Herpes simplex virus (HSV) is the most common cause of sporadic encephalitis worldwide. The high mortality rate (70–80 %) of herpes simplex encephalitis (HSE) can be reduced to 20–30 % by antiviral therapy. However, normocellular CSF can lure physicians to look for non-infectious causes, resulting in delayed treatment. This study aimed to investigate, characterize and differentiate HSE patients, with normocellular and pleocytosis CSF, according to neuroimaging patterns, underlying disease, CSF viral load and clinical outcome. Patients with proven (by PCR positive CSF) or presumed viral infections of the CNS admitted to King Chulalongkorn Memorial Hospital between January 2002 and 2011 were analyzed.3). One patient in this normocellular CSF group had HIV infection. Although this patient had low CD4 counts (<200 cells/mm3), the peripheral WBC counts showed only mild leukopenia. The CSF HSV viral load in the pleocytosis group was higher than the normocellular group, with an average of 12,200 vs 3027 copies/ml respectively. There was no correlation between the viral load and the clinical outcome. With respect to neuroimaging, 4 (66.7 %) patients in the normocellular group had unremarkable/non-specific results.HSV was detected in the CSF of 43 patients but only 23 patients had encephalitis. Among these 23 patients, 6 cases (26.1 %) had normal CSF WBC (<5 cells/mmNormocellular CSF in HSE is not rare, and can be seen in normal as well as immunocompromised hosts. Clinicians should not exclude CNS infection, especially HSE, merely based on the absence of CSF pleocytosis and/or unremarkable neuroimaging study. Herpes simplex encephalitis (HSE) caused by herpes simplex virus (HSV) is the most common sporadic viral encephalitis among adults worldwide , 2. It hHSV-1 is responsible for more than 90 % of HSE in adults . Recent HSV can reside in the nerve and can either replicate or establish a latent infection . Its DNAThe cerebral inflammation from glial cells activation, due to the presence of HSV, includes increase in expression of pattern recognition receptors, especially Toll-like receptors (TLRs) and interferon regulatory factors (IRFs) . GeneticThe characteristic symptoms of HSE are abnormal behavior/psychosis (66 %), confusion/disorientation (81 %) and speech disturbance (66 %), which are justifiable due to the preferential involvement of the frontal and temporal lobes. Other symptoms of HSE include fever (76–90 %), headache (70–90 %) and seizure 50–55 %) . Most of HSE patients were previously healthy except 2 (8.7 %) with diabetes, 2 (8.7 %) hematologic malignancies, 1 (4.3 %) inactive SLE, 1 (4.3 %) multiple sclerosis (MS) and 1 (4.3 %) seropositive for human immune deficiency virus (HIV). Most of the patients were from central Thailand. The patient with HIV infection was diagnosed after hospitalization with acute HSE. Prior to hospitalization, the patient had been in good health, never received any highly active antiretroviral therapy (HAART), and did not have any opportunistic infection. The patient’s CD4 count was 200 cells/mmOf 23 HSE patients, 16 cases (69.6 %) had fever as a prodrome. The most common presenting symptoms were seizures , behavioral changes , alteration of consciousness , focal neurological deficits , and worsening of headaches . All patients received intravenous administration of acyclovir. Six patients (26.1 %) recovered completely , 14 (60.9 %) partially recovered (mRS 2–3), and 3 patients (13 %) died. Demographic data, underlying diseases, and presenting symptoms of each group, classified according to CSF WBC count, are presented in Table 3, with lymphocytic predominance.All 23 cases had serum creatinine in the range of 0–3 mg/dl and normal liver function test. Complete blood count data was available in 20 patients, of whom 14 70 %) were within the normal limit, 1 (5 %) had pancytopenia (patient no. 17 in pleocytosis group), and 1 (5 %) had leucopenia (patient no. 6 in normocellular group). Seventeen of 23 patients (73.9 %) had CSF examination within 2–10 days after neurological symptoms onset, the golden period for CSF PCR examination for HSV [0 % were 3). One patient in this normocellular CSF group (16.7 %) had HIV infection. Although this patient had low CD4 counts (<200 cells/mm3), the peripheral WBC counts were 3150 and 3570 cells/mm3 in two consecutive tests.Six cases (26.1 %), with CSF examinations within 10 days after onset of illness (an average of 3 days), had normal CSF WBC (<5 cells/mm3), 3 of whom had CSF WBC count greater than 500 cells/mm3. Thirteen patients (76.5 %) were previously healthy. None of them had HIV infection. CSF HSV viral load in this group was higher than the normocellular group, with an average of 12,200 vs 3027 copies/ml respectively. Of all 14 specimens received after 2007, 10 cases  were positive for HSV1 and 4 cases  were positive for HSV2 encephalitis. Other CSF parameters are described in Table Seventeen patients had CSF pleocytosis with CSF WBC ranging between 15 and 730 cells (mean 179 cells/mmTwenty-two patients had complete neuroimaging (either computer tomography (CT) or MRI) records. Eleven (50 %) patients had the predominant temporal or fronto-temporal lobe lesions and 2 (9.1 %) had fronto-temporal lesion coexisting with thalamic lesions. Another two patients had cortical/subcortical  lesions. Two patients (9.1 %) had bead-like appearance resembling vasculitis on MR angiography, whereas 5 (22.7 %) had normal or non-specific findings. Brain imaging data altogether with CSF HSV viral load of normocellular and pleocytosis groups are presented in Tables In this study, 6 of 23 HSE patients 26.1 %) had normal CSF WBC counts as shown in Tables  % had noOur study showed that HSE with normocellular CSF is more common than what was generally known in the past. The CSF protein levels, number of cases with abnormal neuroimaging, and CSF viral loads in the pleocytosis group were higher when compared to the normocellular group. It was noted that CSF viral load was up to four times higher in CSF pleocytosis group than normocellular group , potentially due to increased inflammatory response in pleocytosis group. Viral load alone may not entirely explain the magnitude of CSF cellular response, as patients with nonspecific MRI in the normocellular group also had high copies of viral load. When looking at individual data in normocellular group Table , the patA retrospective study of 35 HSE patients in Spain over a 15 year period revealed that 8 (22.8 %) had normal CSF WBC counts . HoweverOur study had some limitations, such as small sample size, and lack of complete data in some subjects. However, the study also revealed important data that directly affects the clinical management of HSE. An absence of CSF pleocytosis and/or MRI abnormalities does not exclude infectious encephalitis, in particular HSE. Additionally, examination of HSV DNA by PCR in the CSF is mandatory in the management of encephalitis. Our study indicates that relying on the CSF pleocytosis and/or presence of classical MRI features of HSE may result in a delay in diagnosis and treatment. A larger study is further required to confirm results statistically and analyze inflammatory responses in normocellular HSE, when compared to pleocytosis group, to better understand the pathophysiology of HSE.Absence of CSF pleocytosis in HSE has been described as a rare finding in the past, which can distract physicians from the veritable diagnosis. Our study intended to demonstrate the extent to which normal CSF WBC findings are found in encephalitis associated with HSV. It is not uncommon, and can be found in previously healthy patients. Therefore, clinicians should not exclude CNS infection  based on the absence of CSF pleocytosis and normal neuroimaging.


