Ectomyelois ceratoniae Zeller , is the main pest of pomegranate, Punica granatum L., in Iran and many parts of the world. In this study, the effects of 11 commercial cultivars of pomegranate  were evaluated on life history variables, nutritional performance, and energy reserves of E. ceratoniae under the following laboratory conditions: 30 ± 1°C, 60 ± 5% RH, and a photoperiod of 14:10 (L:D) hours. In addition, biochemical characteristics of the tested cultivars were assessed in order to understand any possible correlation between important demographic parameters and nutritional properties with biochemical features of pomegranate juice. Our research showed that various pomegranate cultivars have significant effects on life history, demographical parameters, nutritional indices, and energy reserves of E. ceratoniae. The shortest development time was observed on Shahvare-Danesefid cultivar and the longest was on Esfahani-Daneghermez and Malase-Danesyah cultivars. The highest intrinsic rate of increase (rm) was observed on Shahvare-Danesefid and the lowest was on Esfahani-Daneghermez. Six major anthocyanin compounds were detected in juice of various pomegranate cultivars. Significant positive or negative correlations were observed between life history variables and nutritional characteristics with biochemical traits of pomegranate cultivars. The results indicated that Shahvare-Danesefid was a relatively susceptible pomegranate cultivar and Esfahani-Daneghermez was the least appropriate (most resistant) cultivar for feeding of E. ceratoniae, which could be useful in the development of integrated pest management strategies for this pest.The carob moth,  Ectomyelois ceratoniae Zeller , has been recognized as one of the most destructive insect pests of pomegranate and other fruits such as date, almond, carob, nuts, walnut, fig, and pistachio, with a broad distribution in Iran and many tropical and subtropical regions (Punica granatum L. (Ponicaceae), is the main host of the carob moth E. ceratoniae in Iran. The larvae feed inside the fruit, resulting in contamination with saprophytic fungi and causing great damage during the cropping season and after harvest, conditions that make the fruit unfit for human consumption  in pomegranate orchards (The use of resistant cultivars could be an effective approach in integrated pest management (IPM) strategies targeting the carob moth  cultivars, including Aban-Mahi, Esfahani-Daneghermez, Gabri, Gorche-Tafti, Malase-Danesyah, Malase-Yazdi, Shahvare-Daneghermez, Shahvare-Danesefid, Tabolarze-Mehrmahi, Tafti, and Toghe-Gardan, which are commercially important in Iran, were selected from mature trees grown in Agricultural and Natural Resources Research Center (ANRRC) of Yazd, Iran . Commercially ripe fresh fruits were randomly selected and harvested from different mature trees to represent the population of the plantation during September 2016. All cultivars were grown within the same geographical zone, using similar agronomic practices. The fruits were transported by a ventilated car to the laboratory, where pomegranates with defects  were discarded. Approximately 10 kg (n = 60) of pomegranate fruit was sampled for each cultivar. Five pomegranates of each cultivar were used for juice extraction, which was kept frozen at −80°C until analysis. The remainder of the fruits was placed in paper bags, stored in refrigerators at 2°C and then used for feeding of larvae.Fruits of 11 pomegranate , between August and October 2016. The pomegranate fruits infested with carob moth larvae were maintained in a growth chamber at 30 ± 1°C, relative humidity of 60 ± 5%, and under a photoperiod of 14 h:10 h (L:D). New adults that emerged from infested fruits were caught with an electric aspirator and transferred to the mating cages (50 cm × 50 cm × 100 cm) for 24 h . In order to ventilation, the middle part of the lid of Petri dish was cut in a circle (diameter 3 cm) and then covered with a cloth mesh. The Petri dishes contained a part of pomegranate fruit for feeding of larvae. The fruit pieces were replaced with fresh ones every 2 days during the larval development. The fifth instar larvae of E. ceratoniae reared on each cultivar were weighed separately 24 h after their emergence. The Petri dishes were checked daily, and the duration of immature stages , and immature survival rate were recorded. Pupal weight of E. ceratoniae was measured within 24 h of pupation on each pomegranate cultivar . All experiments on pomegranate cultivars were carried out under the following laboratory conditions: 30 ± 1°C, relative humidity of 60 ± 5%, and a photoperiod of 14:10 (L:D).The experiments on each pomegranate cultivar started using same-aged eggs (within 24 h after oviposition) laid by