can be valuable for planning and interpreting related research. The open cohort design yielded new information regarding survivors who did not participate in the first survey wave conducted soon after the attack but joined the study later. In previous studies, which generally use closed cohorts, these survivors would have been non-participants. Prior post-disaster studies have also commonly lacked a clear definition of the study population, whereas our study population was clearly defined by the geographical constriction of the island. Past studies on children and adolescents have often only collected data from parents and included only one parent, typically the mother \[[@CR3]\]. We obtained data from adolescents and parents, who represented directly (i.e., were present during the attack) and indirectly (i.e., had children who were at risk of being killed during the attack) exposed samples, respectively. In addition, our study included maternal and paternal reports for most of the survivors. Moreover, the study included in-depth interviews with little missing data. The study also had several limitations. The study did not provide active intervention or treatment. However if unmet needs were revealed during wave 1, the interviewers were recommended to assist the participants in acquiring suitable care. Therefore, wave 1 participants may have been more likely to receive timely support than non-participants because of study participation, which may have contributed to the lower levels of symptoms in survivors who participated in both waves compared with those who only participated in wave 2. Furthermore, in the preparation of the study, we endeavored to design the questionnaires to suit both adolescents and young adults. Nevertheless, the age range of survivors might have led to variation in the ability to respond to the questionnaire. In addition, we do not know how many parents were invited to participate because their contact information was acquired from the survivors. This fact could have increased the likelihood of selection bias with respect to parental participation. There might also have been variation in the efforts of the interviewers to acquire contact information for both parents. In the instructions to the interviewers, the wording on parental invitation for survivors aged less than 16 years differed slightly from that for those aged ≥16 years. The interviewers were requested to ask survivors under 16 years old for the telephone number of one of the parents to request consent for their offspring's participation. In contrast, they were requested to ask those aged ≥16 years for contact information for both parents. This approach might explain why only half of the survivors under 16 years old had at least two parents in the study, whereas two-thirds of survivors aged ≥16 years had two or more parents in the study (data not shown). Because there were only 29 survivors aged \<16 years in our study, this circumstance is unlikely to have substantially affected the results. We had little data on non-participants and lacked information regarding ethnicity, exposure, and symptoms among non-participants. These factors were associated with loss of follow-up or initial non-participation, and we cannot determine whether the same associations applied for non-participation in both waves. Additionally, the study was based on self-reports and lacked pre-disaster data. Finally, our analysis might have failed to detect significant differences because of a relatively small sample size (i.e., type II error) (Additional file [3](#MOESM3){ref-type="media"}). Conclusions {#Sec9} =========== Compared with survivors who participated longitudinally, those survivors who were lost to follow-up after wave 1 were more likely to be non-Norwegian and report higher exposure. In contrast, those survivors who entered the study at wave 2 reported more posttraumatic stress, anxiety/depression, and somatic symptoms. Therefore, it is beneficial to avoid exclusion of participants who do not participate longitudinally, for instance, by collecting longitudinal data through a linkage to registers. In addition, an open cohort design that enables survivors to join the study in a later stage despite initial non-participation might improve the response rate among survivors with increased symptoms levels and yield a more representative sample. Finally, the factors associated with maternal and paternal participation differed. Parental non-participation was associated with non-Norwegian origin, somatic symptoms and less social support. Additionally, paternal non-participation was associated with having divorced parents, and maternal non-participation was associated with higher age, not living with parents, more posttraumatic stress and anxiety/depression symptoms. Consequently, it is important to consider a potential differential sample selection of mothers and fathers in the analysis and interpretation of the parental data. Additional files {#Sec10} ================ 10.1186/s13104-016-1873-1 Survivor characteristics by maternal and paternal participation in wave 1 among survivors aged 13--32 years who participated in wave 1 or 2 (n = 348).10.1186/s13104-016-1873-1 Survivor characteristics by maternal and paternal participation in wave 2 among survivors aged 13--32 years who participated in wave 1 or 2 (n = 348).10.1186/s13104-016-1873-1 STROBE checklist. LES contributed to the design and data analyses and wrote the manuscript. GD was the study's project leader and contributed to drafting the manuscript. Both authors read and approved the final manuscript. Acknowledgements {#FPar1} ================ We sincerely thank all of the participants. The study received funding from the Norwegian Health Directorate and the Research Council of Norway. Competing interests {#FPar2} =================== The authors declare that they have no competing interests.

Introduction {#sec1} ============ Infectious endocarditis (IE) denotes infection of the endocardial surface of the heart and implies the physical presence of microorganisms in the lesion. Although the heart valves are most commonly affected, the disease may also occur within septal defects or on the mural endocardium but also on intavasculary implanted foreign materials such as valvular prostheses or pacemaker electrodes. Despite medical advances, IE remains a life-threatening disease with substantial morbidity and mortality. Its diagnosis and treatement are still a major challenge in clinical practice. In developped countries, Chronic rheumatic valvular disease has been supplanted by mitral valve prolapse with mitral regurgitation and degenerative aortic valve disease as the leading cardiac conditions predisposing to bacterial endocarditis in adults \[[@cit0001]\]. Nosocomial endocarditis associated with therapeutic interventions such as implanted devices, dialysis shunts is increasing in frequency. Many studies found that Staphylococcus aureus was the most common pathogen throughout much of the world \[[@cit0002]\]. In Sub-Saharan Africa, IE is recongized as a disease associated with rheumatic heart disease. Although many factors have altered the epidemiology of IE while maintaining its incidence: an aging population with degenerative valvular disease, increasing number of valve replacements and medical intervention \[[@cit0003]\]. The objective of this work was to study the epidemiological, clinical features, diagnostic techniques currently used in medical practice and the range of micro-organisms that are responsible. Methods {#sec2} ======= This was a retrospective study conducted at the cardiology, paedriatrics, Internal medicine, Infectious diseases and Intensive Care Unit (ICU) Departments of Principal Hospital of Dakar over a period of 10 years (January 1^st^2005 to December 31^st^ 2014). We include all patients who were admitted with manifestations of definite or possible IE according to the extended DUKE criteria \[[@cit0004]\]. We collected and analyzed epidemiological, clinical, paraclinical and outcomes data of patients. We studied data on age, gender, past history including history of medical interventions, predisposing heart disease and underlying disease. We sought the presence of fever, cardiac murmur and extracardiac symptoms of EI such vascular and immunological phenomena (Osler\'s nodes, Janeway lesions, splinter hemorrhages etc.). All patients had a complete physical examination and a laboratory assessment. Blood cultures were done for most of patients and the other tests included urine examination, renal function and the usual laboratory parameters of inflammation such as C-Reactive Proteine, Erythrocyte sedimentation rate and fibrinogen. On the ECG we looked for chamber enlargement, rhythm and conduction abnormalities. Doppler echocardiography were performed for all patients for the demonstration of endocardial involvement and the assessment of left and right ventricular function and tissue destruction. Current aspects of antibiotics therapy were evaluated as well as evolution during hospitalization. The studied parameters were entered into an electronic questionnaire using Epi Iinfo version 6.0 of the World Health Organization. Data analysis was performed using SPSS (Statistical Package for Social Sciences). Quantitative data were expressed as mean± standard deviation. Qualitative data were expressed as percentage. Results {#sec3} ======= We included 42 patients. Hospital prevalence of IE was 0.078% (42/53711). 27 patients (64.29%) were females and 15 patients (35.71%) were males giving a sex ratio (M/F) of 0.55. The mean age of patients was 27.5+/- 8 years with a range of 8 months and 72 years. Most of patients (54.76%) was under 25 years. Most cases were encountered in the department of cardiology (54.8%), paedriatrics (26.2%) and ICU (11.9%). The Diagnosis latency ie, the interval from the onset of symptoms to the definite diagnosis of IE was 29.97 days. Predisposing conditions to IE in our patients ([Table 1](#t0001){ref-type="table"}) were dominated by rheumatic valvular disease (64.3%). Six patients had endocarditis previously, 4 patients had a valvular prosthesis (9.5%) and 6 patients had congenital heart defects (14.3%). Predominant causes of bacteremia that were found in 27 patients were: dental caries (26.2%), urosepsis (26.2%), skin infections (14.2%) and Ear Nose Throat (ENT) infections (9.5%). For other patients no cause was found. ###### Predisposing conditions to Infective endocarditis Predisposing conditions number Percentage ---------------------------------------- -------- ------------ Rheumatic heart valves 27 64.3% **Congenital heart defects** Tetralogy of Fallot 1 2.4% ventricular septal defect 3 7.1% Atrio-ventricular septal defect 1 2.4% Patent ductus arteriosus 1 2.4% Degenerative valve disease 2 4.8% **Valvular prosthesis** mitral 3 7.1% aortic 1 2.4% Other (diabete, hodgkin, Basedow, HIV) 4 9.5% The most common presenting symptoms were fever (90%), cardiac murmurs (81%) and dyspnea (42.9%). The fever was above 38°5C in 86.8% of cases with high remitting pyrexia often associated with chills and night sweats in 6 patients. Weight loss was found in 22 patients (52.4%). Extracardiac clinical manifestations were dominated by vomiting (26.2%), abdominal pains (14.28%) and seizures (7.14%). Clubbing was found in 2 patients and 1 patient had splenomegaly. Mild to moderate anemia was commonly present (82.1%) with a hypochromic, microcytic picture. Neutrophil Leukocytosis was common (90%) and the Erythrocyte sedimentation rate (ESR) and C-reactive proteine (CRP) were elevated in 90% of patients. Renal function test results showed an elevated creatinine level in 17.1% of patients, proteinuria in 12% and microscopic hematuria in 1 patient. Blood cultures were negative in 50% of cases and positive in 21.4% of cases. The main organism found were Staphylococcus aureus (55.5%), Enterococcus faecalis (22.2%), Klebsiella pneumoniae (11.1%) and Escherichia coli (11.1%) [Table 2](#t0002){ref-type="table"}. Echocardiography found vegetations in 95.2% of cases, Chamber enlargement in 73.8% and mitral regurgiation in 83.3%. ###### Positive blood cultures Micro-Organisms Number Percentage ----------------------- -------- ------------ Staphylococcus aureus 5 55.5% Enterococcus faecalis 2 22.2% Klebsiella pneumoniae 1 11.11% Escherichia coli 1 11.11% The mitral and aortic valves, in respectively 23 patients (57.5%) and 8 patients (20%), were the most frequent affected sites; there was 1 case (2.5%) of tricuspid valve endocarditis, and 1 patient (2.5%) had two valves affected; there was 3 cases (7.5%) of septal defects endocarditis and 2 cases(5%) were prosthetic valve infections. There was 4 cases of rupture of chordae. Concerning treatment, antibiotics, administred parenterally in sufficient doses, were used in 41 patients (97.6%). The average duration of antimicrobial treatment was 17.48 days. Penicillins (68.2%), cephalosporins (68.2%), aminoglycosides (87.8%) and glycopeptides were the most used. No surgical treatment was performed. The evolution during hospitalization after a mean hospital stay of 21.45 days was favorable in 28 patients (66.6%). Complications were dominated by Congestive heart failure (CHF) (47.6%), neurological complications (31%) and major systemic emboli in 14.3% of cases. The complications are summarized in [Table 3](#t0003){ref-type="table"}. Thirteen (13) deaths (31%) were recorded, 8 of which were females (61.5%) and 5 were males(38.4%). ###### Complications Complications Number Percentage ------------------------ -------- ------------ heart failure 20 47.6% **Neurologic** Stroke 6 14,3% Cerebral abscess 4 9.5% Meningitidis 1 2.4% Cerebral hemmorrhagies 2 4.8% Acute renal failure 2 4.8% **Embolic events** Splenic infarction 3 7.1% Renal infarction 1 2.4% Femoral artery emboli 2 4.8% Septic arthritis 1 2.4% In patients with negative blood cultures, mortality was 69.2% against 30.8% in patients with positive blood cultures. Mortality according causative organisms was 50% for Staphylococcus aureus, 25% for Enterococcus faecalis and Klebsiella pneumoniae respectively. Ten (10) deaths were due to CHF, 2 deaths to neurological