The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug  terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and contributes to the increase of morbidity and mortality in the aging population around the world356Kur , IKACh (acetylcholine-activated K+ current) and also small conductance calcium-activated potassium current  are predominantly expressed in atria and are considered to be atrial-selective targets for developing atrial selective anti-AF drugs;28Snow lotus), can uniquely inhibit atrial potassium currents including atrial IKur and IKACh, as well as cardiac Ito (transient outward potassium current)1011Ca channels expressed in HEK 293 cells10Several atrial-selective ion channel currents including Iin vivo, and can effectively terminate AF induced by vagal nerve stimulation in beagle dogs.The prodrug strategy is commonly employed to overcome poor water solubility of hydrophobic drugs in the early stage of drug discovery, especially those that cannot perform very well by formulation strategies. To improve the water solubility of parent drug in the prodrug design, the conjugation of some polar functional groups, e.g. carboxylic, hydroxyl, amine, phosphate, carbonyl, or thiol groups directly or through a linker motif, is a popular choice151630H25O8P (544.49). EI-MS: m/z 545.14 [M+]. 1H NMR : δ = 12.81 , 7.83 , 7.03 , 6.84 , 6.62 , 6.54 , 5.19 , 5.17 , 3.91  ppm. The NMR spectra of compound 2 is shown in Acacetin prodrug was synthesized through three steps as shown in 16H13O8P (364.24). EI-MS: m/z 365.04 [M+], 285.07 [M + H-P(O)(OH)2]+. 1H NMR : δ = 12.91 , 8.06 , 7.11 , 7.01 , 6.96 , 6.62 , 3.87  ppm. 13C NMR : δ 182.04, 163.74, 162.47, 161.15, 159.58, 156.54, 128.37, 122.74, 114.63, 106.05, 103.83, 102.64, 97.64, 55.55 ppm. The NMR spectra of compound 3 is shown in 1H NMR , phosphate ester of acacetin (compound 3), and acacetin prodrug (compound 4) was determined in HKur and IKACh) as well as cardiac Ito1011Kur) or hKv4.3 gene (encoding for Ito), and in rat atrial myocytes. KACh elicited by 5 μ M carbachol in the absence and the presence of 10 μM acacetin (1011KACh (KACh (at −100 mV). Acacetin (10 μM) decreased hKv1.5 current (measured from 0 to the current at end of the voltage step) by 79.7 ± 4.1% , hKv4.3 current (measured from 0 to the current peak) by 69.3 ± 5.1% , and IK.ACh (measured carbachol sensitive current from 0 to the current at end of voltage step) by 66.5 ± 3.1% . However, no significant inhibition of hKv1.5, hKv4.3, or IK.ACh was observed with 30 μM acacetin prodrug . One the other hand, our previous study showed that 30 μM acacetin had ~50% inhibition of hERG current1518ex vivo and needs to be converted into the parent active compound by a specific enzyme. The bioconversion of acacetin prodrug ex vivo and in vivo was verified as below.To determine whether acacetin prodrug, as the parent compound acacetin, would exert inhibitory effects on atrial-specific potassium currents , Tmax (time to reach Cmax), T1/2 , CL (systemic clearance), AUC∞ (area under the respective first moment–time curve from time zero to infinity), AUClast, and other pharmacokinetic parameters of acacetin are summarized in The pre-pharmacokinetic profile of acacetin was analyzed with the program  in beagle dogs with an intravenous bolus of acacetin prodrug 6 mg/kg, . The sta−α∙T + Be−β∙T, in which the A is zero-time intercept of the distribution slope in the compartment model; the B is zero-time intercept of decline in plasma concentration of drug; the α is distribution rate constant; the β is elimination constant. The CL obtained from non-compartmental and compartmental pharmacokinetic analysis is comparable, as is 325 and 287 mL/min/kg, respectively. These results indicate that the metabolization and/or elimination of acacetin are rapid in beagle dogs, and continuous infusion is required for anti-AF experiments.The concentration-time curve of acacetin was best fitted to a two-compartmental model, which could be expressed as: concentration (acacetin) = AeIn anti-AF experiment one third dose bolus of acacetin prodrug was applied in 2 min to obtain the immediate therapeutical concentration followed by continuous infusion of two third dose to maintain the potential effective therapeutic concentration. Three doses of acacetin prodrug  were employed for anti-AF experiments in beagle dogs.th repeat (vs. vehicle) and 71.4%  in animals treated with 3, 6 and 12 mg/kg acacetin prodrug, respectively. The AF duration in animals with effective AF termination was not significantly different in animals with different dosages of acacetin prodrug . In addition, AF induction was repeated at 40 min after discontinuous prodrug infusion. AF was terminated at 25.3 ±1.8 min after re-induction (P < 0.01 vs. vehicle) in these animals (n = 15) with drug treatment, but not in animals treated with 5% glucose vehicle (30 min duration). However, the duration of AF was longer than during the initial drug administration . These results indicate that intravenous infusion of acacetin prodrug can effectively terminate the AF in beagle dogs induced by vagal nerve stimulation and burst atrial pacing.The anti-AF effect of acacetin prodrug was investigated in AF model (with AF duration of 30 min), which was established in beagle dogs with modified procedure as described previously1014h repeat . Acacetih repeat . Blood sQTc (corrected QT) interval of ECG was analyzed in anesthetized beagle dogs before and at 35 min after drug administration. QTc interval was not altered  in vehicle animals  and animals treated with 3 mg/kg , 6 mg/kg , or 12 mg/kg  acacetin prodrug. The results indicate that acacetin prodrug has no effect on QT interval.The plasma concentration profile of acacetin was determined with LC/MS/MS (liquid chromatography-tandem mass spectrometry) in animals with AF treatment. 50) calculated using Bliss method2250 with 95% confidence level was 685.1 to 760.2 mg/kg. By translating the dose based on body surface area2450 would be 108.3 mg/kg in dogs, which is much greater than the therapeutic dose of 3~12 mg/kg in beagle dogs. These results indicate that acacetin prodrug is a highly promising safe drug candidate with a wide therapeutic window to treat AF.The experimental record is illustrated in in vivo chemical or enzymatic transformation. Thousands of patents were granted for the development of prodrugs, and more and more prodrugs have been approved for clinical application in treating different disordersex vivo beneficial effectsProdrug is a well-established versatile strategy which provides possibilities for bioactive compounds to overcome various barriers to drug formulation and delivery, e.g. poor aqueous solubility, chemical instability, insufficient oral absorption, rapid pre-systemic metabolism, inadequate brain penetration, toxicity and local irritation, and also improves drug targeting of existing drugs with improved properties15in vivo, the stability of the prodrug and importantly the original pharmacological effect. The parent drugs could be modified by a polar, most often an ionizable promoiety, to increase water solubility. Phosphate esters-based prodrugs show good chemical stabilityin vivo via alkaline phosphatases and are widely used to synthesize prodrugs for improving the aqueous solubility, not only for oral drugs, but also for drugs intended for parenteral administration (e.g. intravenous administration)15181529There are several considerations in the design of “developable” prodrugs, including the kinds of functional groups involved, the complete bio-conversion rate 2O and clinically acceptable solutions of 5% glucose and 0.9% NaCl. Because acacetin is a unique flavone with selective blockade of cardiac K+ currents101112ex vivo cell models. As many other prodrugs1518ex vivo pharmacological tests. It (30 μM) had no blocking effect on hKv1.5, hKv4.3, or IKACh, whereas acacetin at 10 μM remarkably inhibited these ionic currents. The bioconversion was therefore required for the prodrug into the parent compound. Indeed, we observed that acacetin prodrug was gradually converted into acacetin in ex vivo incubation (37 °C) of rat plasma. The quick conversion was also observed in rat microsome incubation or in whole-animal (beagle dog) administration.The aqueous solubility of acacetin, as other flavone compoundsKur and IKACh, as well as cardiac Ito. Intravenous infusion of acacetin prodrug (3~12 mg/kg) terminated AF induced by atrial bursting pacing and vagal nerve stimulation with efficacy of 50~75% and the effect can still be observed 40 min after discontinued drug infusion. This suggests that acacetin prodrug may be a promising drug candidate that can be used in clinic to treat patients with acute AF.With intravenous administration of acacetin prodrug, the pre-pharmacokinetic profile of acacetin analysed with the standard non-compartmental and compartmental parameters showed that the metabolization and/or elimination of acacetin are rapid in beagle dogs. We therefore infused continuously acacetin prodrug in anti-AF experiment, in which the dose regimen was made with one third dose bolus within 2 min to obtain the immediate therapeutical concentration followed by continuous infusion of two third doses within 18 min to maintain the effective therapeutic concentration of acacetin to inhibit atrial I50 of 721 mg/kg with intravenous administration. With the dosage translation based on the body surface area50 in dogs would be 108 mg/kg (i.v.), which is far less than the therapeutic doses (3~12 mg/kg), suggesting that acacetin prodrug is a safe drug candidate.It is interesting to note that acacetin prodrug had no effect on QTc interval. On the other hand, the acute toxicity assessment in mice demonstrated a high LDOne of limitations was that the acacetin prodrug underwent a rapid metabolism after a bolus injection. The intravenous infusion application may maintain consistent therapeutic plasma levels; however, more effort is required pharmaceutically to slow the metabolic release from the prodrug. In addition, though rapid atrial burst pacing-induced AF model with vagal nerve stimulation is widely used to test the efficacy of anti-AF drugs, it should be noted that this model may not represent general mechanisms of AF in humans. More effort is required for a next step to test efficacy of the acacetin prodrug against AF induced by other AF models and to obtain more comparable data for a clinical scenario. Nonetheless, these would not affect the conclusion that the synthesized water-soluble prodrug of acacetin is effective in terminating AF induced by vagal nerve stimulation.The present preclinical study demonstrated for the first that the synthesized highly water-soluble acacetin prodrug could be converted into the parent compound acacetin and exert an effective action in terminating the experimental AF induced by atrial burst pacing with vagal nerve stimulation in beagle dogs. Acacetin prodrug may be a promising drug candidate for clinic trial in treating patients with acute AF.4, followed by mixture evaporation, and column chromatography on silica gel using n-hex/EtOAc/CH2Cl2. Phosphate ester of acacetin  was synthesized with acacetin  with high performance liquid chromatography (HPLC) as described in The aqueous solubility of acacetin, the intermediate phosphate ester of acacetin, or acacetin prodrug was determined at room temperature (23–25 °C) in dHKACh was determined in isolated rat atrial myocytes as described previously1231Whole-cell currents of hKv1.5 and hKv4.3 were recorded in established HEK 293 cell lines stably expressing hKv1.5 (KCN5A) and hKv4.3 (KCND3) and Iex vivo bioconversion of acacetin prodrug was determined in rat plasma or rat hepatic microsome that contains high content alkaline phosphatasein vivo bioconversion of acacetin prodrug was determined in beagle dogs as described in The The pre-pharmacokinetic parameters of acacetin were calculated in beagle dogs with intravenous administration of 6 mg/kg acacetin prodrug using non-compartmental analysis and compartmental analysis using WinNonlin®1.3  as described in Experimental AF was induced by vagal nerve stimulation in beagle dogs with modified procedure as described previously101450 was calculated by the Bliss method22The acute toxicity of acacetin prodrug was assessed in ICR mice (weighing 18~22 g) as described in t test or repeated ANOVA where appropriate. Categorical data were analyzed with the χ2 test. A value of P < 0.05 was considered statistically significant.All results are expressed as mean ± SEM. Statistical comparisons were analyzed by paired Student How to cite this article: Liu, H. et al. Synthesis of a highly water-soluble acacetin prodrug for treating experimental atrial fibrillation in beagle dogs. Sci. Rep. 6, 25743; doi: 10.1038/srep25743 (2016).