complications (meningitidis and cerebral abscess) and 1 death was due to anaphylactic reaction following blood transfusion. Discussion {#sec4} ========== In our study the prevalence of IE was 0.078%. In Africa, different series show a higher prevalence around 1.6% \[[@cit0005], [@cit0006]\]. This difference is difficult to explain because criteria for diagnosis and methods of reporting vary with different series. Also, in our study all cases were definites IE, while in other series, only a small proportion of clinically diagnosed cases are categorized as definite. HASE \[[@cit0007]\] in Japan found a prevalence of 0.19% over 12 years-study. In France its incidence (0.0032%) has not changed appreciably over the past years according to REVEST\'s study \[[@cit0008]\]. These low incidences are probably due to a decline in the incidence of rheumatic heart disease and antimicrobial prophylaxis before selected dental and invasive procedures in most developped countries. However, the use of antibiotics for IE prophylaxis in procedures that can cause bacteremia is controversial. The mean age of our patients was 27 years. Our study confirms young age of patients with IE as has been emphasized in previous African works. IKAMA \[[@cit0009]\] and NEBIE \[[@cit0005]\] found, respectively, a mean age of 30.6 years and 32.6 years. In European an American series, patients with IE are typically older with a mean age around of 60 years \[[@cit0010]\]. A number of factors may relate to this difference in age distribution. First, there has been a change in the nature of the underlying heart disease owing to a decline in the incidence of rheumatic heart disease. Second, the age of the population has been steadily increasing, and people with rheumatic or congenital heart disease are surviving longer. In addition, such patients increasingly undergo prosthetic valve surgery, an important risk factor for IE \[[@cit0011]--[@cit0013]\]. Our study confirms female predominance as has been emphasized in previous african works \[[@cit0009], [@cit0014]\]. This female predominance relate to the higher incidence of rheumatic disease in women as noted in African literature \[[@cit0015]--[@cit0017]\]. In Europe and America men are more commonly affected than women with a sex ratio up to 2. This is close to what is found in some africans country like Tunisia \[[@cit0018]\]. Predisposing conditions to IE in our patients were dominated by rheumatic valvular disease (64.3%). Indeed, most of Africans series on the occurence of IE, have rheumatic valve as a dominant underlying disease \[[@cit0014], [@cit0019]\]. In high income countries, currently, patients with degenerative valve disease, congenital heart disease, implantable devices and users of illicit intravenous drugs rather than those with rheumatic heart disease, account for the majority of cases of IE \[[@cit0010], [@cit0020], [@cit0021]\]. Predominant causes of bacteremia that were found in our patients were: dental caries (40.7%), urosepsis (40.7%). Dental carries as a cause of bacteremia is more common in african series than urosepsis \[[@cit0009], [@cit0019]\]. In general, risk of IE is considered to be highest for oral or dental procedures in which the oral mucosa is penetrated and in which gingival or mucosal bleeding is likely to occur. The risk of bacteremia is substantially lower for invasive genitourinary procedures. Clinically, IE in Africans does not appear much different from that of high income countries. This has been emphasized by several authors \[[@cit0008], [@cit0014], [@cit0022], [@cit0023]\]. The picture of IE is dominated by fever and cardiac murmurs, found in our study, respectively in 90% and 81% of cases. Blood cultures remain the definitive procedure for diagnosing IE. Blood cultures were negatives in 50% of cases in our study. Similar findings were noted by LAKHDHAR and YAMEOGO \[[@cit0019], [@cit0024]\] versus 5% found by HASE in Japan \[[@cit0007]\] and 9% by HOEN in France \[[@cit0025]\]. These high rates of negative blood cultures in african series may relate to prior antibiotic therapy and also by the method employed. IE is usually caused by Stapylococcus aureus. This has been emphasized by several authors both in Africa and Europa \[[@cit0019], [@cit0025], [@cit0026]\]. In our study, Staphylococcus aureus was the main pathogen and was responsible of 50% of death. This result confirms data from the literature. The utility of other blood laboratory tests in the diagnosis of IE is limited. Hematologic parameters are often abnormal in IE, but none is diagnostic. Anemia is nearly always present and usual parameters of inflammation elevated. Echocardiography has become paramount in the process of evaluating IE. It is crucial in detecting vegetations, echogenic distinct masses from the adjacent valve with independent motion from the valve itself. In our study, vegetations were found in 95.2% of cases. This percentage is close to what is found in Africa, Japan and France \[[@cit0007], [@cit0008], [@cit0019]\]. As in many african studies \[[@cit0017], [@cit0019]\], mitral valves (57.5%) were the most frequently affected sites weither it is the aortic valves in occidental series \[[@cit0008]\]. Following the establishment of a diagnosis using clinical, microbiological, and echocardiographic methods, antibiotics should be administered in a dosage designed to give sustained bactericidal serum concentrations throughout much or the entire dosing interval. A prolonged course of therapy is necessary to eradicate microorganisms growing in valvular vegetations \[[@cit0027]\]. In our study, almost all patients was under antibiotics. Penicillins and aminoglycosides were the most used. Duration of treatment was shorter than indicated. The reasons for this were due to many factors including lack of avalaibility and inability of some patients to meet the high cost of certain antibiotics. No surgical treatment was performed in our study as in many other african series while it has become an important adjunct to medical therapy in the management of IE and is now used in at least 25% of the cases in occident \[[@cit0008]\]. Favorable outcomes, as seen in our work and as noted in literature, seems higher in patients with positive blood cultures than those with negative ones. As usual in african series \[[@cit0014], [@cit0019], [@cit0028]\], complications were dominated by cardiac failure (47.6%) and was responsible of 76.9% of death. Neurologic manifestations occur in 20% to 40% of the cases and may dominate the clinical picture, especially in staphylococcal IE. Stroke is the most common neurologic complication of IE, occurring in 9.6% of patients \[[@cit0029], [@cit0030]\]. In our work stroke was present in 14.3% of cases. The development of clinical neurologic deterioration during IE is associated with a two- to fourfold increase in mortality. Less common noted in literature and found in our study were 4 cases of cerbral abscess (9.5%) and 1 case of meningitidis \[[@cit0031], [@cit0032]\]. Mortality remains high in Africa around 31% \[[@cit0023], [@cit0024]\]. Conclusion {#sec5} ========== Because of the variability in clinical presentation and the heterogeneity in both microbiologic etiology and the patient population, IE remains a challenging disease for clinicians. In Africa, where most health facilities are understaffed, the morbidity and mortality of IE remains high due to many reasons including inability to meet high cost of antibiotics and cardiac surgery which are rarely available. In addition, the costly treatment presents a huge financial burden on both the patients and the health system. Therefore, antimicrobial prophylaxis before selected dental and invasive surgical and diagnostic procedures should become standard and routine. What is known about this topic {#sec5.1} ------------------------------ - In Sub-Saharan Africa, IE is recongized as a disease associated with rheumatic heart disease and Staphylococcus aureus is the main pathogen; - Patients affected are young with a female predominance; - complications are dominated by congestive heart failure and mortality remains very high. What this study adds {#sec5.2} -------------------- - The Diagnosis latency IE, the interval from the onset of symptoms to the definite diagnosis of IE remains long (29.97 days); - Favorable outcomes, seems higher in patients with positive blood cultures than those with negative ones which represent about 50% of cases; - Despite medical advances, No surgical treatment was performed in our study while it has become an important adjunct to medical therapy in the management of IE. Competing interests =================== The authors declare no competing interests. Authors' contributions ====================== Djibril Marie BA, Mouhamed Cherif MBOUP, Nafissatou ZEBA and Sara Boury GNING designed the study protocol, participated in the data collection and contributed in analyzing the data and writing of the draft manuscript. Khadidiatou DIA, Awa Ndaw Fall, Fatou FALL and Pape Diadie FALL participated in data analysis and critically revising themanuscript for important intellectual content. All authors have read and approved the final version of the manuscript.

Palmer VJ, Chondros P, Piper D, *et al*. The CORE study protocol: a stepped wedge cluster randomised controlled trial to test a co-design technique to optimise psychosocial recovery outcomes for people affected by mental illness in the community mental health setting. *BMJ Open* 2015;5:e006688. One of the authors' names in this paper was misspelt. Konstancja Densely should be Konstancja Densley.

1.. Introduction   {#sec1} ================== As the study of molecules, chemistry is concerned with uncovering the molecular structures of compounds of interest. X-ray crystallographic analysis is the only method that provides direct information on molecular structures at the atomic level. However, this method has the intrinsic limitation that the target molecules must be crystalline, and high quality single crystals must be prepared before measurement (Blake, 2009[@bb4]). This limitation has often been a considerable bottle-neck for the development of molecular chemistry, in particular when the structural determination of the target compound is the major concern of the research (*e.g.* structural determination of isolated natural products, total synthesis studies and product analysis in new reactions). In 2013, we reported a new technique for single-crystal X-ray diffraction analysis that does not require the crystallization of samples in the sample preparation (Inokuma *et al.*, 2013[@bb15]). This method, later called the crystalline sponge method (Vinogradova *et al.*, 2014[@bb42]), uses crystals of porous metal complexes (Hoskins & Robson, 1989[@bb13]; Fujita *et al.*, 1994[@bb9]; Subramanian & Zaworotko, 1995[@bb37]; Yaghi & Li, 1995[@bb44]; Kondo *et al.*, 1997[@bb20]; Mori *et al.*, 1997[@bb24]; Zhou & Kitagawa, 2014[@bb47]; Zhou *et al.*, 2012[@bb48]) \[crystalline sponges (Biradha & Fujita, 2002[@bb2]; Inokuma *et al.*, 2010[@bb14]) Fig. 1[▸](#fig1){ref-type="fig"}\] capable of absorbing guest compounds from solution in a common solvent. The guests are efficiently trapped and concentrated at several binding sites in the porous complexes, and the periodic array of the binding sites renders the absorbed guests oriented and observable by common X-ray diffraction studies (Kawano & Fujita, 2007[@bb18]; Biradha *et al.*, 2002[@bb3]; Suh *et al.*, 2002[@bb38]; Ohmori *et al.*, 2004[@bb27]; Ohmori *et al.*, 2005[@bb26]). Since only one tiny piece of crystal, weighing approximately 1 µg, is enough to perform the experiment, the required amount of the guest is on the nano-to-microgram order. Owing to the removal of the crystallization step from the sequential procedure of single-crystal X-ray experiments, considerable attention has been paid to the crystalline sponge method by the communities of organic synthetic chemists, natural product chemists, coordination chemists and crystallographers, as well as researchers in the pharmaceutical, agriculture and food industries. Following our report, the utility of the method has been independently demonstrated by several reports from other groups (Vinogradova *et al.*, 2014[@bb42]; Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]; Sanna *et al.* 2015[@bb32]) in which the crystalline sponge method and related techniques were used to determine the structures of small molecules. Useful applications of this method for absolute structure determination (Yoshioka *et al.*, 2015[@bb45]) and metabolite analysis (Zigon *et al.*, 2015[@bb49]) have been recently reported by our group and others (Kamimura *et al.*, 2013[@bb16]; O'Brien *et al.*, 2014[@bb25]; Tashiro *et al.*, 2012[@bb39]; Kubota *et al.*, 2014[@bb21]; Turega *et al.*, 2014[@bb41]). In our original report, however, the data quality of the trapped guest compounds was not very high and the use of restraints and constraints based on chemical information was necessary to refine the guest structures. This was not due to insufficient knowledge or techniques of crystallographic analysis but instead due to unoptimized experimental protocols throughout the method (Inokuma *et al.*, 2013[@bb15]); the data quality depends on all the steps in the crystalline sponge method, including the synthesis of high-quality sponges, solvent exchange, guest-soaking, data collection and crystallographic refinement of the post-absorbed molecules. These steps were largely unfamiliar to all researchers and the method was still at a proof-of-concept stage. To develop the crystalline sponge method from basic science into a reliable new technology that may innovate all of the molecular sciences, much effort is needed to be put into improving the data quality. In addition, the crystallographic scope and limitations in the refinement of structures with large pores \[so-called metal--organic framework (MOF) structures\] should be carefully considered (Spek, 2015[@bb36]). Over the last 2 years, therefore, our group has made considerable efforts to improve the data quality and to uncover the crystallographic scope and limitations for the refinement of guest structures obtained using the crystalline sponge method. In this article, we describe certain improved procedures in crystal soaking and crystallography of the crystalline sponge method, without which the method cannot be used by the general working chemist. The updated methods and protocols are applied to most of the compounds reported in our original paper (Inokuma *et al.*, 2013[@bb15]). It is also important to understand that, unlike scientific principles, there are no clear right or best answers for the experimental protocols because they are highly dependent on the nature of the samples, the purpose of the analysis, and the skill or taste of the users. Recently, Clardy reported practical guidelines for our crystalline sponge method, in which some modified or improved procedures on our original method are recommended (Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]). Similar attempts to improve the original protocols that may appear in a near future by other groups are welcome. The protocols discussed here, as well as those suggested by the others, are still tentative and they should be ideally elaborated through future discussions among users and by many successful results. Nevertheless, we also emphasize that, without the procedures outlined in this paper, the crystalline sponge method cannot be generally applied by bench chemists or crystallographers. 2.. Results and discussion   {#sec2} ============================ 2.1.. A standard protocol for sample preparation   {#sec2.1} -------------------------------------------------- After the publication of the original paper, we also published a detailed experimental protocol for the crystalline sponge method (Inokuma *et al.*, 2013[@bb15]) using two standard samples, guaiazulene and 2,6-diisopropylaniline, particularly focusing on the guest-soaking step. Guaizulene is a suitable guest for the crystalline sponge method because the guest-soaking process proceeds relatively smoothly and guest inclusion is clearly indicated by the color change of the sponge crystal. The reported protocol was, however, optimized only for the two standard compounds and cannot necessarily be applied to a wide range of organic guest compounds. The soaking process must be optimized for every compound and is dependent on its molecular size, polarity, flexibility, nucleophilicity, stability, solubility and other properties. In summary, there is no universal protocol in the soaking step that can be applied to any compounds. The following discussion will be carried out based on this understanding. Single crystal samples for the crystalline sponge method are prepared by the following steps: (1) host crystal synthesis, (2) solvent exchange and (3) inclusion of the target molecules to be analyzed. Although the detailed procedures in each step have been described in a previous paper (Inokuma *et al.*, 2013[@bb15]), we have here added some new or updated procedures that have improved the method in terms of data quality, practicability and applicability. ### 2.1.1.. \[(ZnI~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ as a crystalline sponge   {#sec2.1.1} In our original paper, two porous complexes, \[(ZnI~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ (**1**) (Biradha & Fujita, 2002[@bb2]) and \[\[Co(SCN)~2~\]~3~(tpt)~2~·*x*(solvent)\]~*n*~ (**2**) (Inokuma *et al.*, 2010[@bb14]) \[tpt = 1,3,5-tris(4-pyridyl)triazine\], were used as crystalline sponges. Regarding complex **2**, absorbed guest molecules were quite prone to static disorder because they frequently lay on the symmetry elements of the cubic lattice (). In terms of practicability, we are thus currently using the less symmetric (*C*2/*c*) complex **1** in most cases as the crystalline sponge. In addition to the relatively low symmetry of the space group, complex **1** is a suitable crystalline sponge because of the following advantages in host--guest complexation in the pores: (1) The size of the pores (5 × 8 Å measured between van der Waals surfaces in the cross section projected from the *b*-axis) is appropriate for the accommodation of organic molecules of common sizes. (2) The tpt ligand offers flat and electron-deficient binding sites suitable for stacking with aromatic compounds and for CH--π interactions even with aliphatic compounds (Ohmori *et al.*, 2004[@bb27]). (3) The cavity is rather hydrophobic and favorable for the binding of common organic molecules. (4) The I atoms in the ZnI~2~ component are good hydrogen-bond acceptors and the pyridyl protons of the tpt ligand are good hydrogen-bond donors, both providing efficient binding sites through hydrogen-bonding. (5) The framework is relatively flexible and guests are often bound in the cavity by induced-fit molecular recognition. Regarding the pore size (5 × 8 Å), it does not strictly limit the size of guest to be included because the sponge framework is very flexible and guest molecules larger than the portal are often accommodated by expanding the pore size. Typical expansion of the pores by inclusion of guaiazulene and santonin is visualized in Fig. 2[▸](#fig2){ref-type="fig"}. As previously described in detail (Inokuma *et al.*, 2013[@bb15]), crystalline sponge **1** is synthesized by the layer diffusion of a methanolic solution of ZnI~2~ into a PhNO~2~/MeOH (*v*/*v* = 4/1) solution of the tpt ligand at room temperature in a test tube (Fig. 3[▸](#fig3){ref-type="fig"}). Isostructural ZnCl~2~ and ZnBr~2~ complexes, \[(ZnCl~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ (Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]; Batten & Robson, 1998[@bb1]; Heine *et al.*, 2011[@bb10]) and \[(ZnBr~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ (Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]; Kawano *et al.*, 2008[@bb19]) have been reported. These complexes are useful when the strong residual electron peaks around the heavy (iodine) atoms due to the termination effect from moderate data resolution obscure the weak peaks of the nearby guests. ### 2.1.2.. What determines the data quality?   {#sec2.1.2} Unlike common X-ray structure analysis, the data quality for the guest structures in the crystalline sponge method is not simply determined by the crystal quality and X-ray experiments. We emphasize that the following issues are particularly important for obtaining high data quality: (1) smooth and complete solvent exchange in the pores, (2) high guest occupancy in the pores, (3) thorough thermodynamic equilibration of the absorbed guest, and (4) careful data collection particularly at high-angle regions. It is worth noting that tailored soaking conditions for every target guest should be designed based on the understanding of host--guest chemistry as well as on feedback from X-ray crystallographic experiments. As such, unlike for common X-ray crystal structure analysis, sophistication and craftsmanship are required not only in crystallography but also in host--guest and synthetic chemistry for successful analysis. The technique should be ideally performed (at least directed) by scientists who can survey these different fields. We recommend the guest soaking and crystal structure analysis be performed by the same researcher. ### 2.1.3.. Solvent exchange   {#sec2.1.3} The pores of the as-synthesized crystalline sponge are filled with nitrobenzene, which is used as the solvent for the sponge preparation. Target guests are not efficiently absorbed into the as-synthesized sponge because nitrobenzene itself is a good guest with a high affinity to the pores. Therefore, replacing nitrobenzene with a non-interactive inert solvent is a crucial step. Typically, cyclohexane is used as the inert solvent. Pentane is useful when the guest soaking is examined at low temperature (∼ 0°C). The solvent exchanging process can be monitored by IR spectroscopy; Fig. S1 of the [supporting information](#suppinfoanchor) shows that the signal at 1346 cm^--1^, assignable to nitrobenzene, almost completely disappears after soaking in cyclohexane for 1 week at 50°C. The complete solvent exchange can be confirmed by elemental analysis or even by crystallographic analysis. Single-crystal X-ray analysis confirms the presence of ordered cyclohexane molecules in the pores, which indicates successful solvent exchange ([Fig. S2](#suppinfoanchor)). After completing the solvent exchange with cyclohexane, the crystalline sponge is ready to use. Clardy reported that the solvent exchange step can be omitted if the crystalline sponge is synthesized in chloroform--methanol (Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]). This modified method gives a chloroform-filled crystalline sponge and is, therefore, probably effective only if the target compound is a better guest than chloroform or if a large excess of the target compound can be used. The yield of high quality crystals suitable for the crystalline sponge method is actually not very high (\< 5%) and, as in common single-crystal X-ray experiments, researchers often have to carefully choose the top quality crystals from many crystals under a microscope. It is rather an advantage of the crystalline sponge method that this quite inefficient step (crystal selection) can be undertaken before guest absorption. Namely, the required amount of the sample can be reduced to nano-to-microgram quantities by undertaking the crystal selection first and the guest soaking second. In this aspect, the crystalline sponge method is clearly distinguished from cocrystallization methods (Desiraju, 2007[@bb8]; Vishweshwar *et al.*, 2006[@bb43]) such as the pioneering work of Toda (1987[@bb40]) who co-crystallized a crystalline host with a non-crystalline guest using host--guest interactions. In this method, high quality crystals are selected at the last step and hence a substantial amount of sample is necessary to prepare the guest-cocrystallized single crystals. ### 2.1.4.. Guest soaking   {#sec2.1.4} Any guest molecules that can interact with the sponge pores better than cyclohexane can be in principle absorbed. As cyclohexane is a poor solvent for common organic compounds, the minimum amount of a good solvent such as chloroform or dichloromethane is added to dissolve the guest in the guest-soaking step. Excess of the good solvent should be avoided because it will extract the target guest from the sponge crystal. The guest-soaking process is the most crucial step and usually determines whether the method works or not, or whether the data quality is good or bad. The following discussion describes how the soaking method has been improved and elaborated on. The history of the guest-soaking step almost parallels that of the crystalline sponge method itself.(*i*) *Original method*: The prototype of the crystalline sponge method can be ascribed to the observation of single-crystal-to-single-crystal (SCSC) solvent exchange in the pores of porous complex (1), which we reported in 2002 (Biradha & Fujita, 2002[@bb2]). This facile solvent exchange occurs on simply dipping the crystals of (1) in the solvent. Similar SCSC exchange of solvents or guests in porous complexes have been reported by us and others (Kawano & Fujita, 2007[@bb18]; Suh *et al.*, 2002[@bb38]; Kawano & Fujita, 2007[@bb18]; Deiters *et al.*, 2005[@bb7]; Li *et al.*, 2009[@bb23]). Meanwhile, discrete *M* ~6~ *L* ~4~ cages that possess a cavity surrounded by the same tpt ligand used in (1) show remarkable guest binding properties in aqueous solution (Kusukawa & Fujita, 1998[@bb22]; Yoshizawa *et al.*, 2009[@bb46]). Inspired by the highly efficient guest binding by the discrete cage, we examined guest uptake by the crystals of (1) and in 2004 demonstrated the SCSC absorption of large organic molecules, such as pyrene, perylene and triphenylphosphine oxide, from their solutions (Ohmori *et al.*, 2004[@bb27], 2005[@bb26]). These results convinced us of the strong guest-binding ability of the tpt-surrounded cavity and prompted us to use this phenomenon for structural analysis. In these prototype crystalline sponge methods, a substantial amount of the host complex (typically ∼ 10 mg; 1000--10000 crystals) was used (Fig. 4[▸](#fig4){ref-type="fig"} *a*). However, this original method is still useful and can be the method of choice when a large quantity of the target compound is available. There is no technical difficulty in this original method.(*ii*) *A-grain-of-crystal method*: To establish the method for the structural analysis of small molecules, the required amount of the sample should be reduced. This is especially necessary when the target compounds are highly valuable and obtained only in minute quantities; this is the case when analyzing isolated natural products, metabolites and impurities in drugs and foods, or flavor components. Thus, we reduced the experimental scale of the original method to the extreme by performing the guest-soaking experiment with only one grain of the sponge crystal (Fig. 4[▸](#fig4){ref-type="fig"} *b*). From the crude solvent-exchanged sponge crystals, a top quality crystal was chosen and contacted with a small amount (typically, a few milligrams) of neat liquid sample or a supersaturated viscous solution on a glass plate. The guest absorption is usually completed in minutes to hours, although depending on the samples and crystal size it can sometimes take a few days. It is noteworthy that the order of events in the original method (guest-soaking followed by crystal selection) is reversed in the 'a-grain-of-crystal' method (namely, crystal selection followed by guest soaking), and this reversed protocol made it possible to analyze very small amounts of the target compounds. The method itself is technically not particularly difficult because there are only a limited number of parameters that influence the data quality.