Background. Procurement of hearts from cardiopulmonary arrest and resuscitated (CPR) donors for transplantation is suboptimal. We studied the influences of donor factors and regional wait times on CPR donor heart utilization. Methods. From UNOS database (1998 to 2012), we identified 44,744 heart donors, of which 4,964 (11%) received CPR. Based on procurement of heart for transplantation, CPR donors were divided into hearts procured (HP) and hearts not procured (HNP) groups. Logistic regression analysis was used to identify predictors of heart procurement. Results. Of the 4,964 CPR donors, 1,427 (28.8%) were in the HP group. Donor characteristics that favored heart procurement include younger age , male gender , shorter CPR duration , and head trauma (60% versus 15%). Among the 11 UNOS regions, the highest procurement was in Region 1 (37%) and the lowest in Region 3 (24%). Regional transplant volumes and median waiting times did not influence heart procurement rates. Conclusions. Only 28.8% of CPR donor hearts were procured for transplantation. Factors favoring heart procurement include younger age, male gender, short CPR duration, and traumatic head injury. Heart procurement varied by region but not by transplant volumes or wait times. For patients with advanced heart failure awaiting heart transplantation (HTx), donor heart supply remains a limiting factor in offering the ultimate treatment option. Efforts to optimize management of potential heart donors have led to increased utilization of donor hearts , yet thiDespite these encouraging reports, utilization of CPR donor hearts has been less than 30% . The reaWe examined the UNOS heart donor data between 1998 and 2012 and identified 44,744 consented heart donors, of which 4,964 (11%) were from CPR donors. This comprised our study group, which was further divided into two groups based on heart procured (HP) or heart not procured (HNP) for transplantation. Besides donor demographics, data on comorbid conditions, social history, causes of death, and duration of CPR were collected in a deidentified fashion for analysis . Data onP value of less than 0.05 is considered statistically significant. Continuous variables were analyzed with Student's t-test; categorical variables were analyzed with the chi-square test. A logistic regression analysis of 11 donor variables was performed to identify predictors of heart procurement for transplantation from the CPR donor pool. The results of this regression analysis are reported as odds ratios with confidence limits.Numeric data were analyzed and reported as mean, median, and standard deviations. A During the study period of 1998 to 2012, a total of 44,744 consented heart donors were identified in the UNOS database. Of which, 4,964 (11%) donors sustained cardiopulmonary arrest and were resuscitated to spontaneous rhythm and circulation. Only 1,427 (28.8%) of these CPR donor hearts were procured for transplantation; these donors comprised the hearts procured (HP) group.P ≤ 0.0001) and a high proportion of them were male gender . Cause of death influenced the percent utilization of CPR donor hearts: those with head trauma leading to brain death were selected more often for heart procurement compared to those with intracerebral bleed . Similarly, mechanism of death played a role in the selection of CPR donors for heart procurement: those with gunshot wounds (44%), asphyxiation (40%), or blunt trauma (39%) were more often selected for heart procurement compared to those with stroke (15%). CPR donors with a history of hypertension were selected less often for heart procurement, as only 12% of the hearts procured group had a history of hypertension, compared to 35% in the hearts not procured group (P ≤ 0.0001). Ethnicity and the social history of the CPR donor were also factored into the heart procurement for transplantation (P ≤ 0.0001). We noticed that there was a higher prevalence of head trauma (35%), male gender (62%), and blood group O (60%) in Hispanic ethnicity heart donors compared to other heart donors. Donors with a history of tobacco abuse (17%) and heavy alcohol consumption (22%) were less often selected for heart procurement. Duration of CPR also contributed significantly to the selection of donor heart for transplantation. Those donors with a CPR duration of less than 15 min were selected more often for heart procurement than those with a CPR duration of greater than 30 min . Left ventricle ejection fraction (EF) was near normal in heart procured group compared to heart not procured group, 61.4 ± 9.0 versus 48.2 ± 17.3, P < 0.0001.When compared to the hearts not procured group, the hearts procured group were relatively younger . We also evaluated the CPR donor utilization by population per heart transplantation performed in the 11 regions. Region 6 had the highest population per heart transplant , while the lowest was in Region 2 . CPR donor utilizations in Regions 6 and 2 were 26% and 29%, not statistically significant.CPR donor heart utilization varied significantly between the eleven regions, with a low of 24% in Region 3 to a high of 37% in Region 1  and fewer Asian donors (5%) were selected for heart procurement for transplantation following brain death compared to other ethnicities studied . In our The most common blood group among US population is O (44%) followed by A (42%), B (10%), and AB (4%). Since donors and recipients are matched primarily by blood groups, it is expected that donors with O blood group  would be utilized most frequently. Heart procurement for transplantation among CPR donors in our study is consistent with this expectation.Transplantation volumes and duration of wait times on the transplantation list vary at different centers and depend on the prevalence of heart failure within the patient population, the number of competing programs in the region, and, most importantly, the surrounding population. Posttransplantation outcomes have been linked to a center's transplantation volume, with low volume centers showing inferior survival . In a stLimitations inherent to retrospective data analysis are applicable to our study. The primary data source for our study is a composite UNOS database from more than 150 US transplantation centers. Completeness of all data fields is wanted and is not always available in the database. However, its merit lies in the fact that large, pooled data from different transplant centers eliminate biases associated with individual center practices. Thus, certain generalizations can be made of the results with regard to practicality of its usage and reproducibility. Since our focus was on the characteristics of CPR donors only, this limited the study population to 11% of the total donor pool, thus eliminating a majority of donors from our analysis. Since CPR donors comprise a potential group of underutilized heart donors, we felt it is important that we focus on this small but important group of donor hearts. We used the large UNOS database to help define donor characteristics that were associated with heart procurement for transplantation with the hope that in future at least CPR donor hearts with similar characteristics would be utilized more frequently for transplantations. Accepting a heart from an extended criteria donor also depends on the medical condition and acuity of illness of the recipient. The present study has not taken the recipient factors into consideration when defining CPR donor heart utilization. In our previous study of CPR heart donors, we noticed that more CPR donors were matched to recipients with high acuity of illness at transplantation, such as those with status 1A listing, those that were admitted to the hospital at transplantation, and those that were supported in an intensive care unit on life-sustaining support . DespiteCPR donors are an expanding group of potential, yet presently underutilized, heart donors. With improvements in onsite resuscitative measures, more patients are regaining spontaneous circulation after CPR. However, if irreversible neurologic damage is sustained, these patients become eligible for organ donation. Though the clinical outcomes from accepting hearts from these donors are comparable, the overall utilization of these donor hearts is only 28.8%. Donor characteristics such as younger age, male gender, absence of hypertension, smoking history, traumatic head injury, and shorter CPR duration are associated with higher heart procurement rates for transplantation. Regional factors do play a role in donor heart selection, but it does not seem to be associated with either recipient wait times or regional transplantation volumes. It is our hope that in the future CPR donor hearts with attributes associated with successful heart procurement for transplantation will not be passed up and that serious efforts will be made to accept these hearts, thus helping patients waiting desperately for transplantation.


Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.Testis of  Spermatogenesis is the complex biological process by which sperm are produced by successive mitosis and meiosis. This requires extensive intercellular coordination within the testis among different cell types, e.g., germ cells, Sertoli cells, Leydig cells, peritubular cells, etc. Intracellular coordination links to the timely expression of certain genes and their interactions, which collectively play a major role in the successful completion of sperm production.Seminiferous tubules in the testis are surrounded by lymphatic endothelium . The perIn mouse (continuous breeder), various phases of testicular development appears as the animal matures from neonatal to adulthood. At around 5 days of post natal age , the testis consist of undifferentiated spermatogonial cells and more than 50 percent of those remain in the resting phase with no cellular division and differentiation . At 8–10Hemidactylus flaviviridis, commonly known as Indian wall lizard, is a seasonally breeding animal. Based on seasonal variations they have three reproductive phases within a year [As opposed to mouse, most of the lizard species are seasonal breeders, they mate in spring, offspring hatches in summers. Seasonal reproduction is a tactic to make use of energy in an economical manner. The wall lizards have a prenuptial cycle of sperm maturation. Prenuptial reptiles generates sperm prior to or during the mating period . Hemidacn a year , namely-n a year . This isn a year . In lizan a year ,9,11. StWe have previously reported the differential gene expression in testis of three different reproductive phases of wall lizard by microarray analysis . As micrHemidactylus flaviviridis were procured from a local supplier in Delhi, India, and were kept in the animal house of Department of Zoology, University of Delhi, and maintained as described earlier by us previously [Laboratory mouse strain FVB/J was kept in animal house of National Institute of Immunology according to guidelines provided by The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), government of India. Indian wall lizards eviously . Adult meviously . For a sTestes isolated from various phases of the reproductive cycle of wall lizards and different postnatal days of mice were fixed using 10 percent neutral buffered formalin, pH 7. Paraffin blocks were prepared, 5 μm sections were made on microtome machine , and hematoxylin-eosin staining was performed. After DPX mounting, the sections were observed and testicular diameters were measured under bright field upright microscope .H. flaviviridis. PCR amplification was terminated in different cycles  and all PCR products were run on gel to validate the subtraction efficiency. The secondary PCR products, contained partial cDNAs, were ligated to a TA cloning vector in multiple cloning site , and were transformed into E. coli. From each SBactive and SBregressed sample, randomly130 positive colonies were selected to perform colony-PCR to verify the insert size in TA cloning vector. Colonies showing higher molecular mass with a single amplification product in the colony-PCRs were considered; 48 different colonies, of each SBactive and SBregressed sample were selected. pDNA from those selected colonies were isolated with GenElute plasmid miniprep kit . The region of pDNA having partial cDNAs were sequenced . A flowchart regarding the above-described subtractive hybridization process is illustrated in Subtractive hybridization was done according to the manufacturer’s protocol . In brief, 2 μg of RNA isolated from testes in active and regressed phases were used to generate double-stranded cDNAs, which were digested with RsaI enzyme, to generate blunt–end partial cDNAs. The digested resultant of each phase was divided in two portions named tester and driver. Testers from the active and regressed phases were again divided in two halves; the one half was ligated with adaptor1 and to another adaptor2 (provided with the kit). The efficiency of adaptor ligation was determined as suggested in the kit protocol. Hybridization was performed between the active tester and the regressed driver, where regressed is subtracted from active (hereafter will be referred to SBactive) and the regressed tester and the active driver, where active is subtracted from regressed (hereafter will be referred to SBregressed). The hybridized samples were used for primary and secondary subtraction PCRs with primers provided with the kit. Subtraction efficiency was evaluated between the subtracted and un-subtracted samples of the active phase using primers specific to To find the identity of the partial cDNAs, the Basic Local Alignment Search Tool (BLAST) was used to align with nucleotide sequences deposited in the NCBI databases. Identities of partial cDNAs were assigned from the best aligned sequence having highest identity score and query coverage with least E value.A phylogenetic tree was constructed with the partial cDNAs and their corresponding ortholog sequences in other species. MEGA5 software was used for generation of the phylogenetic tree by neighbor-joining statistical method and the bootstrap phylogenetic tree construction method, having a bootstrap replication of 1000 .15 as primer in a final volume of 25μl. cDNA was diluted 3 times with nuclease-free water, and subsequent PCRs were carried out using 1μl of the diluted cDNA as template.1 μg of RNA, isolated from the testes samples of lizards and mice, was treated with DNaseI according to the manufacturer’s protocol . To generate cDNAs form the treated RNAs, M-MLV reverse transcriptase  were used with oligo (dT)http://www.ncbi.nlm.nih.gov/tools/primer-blast/ as on 22 July, 2015) and GeneRunner . Reference sequences for gene-specific primers were acquired from NCBI database for mice and partial cDNA sequences, obtained from subtractive hybridization, for wall lizards. Primer sequences are listed in Gene-specific oligo-primers for real-time PCR were designed using software tools: PrimerBlast  for wall lizard and Ppia (Peptidylprolyl Isomerase A) for mice was used as reference gene for normalizing the target genes expression. Calculation for relative fold change with respect to the active phase in wall lizards and 60 days in mice was performed by 2-ΔΔct method as previously described by us [Real-time PCR was carried out with syber green using Mesa-green master mix kit  in Eppendorf Realplex mastercycler machine . Reaction setup and PCR primer efficiency calculations were done by previously described procedure by us ,16. Primed by us .http://discover.nci.nih.gov/cimminer/home.do as on date 22 July, 2015) to represent the gene expression profile between wall lizards and mice contemporary testis. For the generation of heat map methods like euclidean, average linkage, and quantile were used as distance method, cluster algorithm, and binning method, respectively.A clustered image heat-map was generated with the relative fold change values, using online tool CIMminer (-ΔΔct method) of testicular samples against their corresponding testis-weight in percentage on x axis. A linear regression line was drawn by connecting the expression values plotted on the graph. In the graph, independent linear regression line was generated for wall lizards and mice.Testes weights (mg) of nine testicular samples from wall lizards  were assigned percentage values by considering the highest value amongst all of them as 100. Here, the highest value came from one biological replicate of the active phase of wall lizard. In a similar manner, mice testicular weights from nine testicular samples of mouse  were assigned percentage values by considering the highest value amongst all of them as 100. A graph was generated for each gene, where y axis was assigned to the relative fold change values (obtained by 2The relative fold change in gene expression data were represented as ±SEM (n = 3). Statistically significant difference among the samples were analyzed by one-way analyses of variance (ANOVA) followed by Newman-Keuls multiple comparison test. Difference in gene expression was considered significant when the p value was found to be less than 0.05. Correlations were determined between testis wt. and relative gene expression by Pearson's correlation coefficient analysis. p<0.05 was considered statistically significant correlation. All statistics and graph preparation were done by GraphPad prism software (USA) version 5.0.Cross section of testis from the regressed phase of a wall lizard showed small seminiferous tubules having small lumen, consisting mostly Sertoli cells and very few early germ cells . The 5-dSubtractive hybridization was done to identify genes that are differentially expressed between active and regressed phases of the testis. To get transcripts that were highly expressed in the active phase in comparison to the regressed phase, the testicular RNA from regressed phase was subtracted from the testicular RNA of active phase (SBactive). For identification of transcripts expressed more in the regressed phase compared with the active phase, the active phase testicular RNA was subtracted from the testicular RNA of regressed phase (SBregressed).Hk1, Nme5, Akap4, Tubb4b, Arih1, Rassf7, Hmgb1 and Bco2) of the wall lizards were found to have high homology with ortholog sequences in mice . Out of these, 27 sequences were from the active phase (SBactive) and 8 were from the regressed phase (SBregressed). Out of 27 sequences in the SBactive sample, 10 sequences were found to have similarity with known gene sequences from different species . Out of  in mice . These 8Arih1 (JZ822545) from H. flaviviridis was found to be nearest to Anolis with the highest homology, and formed an altogether different node. The sequences of Nme5 (JZ822546) and Tubb4b (JZ822551) were in greater homology with Z. albicollis. Sequences Bco2 (JZ822548), Rassf7 (JZ822549) andHmgb1 (JZ822550) were in greater homology with Xenopus. The sequence similarities of Hk1 (JZ822542) and Akap4 (JZ822547) found to be higher with Drosophila.A phylogenetic tree was constructed using the bootstarp method, with known homolog sequences found in NCBI databases . The seqHk1, Nme5, Akap4, Arih1, Rassf7, Tubb4b, Bco2and Hmgb1in different testicular phases of wall lizard and mice, real-time PCR was performed using testicular RNA. Except Hmgb1, rest other 7 genes were significantly high in the active phase of testis as compared to the regressed and recrudescent phases of wall lizards’ testis . Earlier we have compared the differential transcriptome of three reproductive phases of testis from wall lizard . Since lThirty five unique partial RNA sequences were identified in testis of wall lizard. The 17 and 8 partial sequences from active and regressed phase, respectively, showed no significant similarity with known mRNA sequences in the database. The reason for the lack of similarity of these partial sequences with known mRNA sequences may reside in the lack of longer sequence reads (eg. JZ822572 and JZ822543), which is necessary to attain a significant match. There were other sequences, which were found to be relatively longer and showed partial similarity to short region of more than one mRNAs ; the query coverage for these sequence alignments were also very low (below 40%). Therefore, these 17 and 8 sequences could not be reliably identified as mRNA sequences of known genes. Hence, in the present study we could not consider these partial sequences for further experiments.Hk1, Nme5, Akap4, Arih1, Rassf7, Bco2 and Tubb4b were found to be uniformly associated with testis with active spermatogenesis in both the species. Expression of 6 genes Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b showed positive correlations with increased testicular weight in both wall lizards and mice, that represents their conserved pattern of expression between these two species. Specifically higher expression of Hmgb1 in testis of 5 days old mice suggested that the regressed testis cannot be considered as neonatal testis in terms of developmental status.Ten partial RNAs among 35, showed sequence homology with mRNA sequences of orthologus genes from other species and found to be abundant in the testis during active phase compared to regressed phase of wall lizards. Out of 10 sequences, 8 showed sequence similarity with corresponding mice gene sequences. In order to evaluate testicular phase related similarity in the expression pattern between two species, we analyzed the expression profiles of these 8 genes using real-time PCR in different phases of mice testicular development and lizard reproductive cycle. 