(*iii*) *Slow evaporation method*: In the above method, the quality of the crystals often deteriorates severely during guest absorption if the host--guest interaction is too strong and the host framework is mechanically stressed. To perform the guest absorption with \[(ZnI~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ (**1**) under much milder conditions, the crystals can be soaked in a diluted solution of the guest (Fig. 4[▸](#fig4){ref-type="fig"} *c*). The use of a poor solvent is essential because otherwise the guest is extracted from the crystal; cyclohexane with a minimum amount of CH~2~Cl~2~ is typically used as the solvent. In this method, the sponge crystal is placed in a vial and soaked in the solution of the guest. The vial is equipped with a needle, through which the solvent slowly evaporates. During this slow evaporation, the guest is gradually concentrated, finally reaching the saturation point, and is forced to diffuse into the crystal. The data quality in the crystalline sponge method depends highly on this guest-soaking step. As emphasized in the general discussion of the standard protocols, there is no all-purpose protocol for this soaking step that can be applied to all compounds. The guest-soaking time ranges from minutes to days, or sometimes even weeks.Typically, a set of several conditions are examined. The parameters include soaking temperature, soaking time, solvent, concentration, crystal size, evaporation rate (controllable by changing the thickness of the needle). Guest absorption is, in general, faster at higher temperatures but the guest seems to be more concentrated and ordered in the crystal after soaking at lower temperatures. Annealing of the crystals during the guest-soaking is sometimes effective (*e.g.* 50°C, 2 d → room temperature, 2 d → 4°C, 2 d). The guest concentration in the crystal can be roughly estimated by microscopic IR measurement and this helps in the optimization of the soaking conditions. Although this slow evaporation method has considerably expanded the scope of the crystalline sponge method, it is technically difficult because every step in the method is unfamiliar and the optimization of the soaking conditions requires experience. We recommend that those new to the crystalline sponge method experience the guest-soaking techniques in the order described here \[first (i)[](#l1li1){ref-type="other"}, then (ii)[](#l1li2){ref-type="other"} and finally (iii)[](#l1li3){ref-type="other"}\]. A benchmark test discussed later may help researchers to evaluate their level of expertise with the method.(*iv*) *High-throughput method*: If satisfactory data are not obtained under a few standard conditions, other soaking conditions are examined. The soaking conditions can be optimized by using a high-throughput method, in which dozens of vials, each containing a sponge crystal, are used. For the guest-soaking, *m* different conditions (typically, *m* = 5--10) are applied, for each of which *n* sponge crystals (typically, *n* = 3--5) are used. Namely, *m* × *n* sponge crystals are subjected to guest soaking at once. The *m* × *n* guest-absorbed sponge crystals are transferred to a 96-well cell plate and screened by an X-ray scanner (Fig. 4[▸](#fig4){ref-type="fig"} *d*). Every scan takes approximately 5 min and all the crystals are examined in 1--2 h. Cracked crystals are removed before the screening. Crystals with weak or spelt diffraction are visually checked and eliminated. Small invisible cracks and inhomogeneous inclusion of guests lead to large mosaicity. The best-diffracting crystal that shows sharp spots with the highest resolution is then selected for further study. This high-throughput method considerably shortens the trial-and-error process for optimization of the soaking conditions and is quite effective when representative standard conditions do not work well.(*v*) *Other methods*: Some variations of these guest-soaking methods are still being developed. Like in protein ligand-soaking experiments, the use of micrometer-sized crystals (10--20 µm in size) has shown some excellent advantages over the standard method, which uses 100--200 µm sized crystals (micro-crystal method). For example, nanogram quantities of samples can be measured with faster and more homogeneous guest absorption, and without deterioration in the crystals. The details of the micro-crystal method will be discussed in a future report. 2.2.. Crystallographic analysis   {#sec2.2} --------------------------------- ### 2.2.1.. Guest occupancy   {#sec2.2.1} One of the most important parameters that determine the data quality is the occupancy of the guest. The occupancy of the guest can be roughly estimated by microscopic IR spectrometry. [Fig. S3](#suppinfoanchor) shows the relationship between soaking time (or guest site-occupancy) and *F* ~o~ map for the inclusion of (5) in **1**. In one of our trials, the amount of absorbed guest after 2 d of soaking time was low and we could not clearly observe the guest electron density ([Fig. S3*a*](#suppinfoanchor)). By simply extending the soaking time to 7 d, however, the guest electron density was clearly observed after least-squares treatment of the host framework ([Fig. S3*b*](#suppinfoanchor)). By using a large excess of the guest, the guest site occupancy after 7 d reached almost 100%, and we could obtain better data for (5) ([Fig. S3*c*](#suppinfoanchor)). Since the soaking time is not a highly reproducible parameter (because it depends on crystal size that cannot be precisely regulated), we can discuss only the following tendency from our observations: At low concentrations, ordering of the guests in the crystal is difficult because they cannot interact with each other. However, above a certain concentration, guest molecules seem to start to order in the crystal. In this regard, we suggest that the best description for the principle of the crystalline sponge method is 'crystallization within crystals' or 'post-crystallization'. ### 2.2.2.. Molecular recognition sites   {#sec2.2.2} Two channels (*a* and *b*) are found in the views along with \[010\] and \[101\] directions, respectively, in the sponge crystal (Figs. 1[▸](#fig1){ref-type="fig"} *b* and *c*). The faces indexed as (010) and (101) are not surfaces of a spontaneously grown crystal of **1**. However, (010) is considered as a cleavage face because a surface indexed as (010) is found in many split crystals. A typical crystal of **1** with face indexes is shown in Fig. 5[▸](#fig5){ref-type="fig"}. As rod-like molecules often lie along the channel *b*, most guest compounds seem to penetrate into the pore *b* from the (101) face of the crystal. Several different sites and modes of guest binding are observed in the pores of the crystalline sponge (Fig. 6[▸](#fig6){ref-type="fig"}). The most common feature of the guest binding is the efficient host--guest stacking on an electron-deficient tpt ligand. A typical example is the binding of **5** (Fig. 6[▸](#fig6){ref-type="fig"} *a*). The pyridyl protons on the tpt ligand are good hydrogen-bond donors and are often located within a hydrogen-bond distance from the nitrogen and oxygen lone pairs of the substrates (Fig. 6[▸](#fig6){ref-type="fig"} *b*). Even the Br atom of the guest can be a hydrogen-bond acceptor (Fig. 6[▸](#fig6){ref-type="fig"} *c*). The iodine atom of the ZnI~2~ center seems to be a good hydrogen-bond acceptor (Fig. 6[▸](#fig6){ref-type="fig"} *d*). The host network is relatively flexible and is quite often deformed before and after the guest absorption, which results in efficient induced-fit molecular recognition (Figs. 6[▸](#fig6){ref-type="fig"} *e* and *f*). When the sponge crystal is soaked in a guest solution, the guest molecules can freely diffuse into the solvent-filled pores and find the most suitable binding sites *via* reversible host--guest complexation. The binding sites in the pores vary case-by-case and are unpredictable for any guest. When a large association constant is gained at the binding site, the guest is trapped and concentrated. In other words, finding at least one suitable strong binding site is essential for every guest to be ordered and crystallographically analyzed. ### 2.2.3.. A benchmark experiment   {#sec2.2.3} Although the details of the structural analysis of the guest-soaked crystalline sponge vary case-by-case depending on the guest examined, common and general methods for the analysis can be suggested. Taking guaiazulene as an example, a general procedure for the analysis is described. We recommend that the data for guaiazulene be used as a benchmark for evaluating guest soaking and crystal structure analysis in the crystalline sponge method. Another benchmark test for this method toward a chiral molecule is also proposed with a santonin guest. The obtained crystallographic data represents the potential ability of this method for reliable absolute structure determination. ### 2.2.4.. X-ray diffraction data collection   {#sec2.2.4} Cyclohexane-filled crystalline sponge **1** is labile and rapidly deteriorates when it is taken out of the cyclohexane solvent because the solvent is lost from the pores by rapid evaporation. Upon inclusion of non-volatile guests, however, the crystals become relatively stable and can be treated with various protectants. After coating with an appropriate protectant, the guest-absorbed crystal is subjected to data collection. For the guaiazulene-containing crystals, we used Hampton Type A low viscosity oil as a protectant. Several different X-ray source/detector combinations have been examined for the X-ray diffraction data collection. Data collection with a sufficient number of reflections and a suitable *I*/σ value at the high-angle region (*d* \< 1.0) is particularly important to obtain high data quality for the trapped guest in the crystalline sponge as reflections from a guest is much weaker than those from the host at high-angle region. Although Mo *K*α radiation is commonly used for the X-ray structure analysis of metal complexes because of the weaker X-ray absorption by heavy atoms, Cu *K*α radiation has provided, overall, better results in the crystalline sponge method. The crystalline sponge is a weakly diffracting crystal because of the huge number of solvent molecules (typically cyclohexane) in the pores. Thus, the value of *I*/σ at the high-angle region by the Mo *K*α radiation is typically 1 or lower. However, Cu *K*α radiation provides a much higher *I*/σ value for the high-angle region. For guaiazulene, an exposure time of 90 s per data frame was applied for the high-angle region to satisfy the condition *I*/σ \> 10. Under these conditions, high-resolution data were collected for guaiazulene with sufficient intensity, and the 0.82--0.79 Å shell exhibited a completeness of ∼ 90%. More than 1 d (27 h) was required for complete collection of the above data. Using a synchrotron X-ray source (Ramadhar *et al.*, 2015*a* [@bb30],*b* [@bb31]) is expected to shorten the data collection time. Synchrotron data may improve resolution for a given crystal, but the associated practical problems (*etc.*) mean that it may be less effective than a good in-house facility. Prompt feedbacks from the crystal structure analysis to the soaking experiments are very important to optimize the soaking conditions. Also, beam-times for experiments at a synchrotron facility are usually limited, and highly experienced operations are necessary to find the suitable conditions for data collection (*e.g.* wavelength, detector, attenuator, data scan strategy; see Table S1). All the data in Table 1[▸](#table1){ref-type="table"} were collected with an in-house X-ray diffractometer with a Cu *K*α radiation source. ### 2.2.5.. Space group determination   {#sec2.2.5} The crystalline sponge has the centrosymmetric *C*2/*c* symmetry. This crystallographic symmetry is maintained after inclusion of an achiral guest in many cases. In the case of inclusion of a chiral guest, this space group changes to a non-centrosymmetric one (*C*2) but geometrical distortion in the framework is not obvious. This slight change of framework causes a pseudo-symmetry issue (faint *C*2/*c* symmetry) and may lead to misassignment of the space group. The space group of the guest-included crystalline sponge should be carefully determined based on the extinction rule without excessively relying on an automatic suggestion by a space-group determination program. ### 2.2.6.. Refinement of the framework   {#sec2.2.6} After obtaining an initial structure for refinement using a standard program \[*SHELXT*, (Sheldrick, 2015[@bb34]), *SHELXS* (Sheldrick, 2008[@bb33]), *SIR* (Burla *et al.*, 2015[@bb5]), or *SUPERFLIP* (Palatinus & Chapuis, 2007[@bb28])\], the refinement of the host framework should be performed first. After anisotropic refinement of all non-H atoms in the framework, relatively high residual electron-density peaks are found around zinc and iodine atoms. Since the ZnI~2~ fragments are the most flexible part of the network, disordered typically two ZnI~2~ fragments can usually be modeled and properly refined anisotropically ([Fig. S4](#suppinfoanchor)). After this disorder treatment on the ZnI~2~ fragments, a partial or full guest molecule clearly appears in the difference electron density map. In the analysis of the guaiazulene-absorbed crystalline sponge, the initial structure was obtained as described above. ### 2.2.7.. Refinement of the guest   {#sec2.2.7} If a guest molecule is placed on a general position and its site occupancy factor (s.o.f.) is almost 1, the guest structure can be properly refined using a typical refinement procedure. In the case of the guaiazulene-absorbed crystalline sponge, one guaiazulene molecule (**A**) in the asymmetric unit could be refined with 100% occupancy (s.o.f. = 1; Fig. 7[▸](#fig7){ref-type="fig"}). If the guest is located on a symmetry element (*i.e.* a special position) of the framework, the independent guest molecule and a symmetrically generated one are crystallographically equivalent. Therefore, the occupancy of the guest should be divided by the multiplicity of the special position during the refinement. In the guaiazulene case, the second independent guaiazulene molecule (**B**) was refined using a disordered model of two orientationally different guests with the constraint of the sum of their occupancy being 50% because it was placed on a twofold axis (multiplicity = 2). The refined s.o.f.s for both independent guests are 0.278 (10) and 0.222 (10). This means that the total occupancy of the guest in this site is 100%. If the guest occupancy is considerably lower than 100%, guest molecules should be overlapped with solvent cyclohexane molecules and refined using a disordered model of the guest and cyclohexane. The structure of the guest for refinement is made by selecting some residual electron density peaks in the difference electron density map. The s.o.f. of the guest should be constrained temporarily to a reduced value (typically, 0.3--0.5) to find the overlapped cyclohexane molecules. After refinement of the guest, the overlapped cyclohexane molecules are found as a difference electron density map. Geometries and s.o.f.s of the guest and the cyclohexane are refined using this disordered model. For guaiazulene, the s.o.f. of the third independent molecule (**C**) disordered with solvent was estimated as 0.186 (6) from least-squares refinements. Some restraints are applied in this refinement, and the sum of the s.o.f.s of the guest and solvent is constrained to 100% to refine the s.o.f.s of the guest and cyclohexane. When overlapped cyclohexane molecules are not found, we postulated s.o.f. = 1 to satisfy the 100% sum. In this case, some overestimation is unavoidable if the actual occupancy is slightly less than 100%. ### 2.2.8.. Refinement of remaining solvent molecules   {#sec2.2.8} As mentioned above, filling the remaining void of the pores with inert solvent molecules (cyclohexane in the present case) is an important requirement for efficient guest inclusion. In the crystalline sponge method, less interactive solvents (hydrocarbons) have the advantage of being easily replaced with the guests, but have the disadvantage of being disordered when some solvent molecules remain after guest-soaking. Therefore, refinement of solvent molecules is still challenging in the crystalline sponge method. Unfortunately, the maximum resolution in the case of Cu *K*α radiation is not so high (*d* ≃ 0.8 in our cases) and the present data resolution cannot provide electron densities corresponding to cyclohexane molecules with atomic resolution: broad and obscure electron densities are found as an averaged structure of variously orientated cyclohexane molecules. The observation of unusual conformations (boat or twisted) in an averaged structure for some of the included solvents is unavoidable to some extent. In the original paper, these disordered solvent molecules were eliminated by using the *SQUEEZE* procedure (Spek, 2015[@bb36]) in the program *PLATON*. However, whether it is appropriate to apply such a solvent-masking procedure to the observed electron density is still a matter of debate (Spek, 2015[@bb36]). In the present study, all cyclohexane molecules were found and refined to avoid applying the *SQUEEZE* procedure. Before the guest soaking, 5.5 cyclohexane molecules were refined in the asymmetric unit. The disordered cyclohexane molecules in a guest-soaked crystalline sponge were also refined although some restraints were necessary. Refinement of cyclohexane molecules in the crystal improved the phases of reflections and helped to reduce the number of restraints needed for refinement of a guest structure. Therefore, refinement of guest and solvent molecules in parallel is highly recommended. ### 2.2.9.. Absolute structure determination   {#sec2.2.9} One of the biggest advantages of the crystalline sponge method is the possibility for absolute structure determination of the absorbed guest molecule using anomalous scattering from the heavy atoms installed in the framework (Yoshioka *et al.*, 2015[@bb45]; Zigon *et al.*, 2015[@bb49]). Using this feature, the absolute configuration of a chiral molecule that possesses no heavy atoms can be determined in an efficient way. This would be the only direct method for determining the absolute structure of a trace amount or oily compound. Although current crystallography does not always need heavy atoms for the absolute structure determination (Fujita *et al.*, 1994[@bb9]; Parsons *et al.*, 2013[@bb29]; Hooft *et al.*, 2010[@bb12]) much higher quality data are required and, in principle, the reliability decreases without heavy atom effects. When the crystalline sponge method is used for absolute structure determination, however, we recommend that researchers follow the guidelines described below because there are some important warnings in applying this method. The guidelines are discussed here along with a typical procedure for santonin (**4**), a classical anthelmintic drug, the absolute structure of which was a matter of discussions in the 1950s (Corey 1955[@bb6]). In our original paper, guest-soaking was carried out at 323 K for 2 d (our preliminary standard conditions). To obtain better diffraction data in this study we prepared four vials, each containing a sponge crystal (typically 200 × 100 × 80 µm^3^ in size) with 45 µL of cyclohexane. Then, 5 µL of the guest solution in CH~2~Cl~2~ (2.5 µg μL^−1^) was added to each vial. The vials were pierced with a needle for solvent evaporation and kept at 50°C. After 1 d at 50°C, the vials were kept at 4°C for a further 1 d to facilitate further guest inclusion. The most suitable crystal was selected by preliminary diffraction experiments on all the prepared crystals using an X-ray scanner. For data collection, the use of Cu *K*α radiation is again highly recommended for obtaining better *I*/σ values with a more pronounced intensity contrast between Bijvoet pairs. Single-crystal-to-single-crystal framework deformation concomitant with symmetry lowering from *C*2/*c* to *C*2 or *P*2~1~ has been observed upon chiral guest inclusion in previous studies (Yoshioka *et al.*, 2015[@bb45]; Zigon *et al.*, 2015[@bb49]; Inokuma *et al.*, 2013[@bb15]). For santonin, the space group was changed from *C*2/*c* to *P*2~1~. This space group change was clearly indicated by the preserved extinction rule of 0*k*0 (*k* = odd number) and by the statistics of the normalized structure factor (〈\|*E* ^2^ − 1\|〉 = 0.806 in the present case). For some other chiral guests, the *C*2/*c* symmetry is lowered to *C*2. However, evaluation of centrosymmetry based on the statistics of normalized structure factor often failed because of the very small deformation resulting in the pseudo-symmetry of the host framework. Therefore, the appearance of *h*0*l* (*l* = odd number) reflections, which is evidence for the disappearance of the *c*-glide plane involving the inversion center, should be carefully confirmed. This pseudo-symmetry problem sometimes decreases the reliability of the absolute configuration determined by the crystalline sponge method. For example, when limonene was examined as a chiral guest, the guest molecule was found by crystal structure analysis, but the appearance of *h*0*l* (*l* = odd number) reflections could not be confirmed and the absolute configuration was obscured. One reason that the pseudo-symmetry problem occurred with limonene is presumably its low guest occupancy in the crystalline sponge and its pseudo-mirror symmetrical skeleton. To avoid the pseudo-symmetry problem, a high guest occupancy of above 50% is desired. In the structure refinement, five independent santonin molecules were refined without using restraints or constraints (Table 1[▸](#table1){ref-type="table"}). The Flack parameter calculated using the Parsons' method is −0.0071 (11), which clearly represents the definitive determination of the absolute configuration of santonin. We thus highly recommend santonin as a benchmark for the absolute structure analysis of chiral molecules. The reason why santonin induced significant crystallographic symmetry lowering from *C*2/*c* to *P*2~1~ is considered to be the presence of a relatively large number of host--guest interactions than other molecules. Intermolecular interactions involving guest molecules in **3** and **4** are shown in Fig. 8[▸](#fig8){ref-type="fig"}. The number of host--guest interactions in **4** (25 interactions) is more than double for **3** (12 interactions). Larger expansion of the pores by inclusion of santonin rather than guaiazulene (Figs. 2[▸](#fig2){ref-type="fig"} *b* and *c*) indicates that a considerable number of interactions leads to significant distortion of the host framework and obvious space group change from achiral (*C*2/*c*) to chiral (*P*2~1~). If another enantiomer is available for the target chiral compound, double checking of the absolute configuration by analyzing both enantiomers using the crystalline sponge method is strongly recommended. When the guest occupancy is low, often the Flack parameter does not significantly decrease and the value remains around 0.2, presumably because of the pseudo-symmetry of the guest-absorbed structure. In such cases, obtained absolute configuration should be considered only as reference data. We do not recommend the use of *SQUEEZE* program in the absolute structure determination because the Flack value calculated both in a classical way or the Parsons' method is based on the modeled structure in refinement. In particular, when the electron densities of an un-modeled chiral guest is anticipated in the void space, the Flack value after *SQUEEZE* treatment may be unsuitable because contribution of the anticipated guest is eliminated in the calculation. ### 2.2.10.. Details for other compounds   {#sec2.2.10} (*i*) *Flavonoids*: Three flavonoid compounds \[nobiletin (**5**), tangeritin (**6**), heptamethoxyflavone (HMF, **7**)\] were analyzed with good data qualities (Table 1[▸](#table1){ref-type="table"}). In the flavonoid cases, the improvement in the data quality is to some extent due to the difference in sample purity. In our original paper, the purpose of the original experiments was to demonstrate a proof-of-concept for LC--SCD (liquid chromatography--single-crystal diffraction) analysis and to show potential applications for the crystalline sponge method in the structural determination of natural products. Therefore, all the X-ray experiments were examined with microgram quantities of natural products directly isolated from orange peel, which were contaminated with irremovable impurities. It is noteworthy that the biased electron density (for example, that for **5** shown in [Fig. S3*b*](#suppinfoanchor)) nevertheless gave an acceptable fit to the model, which was constructed based on the chemical knowledge that the compound had a flavone (4H-chromen-4-one) skeleton. Although crystallographically poor, structural information gained from the electron densities is of great help for natural product chemists, particularly when the structural information is supported by or combined with other measurements such as NMR and MS. In the present study, pure chemical compounds **5**--**7** purchased from a reagent company were used. (*ii*) *Cubane*: The structure analysis of cubane (**11**) is worthy of comment. Despite the high guest occupancies in the four sites \[s.o.f. = 1, 0.55 (3) + 0.45 (3); two guests are disordered, 0.5; lie on an inversion center (multiplicity = 2), and 0.646(17)\] and the appropriate treatment of void-filling solvents, the structure analysis resulted in a seemingly worse *R* ~1~ value (0.1296). The reason for this moderate *R* ~1~ value is due to the significant one-dimensional streak-shaped residual electron densities around ZnI~2~ portions in the difference electron density map. These residual densities suggest severe one-dimensional disorder of these parts. In the reciprocal space, diffused scattering along the pore directions \[namely, (010) and (101) directions\] are found. These results suggest one-dimensional periodicity loss involving geometrical uncertainty of ZnI~2~ portions. By applying data treatment as a twinning crystal, crystallographic data was improved (*R* ~1~ reached 0.0530 and 0.0546 for each phase). Reconstructed precession images for **1** and **11** ([Fig. S15](#suppinfoanchor)) represented that no twinning was found in **1**. Guest-induced twinning might be considerable in the crystalline sponge method. (*iii*) *Others*: In our original report, one of the authors attempted the structure analysis of six compounds (**8**--**13**) using the sponge method without any additional chemical information. Although scientifically not particularly important, this attempt at *ab initio* structure analysis did allow us to learn some limitations and warnings for the method. In the analysis of **8**, for example, we were unable to distinguish whether the compound was a phenol, an aniline or a toluene. By combination with MS data, however, an aniline structure was correctly elucidated. In the updated data, the observed C*sp* ^2^---N*sp* ^3^ bond length is 1.33 (3) Å, and is clearly discriminated from the C*sp* ^2^---C*sp* ^3^ bonds in the structure \[1.57 (5) and 1.52 (4) Å\]. For compounds **8**--**13**, the soaking conditions have not been fully optimized; however, acceptable data qualities have been obtained. Presumably, the data qualities can be improved by further optimizing the soaking conditions. 2.3.. Structure analysis of an unknown molecule using the crystalline sponge method   {#sec2.3} ------------------------------------------------------------------------------------- The crystalline sponge method is applicable to determine an unknown molecule in some cases. One actual example is the structure analysis of the unexpected plasticizer (Zigon *et al.*, 2015[@bb49]). The synthesized compound is sometimes polluted by contamination of plasticizer molecules. In the previously reported crystal structure of a guest-included crystalline sponge (CCDC: 1053228), bis(2-ethylhexyl)phthalate, a kind of plasticizer, was properly modeled based on the distances of two non-hydrogen atoms and refined. The amount of this molecule in the synthesized oily guest compound was too low to detect by NMR analysis. However, the crystalline sponge method clearly shows this unexpected molecule in blind analysis. 