7 genes, HK1 is an important enzyme for glucose metabolism. HK1 localizes on the outer membrane of mitochondria [Hk1 in the spermatogenically active testis of wall lizards and mice suggested its requirement for spermatogenic maintenance. Interaction of HK1 with glucose metabolism associated proteins  as well as fructose metabolism associated proteins  based on interactome database, suggested HK1 is important regulator of energy source to actively dividing spermatogonia. NME5 is a NME/NM23 family member protein, also known as Nm23-h5. This is a nucleotide diphosphate (NDP) kinase that catalyses phosphoryl exchanges between nucleoside di- and tri-phosphates [AKAP4, which is a member of the AKAP family, helps in binding to the regulatory subunit of protein kinase A (PKA), which works as a scaffold protein. AKAP4 is found in the fibrous sheath in sperm and helps in giving mechanical support to the sperm tail [Macropus eugenii, located in the cytoplasm of round and elongated spermatids, and in the sperm tail [BCO2 enzyme is a mitochondrial carotenoid-oxygenase, which catalyses asymmetric cleavage of carotenoids and protects the cells from carotenoid induced oxidative stress [Bco2 at 20 days (meiotic) and 60 days (post-meiotic) in comparison to that in 5 days old mice testis. This higher expression of Bco2 in testis showing meiosis can be correlated with the requirement of retinoic acid (RA) for the meiotic initiation of germ cells. When germ cells undergo meiosis, they require retinoic acid [Bco2 may create a balance for maintaining the right amount of beta-carotenoid in the germ cells. ARIH1 can induce aggresome formation and helps in the ubiqutinilation of target proteins [Arih1 in those phases of testis in wall lizards and mice can be justified. In the predicted interactome database, this protein interacts with the JAK1, which is a protein-tyrosine kinase found in human spermatozoa [Xenopus [RASSF7 is a ras association domain-containing protein 7, which negatively regulates stress-induced JNK activation [Rassf8, found to be essential for maintaining adherent junctions (AJ) in epithelial cells and has a role in epithelial cell migration [Rassf7 in the testis of active phase and adult testis of wall lizards and mice, respectively. In the interactome data, it showed interaction with other ras-associated proteins. TUBB4B is a tubulin protein. We found its expression in the adult testis of mice and active testis of lizards, from which we can infer that the requirement of this component of microtubules may be higher in mature testis for providing structural support to large number of germ cells. HMGB1 is a high-mobility group binding protein. The expression of Hmgb1 was found to be comparable during recrudescence and active phase of lizards. This was also comparable in 20 and 60 days old testis of mice. However, this was highest in neonatal testis (5 days) of mice. Interactome data of HMGB1, suggested its interaction with several proteins related to PI3K-Akt signaling pathway, like NFKB1, RELA1, CHUK, and IKBKB, which are known to play important role in cellular growth and differentiation. The Hmgb1 gene was found to be one of the 159 transcripts from testicular biopsy where the expression was found to be high in idiopathic infertile patients of AZFc microdeletion [Hmgb1 in neonatal (immature) testis of mice and testis of infertile patients, led us to hypothesize that higher expression of HMGB1can keep the testis in immature state whereas moderate expression is seen during spermatogenesis.chondria  and facichondria ,20. As gosphates . The mRNosphates ,22, and erm tail . Targeteerm tail . The mRNerm tail . In the e stress . We founoic acid  which isproteins ,30. It iproteins . As the rmatozoa  and help[Xenopus .RASSF7 itivation . It is ativation . The impigration . We obsedeletion . In manyIn our knowledge this is the first report where increased expression of certain genes associated with active spermatogenesis was commonly observed in lizard and mammalian testis. The increase in six commonly identified genes during active phase was observed as compared to regressed state of the testis from lizard and neonatal testis of mice. This not only showed that these genes are conserved across the species but also indicated their strict association with spermatogenesis. Impairment in expression of any of these genes may potentially disturb the process of spermatogenesis leading to infertility. This information may be crucial in divulging unknown causes of idiopathic male infertility and hence should be studied further in detail.S1 FigHk1 (A), Nme5 (B), Akap4(C), Bco2 (D), Arih1 (E), Rassf7 (F), Hmgb1 (G), andTubb4b (H).Phylogenetic tree for the genes, (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


However, the current synthesis leads to agglomerated products with irregular morphologies and wide size distributions. Herein, we report on in vivo rechargeable mesoporous SiO2/ZnGa2O4:Cr3+ (mZGC) afterglow NIR‐emitting nanocomposites that are made by a simple, one‐step mesoporous template method. At less than 600 °C, pores in mesoporous silica nanoparticles (MSNs) act as nanoreactors to generate in situ ZnGa2O4:Cr3+ NIR‐persistent phosphors. The as‐synthesized mZGC preserves defined size, morphology, and mesoporous nanostructure of the MSNs. The persistent luminescence of the as‐synthesized mZGC is recharged in a simulated deep‐tissue environment  in vitro by using red light (620 nm). Moreover, mZGC can be repeatedly activated in vivo for persistent luminescence imaging in a live mouse model by using white LED as a light source. Our concept of utilizing mesoporous silica as nanoreactor to fabricate ZGC PL nanoparticles with controllable morphology and preserved porous nanostructure paves a new way to the development and the wide application of deep tissue rechargeable ZGC in photonics and biophotonics.Near‐infrared (NIR) persistent phosphor ZnGa The optimal used composition was determined at Zn/Ga/Cr = 1/1.997/0.003 by molar ratio . ZGC was formed in the nanochannels of the MSNs after vacuum drying and annealing, as shown in Figure2O4 (JCPDS index no. 01–086–0410). However, impurity peaks start to appear at 700 °C, which indicate the formation of metal silicates  and luminescence measurements were used to confirm the nature of the as‐synthesized nanocomposite. The XRD results Figure1A show2O4, 4A2→4T1, and the 4A2→4T2 transition of Cr3+, respectively.The phosphorescence excitation spectrum of the sample synthesized at 600 °C consists of three ZGC characteristic excitation bands Figure C. These 2.2Figure2 adsorption/desorption was performed to study the influence of the synthesis process on the specific surface area and pore‐size distribution of the MSNs. Apparent mesoporous characteristics of plateau regions can be observed both for the MSN templates and for the as‐synthesized mZGC . To study the in vivo chargeable ability of mZGC, we injected the mZGC saline solution  into a live mouse through the tail vein. After exposure to white LED light, a satisfactory PL imaging picture was obtained, as shown in FigureFigureBased on our observation that the PL of mZGC can be charged by red light under an 8 mm pork layer, for the first time we studied its applications in deep‐tissue imaging by using a white light‐emitting diode (LED) as the excitation light source . The rechargeable PL imaging of the mZGC sample was performed five times under a pork layer of 8 mm without any obvious signal weakening, which implies its potential for use in in vivo imaging applications Figure4. For  200 μL,  mg mL−1 32/ZnGa2O4:Cr3+ persistently luminescent nanocomposites. By using the mesopores of MSNs as a reaction template, the synthesis temperature of the persistent phosphor, ZnGa2O4:Cr3+, was decreased from higher than 750 °C in a solid‐state reaction to only 600 °C. At this lower temperature, both the unique mesoporous attributes, and the uniform size and morphology of the MSNs were retained in the nanocomposites. For the first time, we confirmed that mZGC could be repeatedly charged in situ under a deep‐tissue layer of 8 mm. The deep‐tissue chargeable PL properties of mZGC also ensured its repeatable recharged PL imaging in a live mouse model. It is worth noting that this observation is the first direct evidence that persistent luminescence can be recharged in vivo for multiple times. This concept of utilizing mesoporous silica as nanoreactor to fabricate ZGC PL nanoparticles with uniform morphology and preserved porous nanostructure will be significant in directing the synthesis of mesoporous PL systems with diverse PL phosphors and paves a new way to the wide application of deep tissue rechargable ZGC in photonics and biophotonics.We developed in vivo rechargeable near‐infrared‐emitting mesoporous SiO4Materials: Tetraethoxysilane (TEOS), ethanol, diethanolamine (DEA), ammonium hydrate, cetyltrimethylammonium bromide (CTAB), Ga2O3, Zn(NO3)2·6H2O, Cr(NO3)3·9H2O, and concentrated nitric acid were all of analytical standards, purchased from Sigma‐Aldrich and were used as‐received. Ga(NO3)3 solution was prepared by dissolving Ga2O3 in 1:1 concentrated nitric acid followed by air drying at 105 °C to remove excess nitric acid, and then redissolving in deionized water.Synthesis of MSNs: The MSN synthesis was modified from that in a previous report.Synthesis of mZGC Nanocomposites: A precursor solution was prepared by dissolving the corresponding nitrates in water/ethanol . The final concentration of Zn2+, Ga3+, and Cr3+ was controlled to be 0.5, 0.9985, and 0.0015 mol L−1, according to a stoichiometric ratio of Zn/Ga/Cr , respectively. 200 μL of the precursor solution was mixed with 200 mg mesoporous silica and the mixture was dried in a vacuum oven at 50 °C for 12 h. The samples were then put into a muffle furnace and the temperature was slowly increased by 5 °C min−1.In Vitro and In Vivo PL Imaging of mZGC: In vitro imaging was performed by putting the powder (100 mg) sample into a black 96‐well plate covered with an 8‐mm layer of pork tissue. The PL signal from the covered mZGC was recorded after illumination by using an LED (5000 lumen) for 15 s. The in vivo imaging was conducted by injection of the mZGC dispersion in phosphate‐buffered saline  through the tail vein. The sample was stored in a dark box for 1 day before injection to ensure no preactivation occurred.Characterization: X‐ray powder diffraction (XRD) measurements were performed on a diffractometer equipped with Cu Kα radiation (λ = 1.5418 Å) . The morphology of the samples was inspected by using field‐emission scanning electron microscopy  and transmission electron microscopy  at accelerating voltages of 5 and 100 kv, respectively. High‐resolution TEM (HRTEM) images were recorded by using a JEM‐1200EX II transmission electron microscope. N2 adsorption/desorption isotherms were obtained on a full‐automatic physical and chemical adsorption apparatus . Pore size distribution was calculated from the adsorption branch of N2 adsorption/desorption isotherm and the Brunauer–Emmett–Teller (BET) method. The BET specific surface areas were calculated using the data between 0.05 and 0.35 just before the capillary condensation. The total pore volumes were obtained by the t‐plot method. Total organic carbon analyzer was used to determine the exact loading level of ibuprofen on mZGC by using a CNS elemental analyzer . The spectra and lifetimes of the samples were tested by using powdered samples. The photoluminescence spectra and time‐decay curves were measured by using a fluorospectrophotometer . The absorbance was detected on an UV–vis spectrometer . The PL imaging of mZGC was conducted in a Xenogen IVIS imaging system.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered “vulnerable” while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease.in vivo imaging using fluorescent molecular tomography (FMT), as well as ex vivo fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy.We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques.Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation. Atherosclerosis is a systemic inflammatory disease with plaque formation and progression. Plaque morphology can be broadly divided into two major types, ‘stable lesions’ where the plaque is mainly fibrotic and ‘unstable lesions’ that may rupture causing acute myocardial infarction or stroke. Increased macrophage content is one of the characteristics of unstable plaques, as macrophages contribute to plaque destabilization through multiple mechanisms.The most prominent mechanism is through degradation of the extracellular matrix resulting in a thin fibrous cap that is prone to rupture .Reshaping the extracellular matrix of the plaque microenvironment is mainly controlled by matrix-metalloproteinases and cathepsin cysteine proteases that degrade collagen and elastin , 3]. We. We3]. WIt is now believed that macrophage cell depletion may be an effective approach to avoid the complications of plaque rupture . We receex vivo fluorescence, using a Maestro™ imaging system  at 649/666nm excitation/emission. Samples were incubated for 4 hours with 4% paraformaldehyde/PBS, then overnight in 30% sucrose/PBS at 4°C and embedded in OCT. Frozen samples were cut into slices using a CM 1900 cryotome . Sections, 7μm thick, were stained with primary antibodies against mouse macrophages, F4/80-PE , and fluorescent pictures were taken with an Olympus FV10i confocal microscope . The protocol was approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC).We used a previously described mouse carotid-ligation model , 10], d, d10], d2 [Carotid plaque specimens were collected from patients who underwent carotid endarterectomy at Hadassah—Hebrew University Medical Center with or without a history of cerebrovascular symptoms . The study protocol was approved by the Hadassah Helsinki Review Board  with written consent as described in . The car2   and non-quenched (GB123) ABPs that target the activity of cathepsin B, L and S, as markers for macrophages within atherosclerotic plaques (for enzymatic data and selectivity please see ). Both tIn vivo imaging of mouse carotid arteries using FMT showed a clear signal from the non-quenched probe GB123 in the macrophage-rich ligated left carotids but not in the non-ligated (control) right carotids, at four hours post injection. We also observed signal generated by the probe in the lymph nodes as expected and in the aortic arch and heart as predicted from the disease progression Click here for additional data file.S1 MovieFluorescent tomographical images were acquired by a FMT system of FVB mice treated as in (MOV)Click here for additional data file.S2 MovieFluorescent tomographical images were acquired by a FMT system of FVB mice treated as in (MOV)Click here for additional data file.


Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway. FRET is often employed to verify or refute dynamic interactions between proteins, both  in vivo ,2,3,4. I in vivo ,6. The rFRET measurements are greatly facilitated by the use of brightly fluorescent dyes that emit and absorb in the visible spectrum, such as Alexa Fluor dyes, Atto dyes, BODIPY dyes and cyanine dyes . LabelinAzotobacter vinelandii. This protein is labeled with donor fluorophore Alexa Fluor 488 C5 maleimide (A488) and acceptor fluorophore Alexa Fluor 568 C5 maleimide (A568). These dyes are brightly fluorescent, having a high fluorescence quantum yield (>0.5), are relatively photostable and can be excited with visible light [In this study, we overcome the limitations of FRET determination in ensemble measurements as posed above, by determining the arrival times of acceptor photons upon pulsed excitation of a fluorescent donor. Thus, we determine the “rise time” of acceptor fluorescence, which can be used to quantify the rate of transfer of excited state energy from the donor to acceptor ,14,15,16le light ,17. A488le light ,19,20,21le light ,23).N↔U↔Ioff, in which N and U are native and unfolded protein, respectively, and Ioff is an intermediate that is kinetically off-pathway [Both equilibrium and kinetic (un)folding of apoflavodoxin have been characterized using guanidine hydrochloride (GuHCl) as the denaturant  [Previously, we demonstrated that folding of apoflavodoxin could be tracked using steady-state fluorescence on molecules that were site-specifically labeled with A488 and A568 . Here, wda) ,15,16 ) of folded apoflavodoxin is 0.51 ns at 0 M GuHCl, and 1.57 ns in the case of unfolded protein at 4.12 M GuHCl. The corresponding decay times of acceptor fluorescence ) are 3.85 and 3.71 ns, respectively. The positions of maximum intensity of fluorescence decay curves of erythrosine B and double-labeled apoflavodoxin differ by 0.94 ns for folded protein, whereas they differ by only 0.76 ns for unfolded protein. This apparent contradiction can be explained by taking into account the ratio between corresponding pre-exponential factors of Equation (3) (A−/A+). A−/A+ equals −0.443 for folded protein at 0 M denaturant, and A−/A+ equals −0.134 for unfolded protein at 4.12 M GuHCl.Acceptor fluorescence rise time (τA−) shows a transition between 1 and 2 M GuHCl, whereas the amplitude of the acceptor fluorescence decay time (A+) does not. A−/A+|), which, in contrast to the fluorescence lifetimes, change in a monophasic fashion as a function of denaturant concentration. |A−/A+| is constant between 0 and 1 M GuHCl, where apoflavodoxin is native [s native ,24, and s native ,24 and, A−/A+| at low denaturant concentrations, we estimate that 44% of the detected photons arise from sensitized acceptor fluorescence. Hence, the remaining 56% of the detected photons (with positive decay amplitude) arise from other sources  changes. We previously observed that the folding state does affect both the absorption spectrum, as well as the fluorescence intensity of the fluorophores used [A−/A+| further than what can be expected from a change in the intensity of sensitized acceptor emission. The observed decrease in |A−/A+|  to acceptor rise and decay data of double-labeled apoflavodoxin track protein folding.da; Equation (4); see kt is obtained from the difference between the reciprocal rise time of the acceptor fluorescence (1/τda) and the reciprocal value of the amplitude-averaged donor fluorescence lifetime in the absence of acceptor (1/τd) (see Equation (5)) [d for the denaturant concentrations used in this study were obtained by fitting a three-state model for protein folding to τd determined previously ) . Values  see the . The avekd) and of the fluorescence decay rate of a donor in the presence of an acceptor, as obtained from the rise time of acceptor fluorescence (kda = 1/τda). We determine the FRET transfer rate constant (kt) as a function of denaturant concentration by using Equation (5) (kt is 1.6 (ns)−1 for protein without denaturant. This rate constant decreases to 0.8 (ns)−1 upon increasing the concentration of GuHCl to about 1.3 M. kt increases upon further addition of denaturant, until it reaches a value of 1.1 (ns)−1 at ~1.7 M GuHCl. Subsequently, kt gradually decreases, until it levels off to a value of about 0.25 (ns)−1 when the denaturant is added to 3 M GuHCl and above. This decrease in kt upon the addition of denaturant hallmarks the increase in the distance between the donor and acceptor as apoflavodoxin unfolds.tion (5) . kt is 1rda between donor and acceptor labels attached to apoflavodoxin can be estimated from the values of τd and kt via Equation (7). Determination of distances using kt and τd or from FRET efficiencies requires accurate determination of the Förster radius, R0. Previously, we showed that in the case of apoflavodoxin, R0 changes as a function of denaturant concentration in a folding state-dependent manner [The distance t manner . We recarda) of 40 Å for folded apoflavodoxin without denaturant . Furthermore, we assume that the fluorescence decays of both donor and acceptor are single-exponential, and we excite the donor fluorophores at a wavelength where only donor molecules absorb light, thereby avoiding direct excitation of acceptor molecules. In such a system, we can investigate two time-resolved fluorescence experiments, namely the fluorescence decay of the donor at the maximum emission wavelength of the donor fluorescence spectrum and the time-dependent increase (rise) of the acceptor fluorescence measured at a wavelength where no donor fluorescence photons are detected. Following the solution of differential equations [*D(t)) is directly proportional to the time-resolved fluorescence intensity IDD(t), in which the first subscript D denotes donor excitation wavelength and the second subscript D denotes donor emission wavelength:D* 0 is the excited state concentration of the donor at t = 0. kd is the rate constant of donor molecule de-excitation in the absence of acceptor and is equal to 1/τd, in which τd is the donor fluorescence lifetime, and kt is the rate constant of resonance energy transfer from donor to acceptor. The observed rate constant is kd + kt = kda. 1/kda is equal to τda, which is the fluorescence lifetime of the donor in the presence of the acceptor.Suppose we have a homogeneous donor-acceptor system that exhibits FRET from light-excited donor fluorophores to acceptor fluorophores in the ground state and that does not contain donor labels that are FRET-inactive ), generated via FRET, is directly proportional to the time-resolved fluorescence intensity IDA(t), in which the first subscript D denotes donor excitation wavelength and the second subscript A denotes acceptor emission wavelength:*A(t) is the sum of two exponential components, of which one has a negative pre-exponential factor and the other has a positive pre-exponential factor ) observed at the donor emission wavelength. 1/kda is equal to τda, which is the fluorescence lifetime of the donor in presence of the acceptor. The positive component arises from acceptor de-excitation with rate constant ka and is equal to 1/τa, in which τa is the acceptor fluorescence lifetime.Similarly, the time dependence of the excited state concentration of the acceptor (tails in ). The neA−) and positive (A+) amplitudes is equal to 1. However, because of overlap between the absorption bands of the donor and acceptor, it is, in practice, impossible to selectively excite only donor fluorophores. The decay of acceptor fluorescence then contains an additional term with a positive amplitude, which arises from the detection of photons from directly excited acceptor molecules. Equation (2) must then be rewritten as:A* 0 is the excited state concentration of directly excited acceptors at t = 0. Therefore, the absolute value of A−/A+ (|A−/A+|) of time-dependent acceptor fluorescence is usually smaller than 1.In the ideal case, the acceptor receives energy only from donor molecules via FRET, which implies that the absolute value of the ratio between negative  because of overlapping emission bands. These photons can come from both the FRET-active donors, as well as from FRET-inactive donor molecules that have no acceptors in their proximity. In the latter case, one has a heterogeneous system. The rate constants associated with the fluorescence decay of these FRET-inactive donor molecules might be similar to the decay rate of the acceptor, which makes it impossible to resolve them as separate decay components in the fluorescence signal detected