3.. Conclusion   {#sec3} ================ The crystalline sponge method has been updated to provide a reliable technique for the X-ray structure analysis of non-crystalline compounds in minute quantities. Unoptimized protocols in every step of the method, reported in our original paper, have been thoroughly optimized to be one of an applied technique in X-ray crystallography. The application of the crystalline sponge method in various molecular-based research fields is now highly anticipated. The full optimization of all the steps for every compound is however not always easy. For practical reasons, also depending on the purpose, researchers may have to treat unoptimized data. In such cases, researchers should not forget the important major premise that *the crystalline sponge method is a part of crystallography and the supporting spectroscopic data (NMR and MS) would be helpful for determining the three-dimensional structure of target compounds in general crystallographic structure analysis.* Expected advantages of this method are (i) determination of the absolute structure of a molecule consisting of only light atoms and (ii) direct observation of the molecular structure of an oily compound. The crystalline sponge method would effectively work for those purposes. We also note that the protocols discussed here are still tentative and should be elaborated through future discussions among users and by many successful results. The crystallographic analysis of 'post-crystallized' compounds in the pores with low site-occupancies is quite an unusual situation, and one that past crystallographers have seldom experienced. Researchers may encounter crystallographic phenomena or problems, including space-group changes, the formation of superlattice structure, pseudo-symmetry problems, treatment of guests lying on symmetry elements, and treatment of guests with low site occupancies, all of which were raised upon guest inclusion into the sponge crystal and may be unfamiliar and unexpected to chemists. In addition, the treatment of solvents that fill the void space is a common yet tedious problem in MOF structure analysis (Singharoy *et al.*, 2015[@bb35]). We suggest that researchers undertake all the crystallographic steps from data collection to structure refinement with great care, through collaboration with highly experienced crystallographers. Supplementary Material ====================== Crystal structure: contains datablock(s) cyclohexane, guaiazulene, santonin, nobiletin, tangeretin, heptamethoxyflavone, diisopropylaniline, nitrobenzaldehyde, cinnamaldehyde, Dimethyl_cubanedicarboxylate, cubane_twin_1, cubane_twin_2, bromophenanthrene, vanillin. DOI: [10.1107/S2052252515024379/de5035sup1.cif](http://dx.doi.org/10.1107/S2052252515024379/de5035sup1.cif) Structure factors: contains datablock(s) Dimethyl_cubanedicarboxylate. DOI: [10.1107/S2052252515024379/de5035Dimethyl_cubanedicarboxylatesup2.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035Dimethyl_cubanedicarboxylatesup2.hkl) Structure factors: contains datablock(s) cyclohexane. DOI: [10.1107/S2052252515024379/de5035cyclohexanesup3.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035cyclohexanesup3.hkl) Structure factors: contains datablock(s) guaiazulene. DOI: [10.1107/S2052252515024379/de5035guaiazulenesup4.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035guaiazulenesup4.hkl) Structure factors: contains datablock(s) santonin. DOI: [10.1107/S2052252515024379/de5035santoninsup5.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035santoninsup5.hkl) Structure factors: contains datablock(s) nobiletin. DOI: [10.1107/S2052252515024379/de5035nobiletinsup6.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035nobiletinsup6.hkl) Structure factors: contains datablock(s) tangeretin. DOI: [10.1107/S2052252515024379/de5035tangeretinsup7.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035tangeretinsup7.hkl) Structure factors: contains datablock(s) heptamethoxyflavone. DOI: [10.1107/S2052252515024379/de5035heptamethoxyflavonesup8.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035heptamethoxyflavonesup8.hkl) Structure factors: contains datablock(s) diisopropylaniline. DOI: [10.1107/S2052252515024379/de5035diisopropylanilinesup9.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035diisopropylanilinesup9.hkl) Structure factors: contains datablock(s) nitrobenzaldehyde. DOI: [10.1107/S2052252515024379/de5035nitrobenzaldehydesup10.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035nitrobenzaldehydesup10.hkl) Structure factors: contains datablock(s) cinnamaldehyde. DOI: [10.1107/S2052252515024379/de5035cinnamaldehydesup11.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035cinnamaldehydesup11.hkl) Structure factors: contains datablock(s) cubane_twin_1. DOI: [10.1107/S2052252515024379/de5035cubane_twin_1sup12.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035cubane_twin_1sup12.hkl) Structure factors: contains datablock(s) cubane_twin_2. DOI: [10.1107/S2052252515024379/de5035cubane_twin_2sup13.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035cubane_twin_2sup13.hkl) Structure factors: contains datablock(s) bromophenanthrene. DOI: [10.1107/S2052252515024379/de5035bromophenanthrenesup14.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035bromophenanthrenesup14.hkl) Structure factors: contains datablock(s) vanillin. DOI: [10.1107/S2052252515024379/de5035vanillinsup15.hkl](http://dx.doi.org/10.1107/S2052252515024379/de5035vanillinsup15.hkl) CCDC references: [1418972](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418972), [1418974](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418974), [1418979](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418979), [1418978](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418978), [1418980](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418980), [1418976](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418976), [1418973](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418973), [1418977](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418977), [1418970](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418970), [1418971](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418971), [1442316](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1442316), [1442317](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1442317), [1418969](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418969), [1418981](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1418981) ![(*a*) Preparation of the most potent crystalline sponge \[(ZnI~2~)~3~(tpt)~2~·*x*(solvent)\]~*n*~ (**1**). (*b*, *c*) Packing views of **1** in the (*b*) \[010\] and (*c*) \[101\] directions. Solvent molecules (cyclohexane) filling the void space are omitted for clarity.](m-03-00139-fig1){#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} ![Crystal structure of the guest absorbed crystalline sponge (guest = guaiazulene, **3**). (*a*) The three-dimensional network structure (a projection down \[010\]). The host and guest are represented by stick and space-filling models, respectively. Solvents filling the voids are omitted for clarity. (*b*) Asymmetric unit structure. Three independent guaiazulene molecules (**A**--**C**), along with cyclohexane solvents are observed. Molecule **A**: observed with s.o.f. = 1 without disorder; **B**: fourfold disordered around the twofold axis with the two statistically disordered guaiazulene molecules \[s.o.f. = 0.278 (10) and 0.222 (10), respectively\]; **C**: disordered with solvents with s.o.f. = 0.186 (6). The superimposed blue and yellow colors on some atoms show the use of a PART command and represent the disordered model. (*c*) The guests **A**--**C** (and overlapping cyclohexane molecules) are individually shown.](m-03-00139-fig7){#fig7} {#fig8} ###### Molecular structures of guests **3**--**12** determined by the crystalline sponge method; our original data and the updated data are compared       Data quality[†](#tfn1){ref-type="table-fn"} -------------------- -------------------------------------------- -------- --------------------------------------------- --- \(1\) 0.0279 0.0424 \(2\) 0.0379 0.0859 \(3\) 0.1035 0.3021 \(4\) No Yes \(5\) 1.056 1.097 \(6\) 0 71 \(7\) ∼ 100% ∼ 60%[††](#tfn5){ref-type="table-fn"}           \(1\) 0.0421 0.0511 \(2\) 0.0312 0.0827 \(3\) 0.0781 0.1813 \(4\) No Yes \(5\) 1.020 1.101 \(6\) 0 120 \(7\) ∼ 100% ∼ 100% \(8\) −0.0071 (11) 0.092 (18)[‡‡](#tfn6){ref-type="table-fn"}           \(1\) 0.0332 0.0491 \(2\) 0.0808 0.1065 \(3\) 0.2136 0.2915 \(4\) No Yes \(5\) 1.197 1.052 \(6\) 5 224 \(7\) ∼ 51% ∼ 50%           \(1\) 0.0701 0.0400 \(2\) 0.0768 0.0823 \(3\) 0.1730 0.2283 \(4\) No Yes \(5\) 1.091 1.043 \(6\) 9 342 \(7\) ∼ 100% ∼ 94%           \(1\) 0.0492 0.073 \(2\) 0.0536 0.073 \(3\) 0.1545 0.2184 \(4\) No Yes \(5\) 1.033 0.893 \(6\) 224 102 \(7\) ∼ 60% ∼ 50%           \(1\) 0.0309 0.0459 \(2\) 0.0653 0.1182 \(3\) 0.1541 0.3520 \(4\) No Yes \(5\) 1.128 1.082 \(6\) 78 64 \(7\) ∼ 100% ∼ 75%           \(1\) 0.0342 0.0700 \(2\) 0.0667 0.1130 \(3\) 0.2061 0.3380 \(4\) No Yes \(5\) 1.090 1.217 \(6\) 0 49 \(7\) ∼ 100% ∼ 50%           \(1\) 0.0305 0.0558 \(2\) 0.0416 0.0750 \(3\) 0.1216 0.2631 \(4\) No Yes \(5\) 1.044 1.173 \(6\) 27 45 \(7\) ∼ 100% ∼ 50%           \(1\) 0.0605 0.1216 \(2\) 0.1296 0.1345 \(3\) 0.4390 0.3775 \(4\) No Yes \(5\) 1.574 1.082 \(6\) 18 58 \(7\) ∼ 100% ∼ 70%           \(1\) 0.0343 0.0705 \(2\) 0.0752 0.1162 \(3\) 0.2359 0.3753 \(4\) No Yes \(5\) 1.038 1.448 \(6\) 109 136 \(7\) ∼ 63% ∼ 68%           \(1\) 0.0450 0.0555 \(2\) 0.0733 0.1103 \(3\) 0.2397 0.3402 \(4\) No Yes \(5\) 1.038 1.158 \(6\) 92 9 \(7\) ∼ 100% ∼ 100% Data listed: (1) *R* ~int~; (2)*R* ~1~ \[*F* ^2^\> 2σ(*F* ^2^)\]; (3): *wR* ~2~; (4) *SQUEEZE* treatment; (5) GoF; (6) number of restraints (for the best-resolved guest); (7) occupancy (for the best-resolved guest); (8) Flack (χ) parameter (Flack value calculated by the Parsons' method). Values in the parentheses are σ level. 30% probability. Refinement was performed using *SHELXL*97 (Sheldrick, 2008[@bb33]) program. Only 500 ng of the guest was used to examine the lowest limit of the sample amount required. Classic Flack parameter.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Mammalian gene expression is regulated in *cis* by DNA sequences that serve as binding sites for proteins which support or suppress transcription either by directly interacting with transcription factors or by modifying the surrounding chromatin. Information generated by the ENCODE project \[[@pone.0124588.ref001]\], as well as similar studies have led to the chromatin profiling and functional characterization of several classes of chromatin regulatory sequences, including enhancers, promoters, and more recently chromatin insulators \[[@pone.0124588.ref002]--[@pone.0124588.ref004]\]. Regulatory elements capable of silencing gene expression have also been described in several species (for example, see \[[@pone.0124588.ref005],[@pone.0124588.ref006]\]). However, the number of silencers indentified in the mammalian genomes has been very limited, resulting in a dearth of information regarding silencer-specific chromatin profiles and mechanisms of action. Limited information suggests there are two general categories of silencers: elements that only function in a specific context to repress promoter activity (termed negative regulatory elements \[[@pone.0124588.ref005]\]); and elements that function in an autonomous, context-independent manner. Negative regulatory elements function by recruiting proteins which disrupt or inactivate the formation of functional Pol II transcription complexes at otherwise accessible promoters. This is accomplished by recruiting repressor proteins, blocking the nearby binding of activator proteins, or competing directly with activator proteins for the same binding site (reviewed in \[[@pone.0124588.ref006]\]). Autonomous silencers function by establishing a repressive chromatin state that can be stably inherited \[[@pone.0124588.ref007]\]. This is typically accomplished by recruiting proteins capable of modifying DNA (e.g. methyltransferases) or histone tails (e.g. histone deacetylases) in a manner that supports the formation of heterochromatin, or proteins that help stabilize and propagate heterochromatin (e.g. polycomb group proteins, heterochromatin protein 1). This in turn prevents activators and transcription factors from accessing gene promoters. The availability of a very limited number of autonomous mammalian silencer elements described in the scientific literature has made it difficult to identify common mechanisms of action, as well as common chromatin profiles. Advances toward indentifying and characterizing autonomous silencer elements have also been hampered by the lack of independently-validated functional assays. A question even remains as to whether silencer activity determined by transient transfection of reporter constructs can accurately predict whether a particular sequence is capable of providing silencer activity in the setting of intact chromatin. We report here the use of two functional assays based on stable transfection of K562 cells, which allow for the identification of potential silencers in the human genome. Using these assays we characterize a novel silencer element from the human genome, silencer T39, which is active in mammalian cells. We also use these stable transfection assays to functionally characterize elements previously reported to function as silencers on the basis of transient assays. Finally, we present data indicating that the T39 silencer is functional in combination with three distinct promoters and in three distinct cell lines, and that it physically interacts with other putative regulatory elements within its native locus. Materials and Methods {#sec002} ===================== Cell lines {#sec003} ---------- The following human cell lines were used for these studies: K562 (bone marrow, myelo/erythro-leukemia, ATCC CCL-243), HEK-293 (embryonic kidney, ATCC CRL-1573), HeLa (epithelial, adenocarcinoma, ATCC CCL-2), and HepG2 (liver, hepatocellular carcinoma, ATCC HB-8065). K562 cells were cultured in Iscove\'s Modified Dulbecco\'s Medium supplemented with 10% fetal bovine serum (IMDM/FBS) and the other cell lines were cultured in Dulbecco\'s Modified Eagles Medium supplemented with 10% fetal bovine serum (DMEM/FBS) at 37°C, 5% CO~2~. Drug-resistant colony assay {#sec004} --------------------------- The drug-resistant colony