in the acceptor window (A* 0 in Equation (3) is then composed of both donor and acceptor contributions). In addition, direct excitation of acceptor molecules also leads to a decrease of |A−/A+|. However, the exponential rate constant connected with the rise term in Equation (3) will not be affected, even if the absolute amplitude ratio is significantly smaller than unity. This is the main advantage of rise time measurements, since one can obtain the pure FRET rate constants in a heterogeneous donor-acceptor system.An additional factor that results in the non-unity of the absolute ratio between negative and positive amplitudes is the detection of fluorescence photons from the donor in the acceptor detection window  is proportional to the inverse sixth power of the distance rda between donor and acceptor, which makes it a sensitive parameter for obtaining distances less than 10 nm:R0 is the so-called critical or Förster radius, the distance between donor and acceptor at which 50% of the donor energy is transferred to the acceptor. Through rearrangement of Equation (6), one obtains the following relationship:The transfer rate constant , was first incubated with Alexa Fluor C5 568 maleimide  according to the protocol of the manufacturer in 100 mM potassium pyrophosphate , pH 7.5, in the presence of 1 mM Tris(2-carboxyethyl)phosphine , for 1 h, at room temperature, in the dark. An excess of reduced glutathione was used to quench the maleimide moiety of the unreacted label, prior to unfolding of the protein by the addition of an equal volume of 7.5 M GuHCl. Upon unfolding, FMN is released from acceptor-labeled flavodoxin. A precast Biogel P6DG column , equilibrated in 3.5 M GuHCl in KPPi, pH 7.5, was used to separate protein from FMN and unreacted label. To the fractions containing protein (identified as high molecular weight fractions with the color of the acceptor), 10× molar excess of Alexa Fluor 488 C5 maleimide was added, which was followed by incubation in the dark during 1 h. Subsequently, reduced glutathione was added to quench the unreacted maleimide, followed by the concentration of the protein using a Centricon spin filter with a cutoff of 10 kDa . The concentrated protein was refolded in 100 mM KPPi pH 6.0 on a Superdex 75 HR 10/30 column , and fractions containing monomeric double-labeled apoflavodoxin were snap-frozen in liquid nitrogen and stored at −80 °C until use.Apoflavodoxin has a single, wild-type cysteine at position 69 ,32. ProtThe buffer used in all experiments with purified protein was 100 mM KPPi, pH 6.0, and contained 0.001% Tween-20 to prevent the adsorption of protein to surfaces. The temperature was set to 20 °C. A volume range of protein in 6.5 M GuHCl was added to a volume range of solutions of protein in buffer. Protein concentrations in both solutions were identical. After each addition, the sample was mixed and incubated for 5 min to reach equilibrium before measurement. A similar titration of solutions without protein was prepared to function as the background for time-resolved fluorescence.IRF) was determined using a freshly made solution of erythrosine B in water as reference compound, which has a fluorescence lifetime τref = 89 ps at 20 °C [Picosecond-resolved fluorescence measurements were carried out using mode-locked continuous wave lasers for excitation and time-correlated single-photon counting (TCSPC) as the detection technique, as described previously ,33. The at 20 °C . Fluoreswww.sstcenter.com). Each of the decay curves is described by a bi-exponential decay function (Equation (3)) that was convolved with the IRF. Because of the finite width of the IRF, Equation (3) is rewritten as:iDA(t) is the experimental time-dependent acceptor fluorescence, DAI(t) is the model function as given by Equation (3) and is the convolution operator. Equation (8) forms the basis of the reference convolution method, of which details are given in [2 criterion, which should be equal to 1 for an optimal fit. In addition, the weighted residuals were used for the visual inspection of the agreement between measured (yexp) and model-generated  data. The weighted residuals (WR) in each time point k were calculated as:k = 1, …, 2000 and σ is the standard deviation.Fluorescence rise and decay curves were analyzed using the TRFA data processor  have been reported previously [Determination of acceptor fluorescence rise times enables quantification of FRET between donor and acceptor molecules attached to a protein of interest, yielding inter-dye distances. We demonstrate that this spectroscopic approach tracks protein folding. The key advantage of this methodology is that one only monitors those protein molecules that actually exhibit FRET between donor and acceptor, because FRET-inactive donor populations do not contaminate the measured rise and decay times of acceptor fluorescence.


Cerebrospinal fluid (CSF) contained within the brain ventricles contacts neuroepithelial progenitor cells during brain development. Dynamic properties of CSF movement may limit locally produced factors to specific regions of the developing brain. However, there is no study of in vivo CSF dynamics between ventricles in the embryonic brain. We address CSF movement using the zebrafish larva, during the major period of developmental neurogenesis.tnnt2a mutant fish (tnnt2a−/−) and chemical  treatment. Cilia motility was monitored at these stages using the Tg(βact:Arl13b–GFP) transgenic fish line.CSF movement was monitored at two stages of zebrafish development: early larva  and late larva  using photoactivatable Kaede protein to calculate average maximum CSF velocity between ventricles. Potential roles for heartbeat in early CSF movement were investigated using tnnt2a−/− fish and after BDM treatment. Brain cilia are immotile at the early larval stage.In wild-type early larva there is net CSF movement from the telencephalon to the combined diencephalic/mesencephalic superventricle. This movement directionality reverses at late larval stage. CSF moves directionally from diencephalic to rhombencephalic ventricles at both stages examined, with minimal movement from rhombencephalon to diencephalon. Directional movement is partially dependent on heartbeat, as indicated in assays of These data demonstrate directional movement of the embryonic CSF in the zebrafish model during the major period of developmental neurogenesis. A key conclusion is that CSF moves preferentially from the diencephalic into the rhombencephalic ventricle. In addition, the direction of CSF movement between telencephalic and diencephalic ventricles reverses between the early and late larval stages. CSF movement is partially dependent on heartbeat. At early larval stage, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain. In addition, CSF movement may also provide directional mechanical signaling.The online version of this article (doi:10.1186/s12987-016-0036-z) contains supplementary material, which is available to authorized users. Cerebrospinal fluid (CSF) is contained in the brain ventricles (chambers derived from the brain lumen) and contacts neuroepithelial progenitor cells during brain development and in adulthood. The CSF contains a unique composition of proteins (CSF proteome) –6, smallXenopus have investigated local intraventricular CSF dynamics in the developing brain  and late larva . Our study leads to four conclusions: (1) CSF moves preferentially anterior to posterior from the diencephalic (D) ) to rhombencephalic (R) ventricles. (2) Direction of CSF movement between the telencephalic (T) and diencephalic (D) ventricles reverses from early to late larval stages. (3) Directional CSF movement is partially dependent on heartbeat. (4) At early larval stages no motile cilia are present in the brain and therefore are not likely to direct the CSF movement observed. Together these data indicate that locally restricted CSF factors or physical signals associated with fluid movement may contribute to specialization of the early brain.max from D (D/M) to R ventricles. Reverse movement was present, but with about 25 % of the observed forward vmax. This raises two questions: (1) where does the excess volume of CSF entering the R ventricle go and (2) what mechanisms underlie directional CSF movement? First, with respect to CSF “destination”, the brain-CSF barrier is quite tight at early developmental stages  to late  larval stages. CSF movement is partially dependent on heartbeat. At early larval stages, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain and CSF movement may also provide directional mechanical cues.


Salmonella colonization of food animals is a concern for animal health and public health as a food safety risk. Various obstacles impede the effort to reduce asymptomatic Salmonella carriage in food animals, including the existence of numerous serovars and the ubiquitous nature of Salmonella. To develop an intervention strategy that is non-specific yet effective against diverse Salmonella serovars, we explored the prophylactic use of a cytokine to decrease Salmonella in swine by boosting the host’s innate immune system. Granulocyte-colony stimulating factor (G-CSF) is the major cytokine regulating the production, differentiation, function, and survival of neutrophils. Neutrophils play a critical role in the response to Salmonella; therefore, we evaluated the vectored-delivery of porcine G-CSF as a prophylactic to reduce Salmonella in pigs. Crossbred pigs, 5 weeks of age, were intramuscularly injected with a replication-defective human adenovirus (Ad5) engineered to express porcine G-CSF . Control pigs received the same Ad5 vector lacking the gene encoding G-CSF . Four days later, all pigs (n = 16) were intranasally inoculated with 1 × 107 colony forming unit (CFU) of Salmonella enterica serovar Typhimurium UK1. At 2 and 3 days post-challenge with Salmonella, Ad5-G-CSF-treated pigs shed significantly less Salmonella (~103 CFU/g) in their feces than Ad5-empty-treated pigs . A significant 4-log reduction in tonsil colonization was also observed in the Ad5-G-CSF-treated pigs at 7 days post-challenge (P < 0.05). In the gastrointestinal tract, the Peyer’s patch region of the ileum exhibited a significant 0.5-log reduction in colonization in the Ad5-G-CSF-treated pigs (P < 0.05). The microbiota of all challenged pigs was assessed by sequencing and analyzing the V1–V3 region of the 16S rRNA gene from fecal DNA samples. The microbial community structure of Salmonella-challenged pigs was less disturbed post-challenge in the Ad5-G-CSF-treated pigs than the Ad5-empty-treated pigs. This suggests that Ad5-G-CSF administration mitigated changes in the microbial community structure caused by Salmonella challenge. Collectively, these data suggest that delivery of a targeted immunostimulant to enhance neutropoiesis may be a strategy to reduce Salmonella colonization, potentially during periods of immunological stress. Salmonella . To overcome the complexity of Salmonella, management strategies that target innate immune mechanisms warrant exploration to control the commensal-like state of this human foodborne pathogen in the gastrointestinal tract of animals contributing to our food  supply.Asymptomatically colonized food animals are a major reservoir of the human foodborne pathogen lmonella –3. InterAn animal’s innate immune system offers multiple pathways that can be modulated to fight disease-causing agents without activation of the adaptive immune system, which is the primary target of vaccination strategies. Instead, bolstering an innate immune response during stressful events in animal production or periods of immune dysfunction could reduce pathogen recrudescence and infection susceptibility. One possible intervention to address this vulnerability is the prophylactic use of biotherapeutic proteins, such as cytokines [reviewed in Ref. ]. GranulSalmonella infection  colonization and shedding in swine, as well as decreased Salmonella-induced disturbance of the gastrointestinal microbiota, suggesting prophylactic use of porcine Ad5-G-CSF may serve as a biotherapeutic approach to reduce Salmonella in pigs.Rapid neutrophil influx into the intestines is the hallmark of a nfection , 16. In hallenge , 18. Fureraesuis . Thus, wSalmonella-fecal-negative sows were weaned at 12 days of age and shipped to the National Animal Disease Center, Ames, IA, USA. Siblings from each litter were divided and raised in two isolation rooms. Piglets tested fecal-negative for Salmonella spp. twice over a 2-week period using bacteriological culture with selective enrichment (10 TCID50/pig of a replication-defective human adenovirus (Ad5) engineered to express porcine G-CSF  (n = 7). Four days later, all pigs (n = 16) were intranasally inoculated with 1 × 107 colony forming unit (CFU) of a nalidixic acid-resistant derivative of S. enterica serovar Typhimurium UK1 . Blood samples were collected from the jugular vein at −4, −2, 0, 1, 2, 3, and 7 d.p.i. for enumeration of circulating blood cells by flow cytometry (see below). At 7 d.p.i., all pigs were euthanized and necropsied to obtain tissue samples from the tonsil and the intestinal tract  for quantitative and qualitative Salmonella culture analysis (see below). Procedures involving animals followed humane protocols as approved by the USDA, ARS, NADC Animal Care and Use Committee in strict accordance with the recommendations in the Guide for the Care, and Use of Laboratory Animals of the National Institutes of Health.Sixteen crossbred, conventionally reared piglets from three richment . At 5 we, n = 9) . As prevrium UK1  that hadSalmonella by serogroup antiserum agglutination . The total number of CFU for each quantitative tissue or fecal sample was calculated per gram of sample by obtaining the number of Salmonella per plate and multiplying by the dilution factor.For quantitative bacteriology, 1 g of pig feces was combined with 5 ml PBS, vortexed, and 0.1 ml directly plated to XLT-4 medium  containing 30 μg/ml of nalidixic acid. For tissue samples, 1 g of each tissue was combined with 2 ml of PBS in a whirlpak bag, pounded with a mallet, and homogenized in a Stomacher  for 1 min. One hundred microliters of the resulting solution was aliquoted onto XLT-4 medium containing nalidixic acid. One hundred microliters of a 10-fold dilution of each fecal and tissue sample was also plated, and additional dilutions were performed when CFU reached >300/plate. Following 48 h of incubation at 37°C, black colonies were enumerated and a single colony from each plate was confirmed to be Salmonella was performed as follows: 1 g  or 0.1 ml (homogenized tissue) samples were inoculated in 10 ml tetrathionate broth  for 48 h of growth at 37°C. Following incubation, 0.1 ml of each culture was transferred to 10 ml Rappaport–Vassiliadis medium  and incubated at 37°C for 18–20 h. Cultures were streaked on XLT-4 medium containing nalidixic acid. Colonies suspicious for Salmonella were confirmed by serogroup antiserum agglutination.Qualitative bacteriology of Salmonella shedding in feces (CFU/g) was Log10-transformed and analyzed using a mixed linear model for repeated measures . Covariance structures within pigs across time were tested and modeled using the REPEATED statement to determine the optimal covariance structure. Linear combinations of the least-squares mean estimates were used in a priori contrasts after testing for a significant (P < 0.05) treatment group effect. Comparisons were made between each group at each time point, using a 5% level of significance (P < 0.05) to assess statistical differences. The endpoint data for bacterial colonization (CFU/g) of tissues collected at necropsy were Log10-transformed and analyzed by analysis of variance using a general linear model for unbalanced data. A 5% level of significance (P < 0.05) was used to assess statistical differences.Statistical analysis for P < 0.05) was used to assess statistical differences.White blood cell counts were performed via flow cytometry as previously described . BrieflyAmplicon libraries of the 16S rRNA gene were generated and sequenced according to Kozich et al. , with ouContig assembly, sequence alignment, chimera removal, and non-bacterial sequence removal were performed in the program mothur (version 1.33.3) . SequencS. Typhimurium challenge on circulating neutrophils were determined by enumerating neutrophils in the blood at various days after Ad5-G-CSF administration and S. Typhimurium challenge. S. Typhimurium challenge alone induced a significant approximately threefold increase in circulating neutrophil counts, as values post-challenge were greater when compared to values on the day of challenge (day 0) , which was 4 days after Ad5-G-CSF administration, a significant neutrophilia occurred compared to pre-Ad5-G-CSF numbers . Collectively, Ad5-G-CSF administration induced a significant and sustained ~10-fold increase in the number of circulating neutrophils, and Salmonella challenge also induced significant increases in circulating neutrophils.The effects of Ad5-G-CSF administration and ) Figure A. CirculSalmonella shedding and tissue colonization were compared between Ad5-G-CSF and Ad5-empty-treated pigs. Ad5-G-CSF-treated pigs shed significantly less Salmonella (103 CFU/g feces) when compared to the Ad5-empy-treated pigs at 2 and 3 d.p.i. (104–5 CFU/g feces) . Gastrointestinal tissues  were analyzed at 7 d.p.i., and all tissues were Salmonella positive in both Ad5-G-CSF-treated and Ad5-empty-treated pigs. Of these three tissues, the Peyer’s patch region of the ileum exhibited a significant 0.5-log reduction in Salmonella colonization in the Ad5-G-CSF-treated pigs compared to the Ad5-empty-treated group . By contrast, all seven Ad5-empty-treated pigs harbored Salmonella in the tonsils at an average of ~10,000 CFU/g. These data suggest that prophylactic administration of Ad5-G-CSF can reduce Salmonella colonization and subsequent fecal shedding, including the tonsils that have been implicated in the carrier-status of Salmonella , but the microbiota of pigs that received Ad5-empty treatment was significantly different at day 7 compared to all previous time points . However, the difference between the Ad5-G-CSF-treated and Ad5-empty-treated groups at day 7 was insignificant. The dissimilarity of the microbiotas between days 3 and 7 was visualized via an NMDS plot, which showed the disturbed microbiota at day 7 in the Ad5-empty-treated animals compared to Ad5-G-CSF-treated animals  for Salmonella in pigs in the European Union concluded that interventions should focus on decreasing the level of Salmonella in the feces of infected swine because the vast majority of human risk is derived from a subset of pigs with a high concentration of Salmonella in their feces (≥104 CFU/g)  . In our Salmonella colonization of the tonsils in the Ad5-G-CSF-treated pigs also highlights prophylactic treatment with G-CSF as a potential control strategy for persistently infected pigs. Salmonella can reside in lymph nodes and especially the tonsils . While additional investigations of the efficacy and safety of Ad5-G-CSF administration in swine are warranted, our data suggest that increasing the number of circulating neutrophils via Ad5-G-CSF administration may offer a non-specific yet effective method for reducing Salmonella colonization in swine.The dramatic reduction in  tonsils –31. In tdescence , 35. RedSalmonella, that are capable of taking advantage of an inflamed environment (Salmonella colonization of the porcine gastrointestinal tract causes a disturbance within the gut microbial community (Salmonella during the initial stages of colonization could reduce overall gut inflammation and subsequently prevent the development of a “nutrient-niche” that can be selectively used by Salmonella (Salmonella-induced microbiota disturbance. Nevertheless, neutrophils are a primary player in the inflammatory response, and their contribution to the inflammatory response that provides an optimal environment for Salmonella expansion needs to be considered with an intervention strategy that increases neutrophils in circulation. It may be important to establish an optimal neutrophilia for the greatest Salmonella reduction with minimal neutrophil-stimulated tissue damage that, in itself, could encourage S. Typhimurium virulence factor-induced inflammation (Inflammation-associated intestinal dysbiosis can result in pathogen expansion, especially for microorganisms, such as ironment . We haveommunity  and trigommunity , 18. Intlmonella . In the ammation .As regulatory and public scrutiny necessitates the judicious use of antibiotics in food animals , 40, theSB, BB, CL, HA, DM, and MK conceived and designed experiments. SB, BB, CL, HA, IL, and DM performed the experiments. SB, BB, CL, HA, and MK wrote and edited the manuscript.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer. 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.