assay has been described previously \[[@pone.0124588.ref004],[@pone.0124588.ref008],[@pone.0124588.ref009]\]. The reporter plasmids contain an expression cassette for the G418-resistance gene *neo*, transcribed by a promoter from the human γ -globin gene HBG1 and terminated with a polyadenylation sequence from the virus SV40. High level reporter gene expression is dependent on an enhancer derived from DNase I hypersensitive site 2 (HS2) of the mouse β-globin gene cluster and placed 3′ of the *neo* expression cassette. In order to assess both silencer and enhancer-blocking insulator activity, candidate sequences are inserted immediately 5′ of the *neo* expression cassette and 3′ between the *neo* expression cassette and HS2 enhancer. In order to assess silencer activity only, candidate sequences are inserted immediately 5′ of the *neo* expression cassette and 3′ of the HS2 enhancer. Candidate sequences were inserted by MultiSite Gateway exchange \[[@pone.0124588.ref009]\]. Reporter plasmids were linearized and transfected into K562 cells at a dose of 2 μg per 10^6^ cells using the Amaxa Biosystems Nucleofector II electroporator and Nucleofector V kit (Lonza Group Ltd., Basel, Switzerland). The cells were then resuspended in 5 mL IMDM/FBS and cultured for 2 days to allow for genomic integration of the plasmid. After this, the cells were collected, counted, resuspended at a dose of 5 x10^5^ cells in 5 mL IMDM, 20% FBS, 0.8% low-melting agarose, and 1 mg/mL of the neomycin drug analog G418 (active component), and plated in 60 mm tissue culture dishes in triplicate. These semisolid cultures were incubated another 2--3 weeks and then scored for colony formation. Inserts included the PRE2-S5, MECP2 F3, PDGFA 5′SHS, and T39 putative silencers listed in [Table 1](#pone.0124588.t001){ref-type="table"}, the 1.2 kb version of the prototypic chromatin insulator cHS4 element as a positive insulator control \[[@pone.0124588.ref008]\], and a 308 bp fragment from the bacterial drug resistance gene *zeo* as a neutral spacer control \[[@pone.0124588.ref010]\]. See [S1 Table](#pone.0124588.s003){ref-type="supplementary-material"} for details on datasets. 10.1371/journal.pone.0124588.t001 ###### Putative silencers reported in the literature. {#pone.0124588.t001g} Element Genomic Coordinates Size (bp) Assay Enhancer / Promoter Reporter Topology[^(a)^](#t001fn001){ref-type="table-fn"} Control[^(a)^](#t001fn001){ref-type="table-fn"} Cells Effect Ref. --------------- ----------------------------------------- ----------- ----------------------- --------------------- ----------------------------------------------------------- ------------------------------------------------- ----------------------------------- --------------- ---------------------------- PRE2-S5 Human hg19 chr11:69378896--69379848 953 Transient Luciferase PRE1/ CCND1 P→R-E-S No E No S MCF7 50-fold \[[@pone.0124588.ref011]\] Gal4-CBX4 Yeast 9xGal4 binding site 153 Transient Luciferase none/CMV S-I-P→R With I HEK293 2-fold \[[@pone.0124588.ref016]\] MECP2F3 Human hg19 chrX:153357647--153358631 985 Transient Luciferase SV40/SV40 S-P→R-E No S SK-N-SH, HT1080 CRL1718, HeLa 10-fold \[[@pone.0124588.ref012]\] Myh6 PNR Rat rn5 chr15:37516688--37516719 32 Transient CAT none/Myh6 P-S→R S-P→R No S Cardiomyocytes, HeLa, NIH3T3, Jeg 20-fold \[[@pone.0124588.ref017]\] TSHB 3\' Human hg19 chr1:115571965--115572317 353 Transient CAT none/TSHB S-P→R No S HeLa, HEK293 GH3, TtT-97 15-fold \[[@pone.0124588.ref018]\] OVAL NRE Chicken galGal4 chr2:67779445--67779665 221 Transient CAT none/OVAL none/Tk S-P→R P→R-S No S Primary oviduct cells 8-fold 4-fold \[[@pone.0124588.ref019]\] PDGFA 5\' SHS Human hg19 chr7:560885--560915 31 Transient Luc / CAT none/PDGFA none/Tk S-P→R P→R-S No S BSC-1, HepG2 U87 2-fold 5-fold \[[@pone.0124588.ref013]\] T39 hg19 chrX:11551258--11551578 321 Stable G418^r^ colony HS2/HBG1 S-P→R-E-S Spacer K562 23-fold \[[@pone.0124588.ref004]\] ^(a)^ P→R, Promoter transcribing Reporter gene; S, silencer; E, enhancer, I, barrier insulator. GFP reporter assay {#sec005} ------------------ The GFP reporter plasmids contain an expression cassette for GFP terminated with a polyadenylation sequence from the virus SV40. This cassette is transcribed either by the same HBG1 promoter used for the drug-resistance colony assay reporter plasmid, the promoter from cytomegelovirus (CMV), or the promoter from the human PGK gene. These reporter constructs were either used directly or with the T39 silencer element inserted either 5′ or 3\' of the expression cassette. Reporter plasmids were either left circularized or were linearized with Sca I, and transfected into HEK293 cells, K-562 cells, HeLa cells, or HepG2 cells as for the *neo* colony assay, except that the cells were expanded in DMEM/FBS for 7 days without selection. The cells were then collected, washed, and analyzed by flow cytometry for reporter GFP expression. The level of expression was determined by measuring the mean fluorescence of the cells located within the GFP-positive gate. See [S1 Table](#pone.0124588.s003){ref-type="supplementary-material"} for details on datasets. Informatics {#sec006} ----------- Data involving chromatin profiles, including CTCF binding, Digital DNase footprinting, DNase I hypersensitivity, and CTCF Interactions, where derived from data publically available on the UCSC Genome Browser (<http://genome.ucsc.edu>). Track names used for analysis include the following: CTCF binding: K562 CTCF TFS ChIP-seq raw signal rep 1/2 from ENCODE/UW.Digital DNase I footprinting: K562 DNase I DGF per-base signal rep 1/2 from ENCODE/UW.DNase I hypersensitivity: K562 DNase I HS raw signal rep 1/2 from ENCODE/UW.CTCF interactions: K562 CTCF ChIA-PET Interactions rep 1/2 form ENCODE/GIS. Results {#sec007} ======= Experimental Approach {#sec008} --------------------- In our search for enhancer-blocking insulators from the human genome, we used reporter constructs in which candidate sequences were placed between an expression cassette for the drug-resistance gene *neo* and a potent enhancer ([Fig 1A](#pone.0124588.g001){ref-type="fig"}) as described previously \[[@pone.0124588.ref004],[@pone.0124588.ref008],[@pone.0124588.ref009]\]. If the insert contains an enhancer blocking insulator, it will disrupt communication between the enhancer and promoter, resulting in a decreased level of *neo* gene expression and a decreased rate of colony formation when transfected K562 cells are grown in a semisolid culture in the presence of neomycin drug analog G418. However, this assay cannot distinguish between enhancer-blocking insulators and elements capable of silencing gene expression, since both activities cause a decrease in *neo* gene expression. To distinguish between these two activities, it is necessary to use a second assay in which the candidate element is instead used to bracket the enhancer and *neo* gene expression cassette from the outside ([Fig 1B](#pone.0124588.g001){ref-type="fig"}) as described previously \[[@pone.0124588.ref004],[@pone.0124588.ref008]\]. In this configuration, enhancer-blocking insulators are no longer capable of blocking enhancer-promoter interactions and thus have no effect on *neo* gene expression or the rate of drug-resistant colony formation. In contrast, elements capable of silencing gene expression should still be capable of reducing *neo* gene expression in this setting, resulting in a reduction in the rate of drug-resistant colony formation. {#pone.0124588.g001} Initial characterization of the T39 silencer {#sec009} -------------------------------------------- Based on previous studies \[[@pone.0124588.ref004]\], we expanded our search for enhancer-blocking insulators to include sequences from the human genome that contain multiple CTCF binding motifs located within 50 to 200 bp from each other. In addition, we required that these motifs belong to classes with over 50% probability of being occupied by CTCF as determined by ChIP-seq as previously described \[[@pone.0124588.ref004]\]. One of the sequences we tested, designated T39, was found to exhibit very strong activity in the enhancer-blocking assay. In the experiments shown in [Fig 2A](#pone.0124588.g002){ref-type="fig"}, the frequency of colony formation for the insulator assay construct containing T39 was only 2% of that for the neutral spacer control, compared to 40% for the prototypic enhancer-blocking chromatin insulator cHS4. However, as seen in [Fig 2B](#pone.0124588.g002){ref-type="fig"}, the T39 element also decreased the rate of colony formation to 2% of that for the neutral spacer control in the silencer assay. In comparison, the cHS4 insulator had no statistically distinguishable effect on colony formation in this assay. Taken together, these studies demonstrate that the T39 element is a powerful silencer element. {ref-type="supplementary-material"} for details on datasets.](pone.0124588.g002){#pone.0124588.g002} The T39 element is located on the short arm of the X chromosome (p22.2) in the first intron of the gene ARHGAP6 ([Fig 3A](#pone.0124588.g003){ref-type="fig"}). The sequence of T39 contains three CTCF motifs, all from the same CTCF class 38, which exhibits an average CTCF occupancy of 90.79% \[[@pone.0124588.ref004]\]. Genomic profiles of the T39 sequence suggested that all three sites are bound by CTCF in K562 cells ([Fig 3B](#pone.0124588.g003){ref-type="fig"}), and that binding by CTCF results in distinct footprints detected by digital DNase I genomic footprinting analysis in K562 cells ([Fig 3C](#pone.0124588.g003){ref-type="fig"}). These properties of the T39 element appear to be ubiquitous, since strong peaks of CTCF binding and DNase hypersensitivity are seen in 19 independent cell lines \[[@pone.0124588.ref004]\]. ![Genomic properties of the native T39 locus.\ (A) Chromosome location. The location of the T39 element is shown within the body of the ARHGAP6 gene at p22.2 of the human X chromosome. Below this is shown the primary sequence of the T39 element used for functional studies, with the consensus sequences for the three class-38 CTCF-binding motifs indicated in underlined bold type face (see \[[@pone.0124588.ref004]\] for details of CTCF-binding motif classes). (B) CTCF ChIP-seq profile for the native T39 sequence in K562 cells. The density of ChIP-seq reads are presented as vertical bars. The location of the CTCF binding motifs are indicated underneath the histogram. The putative distribution of CTCF binding is shown as shaded curves centered on each CTCF binding motif. (C) Digital DNase footprint profile for the native T39 sequence in K562 cells. The density of single-base DNase-seq reads are presented as vertical bars. The location of the CTCF binding motifs are indicated underneath the histogram.](pone.0124588.g003){#pone.0124588.g003} Comparison of T39 with other silencers from the literature {#sec010} ---------------------------------------------------------- A partial list of silencers from the literature appears in [Table 1](#pone.0124588.t001){ref-type="table"}. These silencers have been identified in cells of various species using transient reporter assays based on expression of luciferase, chloramphenicol acetyltransferase, or both. Silencer activity was uniformly assessed by transient transfection and ranged from 2-fold to 50-fold. For comparison with T39 we selected three silencers. PRE2-S5 is a silencer studied in a breast cancer cell line (MCF7) and was shown to reduce expression 50-fold compared to a no-insert control \[[@pone.0124588.ref011]\]. MECP2 F3 is a silencer active in several cell lines and was shown to reduce expression an average of 10-fold compared to a no-insert control \[[@pone.0124588.ref012]\]. PDGFA 5′ SHS is a silencer active in several cell lines and was shown to reduce expression 2- to 5-fold compared to a no-insert control \[[@pone.0124588.ref013]\]. When assessed using the enhancer-blocking assay described in [Fig 1A](#pone.0124588.g001){ref-type="fig"}, the PRE2-S5 element reduced the frequency of G418-resistant colony formation 2.4-fold compared to the neutral spacer control, while the MECP2 F3 and PDGFA 5\' SHS elements had no statistically distinguishable effect on the rate of G418-resistant colony formation ([Fig 2A](#pone.0124588.g002){ref-type="fig"}). In comparison, the cHS4 element used as an insulator control reduced G418-resistant colony formation 2.5-fold, and the T39 silencer reduced G418-resistant colony formation 77-fold. When assessed using the silencer configuration, the PRE2 element reduced the frequency of G418-resistant colony formation 2.2-fold compared to the spacer control, while the MECP2 element had no statistically distinguishable effect on the rate of colony formation and the PDGFA element actually increased the rate of colony formation ([Fig 2B](#pone.0124588.g002){ref-type="fig"}). In this configuration, the cHS4 insulator had no statistically distinguishable effect on the rate of G418-resistant colony formation as expected, while the T39 silencer reduced the rate of colony formation 36-fold. Taken together, these studies indicate that PRE2-S5 functions as an autonomous silencer, while MECP2-F3 and PDGFA 5′ SHS do not function as silencers in this integration-based assay. These studies also demonstrate that the T39 element is a far more potent autonomous silencer than the PRE2-S5 element. Chromatin profiling of the PRE2, MECP2, and PDGFA native loci revealed the absence of constitutive DHSs in five independent cell lines used in their original characterizations while the native T39 loci exhibited strong DHS peaks in all five of these cell lines ([S1 Fig](#pone.0124588.s001){ref-type="supplementary-material"}). These profiles suggest only the T39 element should be expected to exhibit constitutive activity. Silencer activity is marginally influenced by context {#sec011} ----------------------------------------------------- In theory, autonomous silencer elements should be capable of exerting their activity on different sources of promoters and within multiple cell lines. The G418-reisistant colony assay and associated reporter construct could not be easily used to assess these properties, since the HS2 enhancer does not function with all promoters, the HBG1 promoter does not function in all cell types, and not all cell types are capable of forming distinct colonies in agarose. For these reasons, we turned to the reporter construct diagramed in [Fig 4A](#pone.0124588.g004){ref-type="fig"}. This construct links various promoters to a GFP expression cassette, with candidate insulators inserted immediately 5\' of the promoter. In this assay, the degree of silencing is assessed by comparing the intensity of reporter GFP expression in GFP-positive cells following stable plasmid transfection. As with the drug-resistance colony assay, this GFP reporter assay provides an opportunity to detect silencer activity in the context of stable integration and intact chromatin. However, the use of GFP provides for a very wide dynamic range that makes it possible to assess reporter gene expression from a wide range of promoters and in a wide range of cell types. Using this reporter system in easily transfectable HEK293 cells, we found that the T39 element was capable of silencing the activity of three very distinct promoters: the HBG1 promoter, a constitutive promoter from cytomegalovirus (CMV), and the constitutive promoter from the human PGK gene ([Fig 4B](#pone.0124588.g004){ref-type="fig"}). The degree of silencing was nearly uniform, ranging from 1.5-fold for CMV to 1.9-fold for HBG1. {ref-type="supplementary-material"} for details on datasets.](pone.0124588.g004){#pone.0124588.g004} We noted that the degree of silencing achieved with the HBG1 promoter in this setting was far less than the 36-fold observed with the same silencer and promoter in the drug-resistant colony assay. We believe this is due to the absence of the HS2 enhancer in the GFP reporter construct, and the use of a non-erythroid cell line, resulting in a substantial reduction in the basal activity of this promoter. This in turn restricts the dynamic range available for detecting the effect of a silencer. Taking this into account, we chose the reporter construct with the highly active CMV promoter for further studies ([Fig 5A](#pone.0124588.g005){ref-type="fig"}). As summarized in [Fig 5B](#pone.0124588.g005){ref-type="fig"}, we found that the T39 element silenced this promoter in erythroid K-562 cells, epithelial HeLa cells, and HepG2 liver cells. In this case the degree of silencing ranged a little more broadly, from 1.2-fold in K-562 cells to 1.9-fold in HeLa cells. In order to determine whether the silencing activity of T39 was affected by reporter construct topology, we placed this element either 5\' only or 3\' only of this CMV-GFP expression cassette ([Fig 5C](#pone.0124588.g005){ref-type="fig"}), and analyzed the effects on reporter GFP expression following transfection of HepG2 cells. As a further control, we also compared circular versus linear plasmids, since the circular topology would, in effect, place the T39 element both 5\' and 3\' of the CMV-GFP expression cassette. As seen in the new [Fig 5D](#pone.0124588.g005){ref-type="fig"}, the T39 element was equally effective at silencing expression when located in either position, with an average degree of silencing of 1.6-fold when the reporter was circular and 2.5-fold when the reporter was linear. Genomic profiling demonstrated that the T39 element is both bound by CTCF and contains a DHS in all three of the cell lines used for these studies ([Fig 6](#pone.0124588.g006){ref-type="fig"}). {ref-type="fig"}, except in this case only the constructs using the CMV promoter with and without the T39 silencer were compared. (B) Assessment of reporter expression in different cell lines. Reporter plasmids were linearized and transfected into the indicated cell lines. After 7 days, the level of reporter gene expression was analyzed by measuring the mean fluorescence of the cells expressing GFP by flow cytometry. (C) GFP reporter constructs for assessment of silencer topology. The reporter plasmids are the same as in panel A, except the T39 silencer was inserted either 5\' only, or 3\' only, of the CMV-GVP cassette. (D) Assessment of reporter expression for assessment of silencer topology. Reporter plasmids were left circular or were linearized and transfected into HepG2 cells. After 7 days, the level of reporter gene mean fluorescence was assessed by flow cytometry as for panel B. Data are shown for the mean ± s.e. from three independent transfections, and are normalized to the mean fluorescence of the construct without the T39 silencer. P values based on one-sided *t*-test comparing constructs with and without the T39 silencer for each cell line. See [S1 Table](#pone.0124588.s003){ref-type="supplementary-material"} for details on datasets.](pone.0124588.g005){#pone.0124588.g005} {#pone.0124588.g006} Discussion {#sec012} ========== The existence of transcriptional silencers has been recognized in the literature for nearly three decades (for example, see \[[@pone.0124588.ref014]\]). However, relatively few silencer elements have been identified and functionally characterized in mammalian cells. A survey of the literature revealed the examples summarized in [Table 1](#pone.0124588.t001){ref-type="table"}. They include silencer elements derived from humans, rats, chickens, and yeast, with sizes ranging from 31 bp to 985 bp. The activities of some of these elements were assessed using the promoters and topological arrangements derived from their native loci, making it difficult to distinguish between context-dependent negative regulatory elements and context-independent autonomous silencers. The activities of other elements were assessed using heterologous promoters, enhancers, and topological arrangements in a manner that did allow an assessment for context-independent, autonomous silencer activity. Only the T39 element was studied in the context of stable gene transfer, where the reporter constructs were allowed to integrated into the target cell genome in a manner that allowed for the formation of intact chromatin; all of the other elements were studied with transient transfection assays. In addition, only the studies with the T39 element included the use of a neutral \"spacer\" sequence as a control, making it possible to distinguish between the direct effects of the candidate silencer element and the indirect effects of changes in spacing between enhancers, promoters, and other regulatory elements. Of the three elements we identified from the studies of others and analyzed in the drug-resistance colony assay, only PRE2-S5 \[[@pone.0124588.ref011]\] exhibited autonomous silencer activity. It remains unclear why the other two elements, MECP2 F3 \[[@pone.0124588.ref012]\] and PDGFA 5′ SHS \[[@pone.0124588.ref013]\], failed to exhibit autonomous silencer activity in the drug-resistant colony assay. Both of these elements had been shown by others to function in the context of heterologous promoters, but they were not previously tested with the HBG1 promoter used in this particular assay. Likewise, neither of these elements had been previously tested in erythroid K562 cells, although they had been shown to function in several other cell lines. Perhaps most importantly, neither of these two elements had previously been tested in the context of stable gene transfer, where they would be required to function in the context of intact chromatin. Chromatin profiling suggests that these elements are not constitutively active and thus unlikely to function in a constitutive manner. Regardless to the reasons for the discrepancies between our results and the results of others, our studies demonstrate that these two elements do not function as fully autonomous silencers. The T39 element functioned as the most potent autonomous silencer out of the four elements analyzed here. As reported here, the T39 silencer reduced the rate of G418-resistant colony formation 77-fold in the enhancer-blocking configuration and 36-fold in the silencer configuration. Although less dramatic, this element also reduced expression of the GFP reporter constructs in the context of three distinct promoters and three distinct cell lines. In addition, this silencing activity can be achieved when T39 is used as a single copy, and when the T39 element is placed either 5\' or 3\' of an expression cassette. From these studies we conclude the T39 element acts as a potent, constitutive, autonomous silencer. The T39 silencer exhibited statistically significant silencer activity in both of the drug-resistant colony assays and the GFP expression assay. However, the magnitude of this effect varied greatly between these two assay systems. Previous studies demonstrated that the drug-resistant colony assay measures the frequency of transfection events in which expression of the *neo* reporter gene exceeds a specific threshold \[[@pone.0124588.ref003]\]. This property allows the drug-resistant colony assay to amplify small differences in gene expression. The GFP expression assay, on the other hand, allows for a direct and linear measurement of reporter gene expression in a manner that does not amplify small differences. In addition, the drug-resistant reporter constructs contain two copies of the silencer element in a flanking arrangement, while the GFP reporter construct only contains one copy of the silencer element. This represents a difference both in the silencer dose and the ability of the silencer to block interactions between tandemly integrated reporter constructs or interactions between integrated reporter constructs and the surrounding genome. The T39 silencer exhibits a distinct chromatin profile, including high occupancy by CTCF and DHS formation in multiple tissues. This silencer is located in the first intron of the ARHGAP6 gene. It has been suggested that silencers located in introns could suppress transcription by blocking transcriptional elongation, by preventing recognition of intronic splice sites or by abrogating basal transcriptional apparatus assembly \[[@pone.0124588.ref005],[@pone.0124588.ref006],[@pone.0124588.ref007]\]. Only the third of these mechanisms could potentially explain the silencing function of T39 in the stable transfection assays we have used. Publically available CTCF interactome data indicate that the T39 element physically interacts with other CTCF sites located at the terminus of the ARHGAP6 gene and within the neighboring gene MSL3, but not with the promoters of these genes ([S2 Fig](#pone.0124588.s002){ref-type="supplementary-material"}). It is of interest to speculate that the T39 silencer may modulate the expression of MSL3, a gene contributing to the X-inactivation complex of Drosophila and humans \[[@pone.0124588.ref015]\]. Future addition/deletion studies will be needed to specifically link CTCF binding to the silencing activity of the T39 element, as has been done in other settings \[[@pone.0124588.ref020]\]. In conclusion, our studies independently validated two integration-based reporter assays for the identification of silencer elements, and use these assays to validate two autonomous silencer elements: PRE2-S5 and T39. The drug-resistant colony assay provides the greatest degree of sensitivity, while the GFP reporter assay allows for a more direct assessment of silencer activity and provides a greater degree of experimental flexibility. These studies also suggest that the T39 sequence functions as a potent, constitutive autonomous silencer in a manner that is independent of the promoter or cell line. This element can be used as a positive control for studies into other silencer elements, as well as chromatin insulators. More studies are needed to dissect the mechanisms underlying silencer function, and to link these activities to specific chromatin profiles. Supporting Information {#sec013} ====================== ###### DHS profile for putative insulators in multiple cell lines. The DNase I hypersensitive site (DHS) profiles are presented for the four putative insulators in the 5 indicated cell lines. The four putative silencer elements T39, PRE2- S5, MECP2-F3, and PDGFA 5′ SH5, are described at length in the main manuscript. The five cell lines were chosen based on their use in the previous characterization of the putative insulators and the availability of publicly accessible DHS profiles. The density of DNase-seq reads are presented as vertical bars, with gray horizontal bars indicating the presence of statistically significant DHS peaks. The locations of the putative silencer sequences are indicated underneath the histograms. Each window is set at 1000 bp. (PDF) ###### Click here for additional data file. ###### CTCF interactome for the native T39 locus. The image shows the genomic organizations of a 710 kb window around the native T39 locus in K562 cells, derived from publically available data on the UCSC Genome Browser. The location of the T39 element and three surrounding genes (ARHGAP6, AMELX, and MSL3) are indicated. Also indicated are the promoters for these genes (P). The middle two tracks show the CTCF and DHS profiles for the region. At the bottom is indicated the CTCF interactome for the region, as determined by chromatin capture technologies. Interactive regions are connected by red arches, with the intensity of the red representing the frequency of the interaction. Note that T39 interacts with a DHS/CTCF element inside the same ARHGAP6 intron, with a DHS/CTCF element at the distal end of the ARHGAP6 gene, and with a DHS/CTCF element in the intron of the adjoining MSL3 gene, but not with any of the gene promoters. (PDF) ###### Click here for additional data file. ###### Summary of Raw Data. (PDF) ###### Click here for additional data file. The authors wish to thank G. Felsenfeld and D.M. Bodine for reagents and help with the plasmid-based insulator and silencer assays. [^1]: **Competing Interests:**Author DWE is an employee of WIRB-Copernicus Group. This employment status does not alter the authors\' adherence to PLOS ONE policies on sharing data and materials. [^2]: Conceived and designed the experiments: HQ ML DWE GS. Performed the experiments: HQ ML. Analyzed the data: HQ ML DWE GS. Contributed reagents/materials/analysis tools: HQ ML. Wrote the paper: HQ ML DWE GS.