females reared as larvae on the same cultivar. Sixty eggs of E. ceratoniae eggs deposited in each tube was recorded. In this way, each pair was daily transferred to a new tube, the number of eggs laid by adults was counted, and the experiments were continued up to the death of the last adult female and male moth. To supply a source of carbohydrate for adult feeding, a small cotton wick soaked in 10% honey solution was inserted into the egg- laying container. In this experiment, adult preoviposition period , total preoviposition period (TPOP: the duration from egg to first oviposition), oviposition period, fecundity (eggs laid during the reproductive period), and adult longevity were recorded. Resulting data were analyzed based on the age-stage, two-sex life table model developed by After emergence of adult moths, a pair of female and male moths was transferred to the same mating cages as described above and maintained for 24 h. Then, each pair was placed in a transparent plastic tube fitted with mesh lids (6 cm diameter and 15 cm depth). Experimental tubes were checked daily and the number of E. ceratoniae fourth-instar larvae on 11 pomegranate cultivars (40 replicates for each cultivar). Nutritional indices were evaluated on the basis of dry weight. Fourth-instar larvae were placed on fruit of each pomegranate cultivar after weighing and transfer into Petri dishes . Larval weight was recorded daily, before and after feeding, for a 1-week experimental period. The fresh food provided, the remaining food, and feces at the end of each experiment were weighed daily. The quantity of ingested food was calculated by subtracting the weight of remaining food at the end of each experiment from the weight of fresh food at the beginning. To obtain the dry weight percentage of the food and larvae, 20 specimens of food and larvae for each cultivar were weighed, oven-dried at 60°C for 48 h, and then weighed again. Nutritional indices of larval E. ceratoniae were calculated using the ∗T; relative growth rate (RGR) = P/A∗T; where A = mean dry weight of larvae over the feeding period (g), E = dry weight of food consumed (g), F = dry weight of feces produced (g), P = dry weight gain of larvae (g), and T = duration of feeding period (day).The gravimetric method described by E. ceratoniae larvae (protein and glycogen contents), we used the whole body of fourth-instar larvae feeding on each pomegranate cultivar. All assays were replicated three times.To measure the energy content of Total protein concentration of individual larvae was assessed using the E. ceratoniae larva was extracted according to the Glycogen content was assayed using the anthrone reagent as described  and wholE. ceratoniae, on the one hand, and biochemical compounds founds in various pomegranate cultivars. All assays (each pomegranate cultivar) were done in three replicates. All biochemical metabolites of pomegranate were measured using pomegranate juice. Before juice extraction, fruits of 11 pomegranate cultivars (five from each cultivar) were washed in cold tap water and dried, and the damaged pomegranates were discarded. The juicy sacs from fruit pericarp were separated by hand and the juices were obtained using a manual juicer via pressing the arils. The juice samples (200 mL) were immediately centrifuged , divided into small vials and frozen at −80°C until analysis . Anthocyanins in juices were determined by high-performance liquid chromatography (HPLC) using an HPLC system equipped with an Empower software, a pump (Waters 600), a Rheodyne 7125i six-way injector with 20 μL sample loop, and a UV–Vis detector (Waters model 2487). Twenty microliters of purified juice was injected onto the HPLC column. Calculation of concentrations was based on the external standard method and anthocyanins were identified by comparison of their retention times with those of pure standards.Protein content of pomegranate cultivars we tested was determined according to the The carbohydrate content was assayed using the Anthrone reagent (0.05% in sulfuric acid) .Condensed tannin content reactive to Sulphosphovanillin reagent (orthophosphoric acid 0.6% aqueous vanillin solution 4:1 v/v) was analyzed according to Total anthocyanin contents were measured by pH differential method using two buffer systems: potassium chloride, pH 1.0 (0.025M) and sodium acetate, pH 4.5 (0.4M) . ResultsTotal phenolic content was determined using Folin–Ciocalteu method described by Total flavonoid content was measured by aluminum chloride colorimetric assay . Total fEvaluation of antioxidant activity was done based on radical scavenging properties of the juice using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method .Total titrable acidity was determined by titration to pH = 8.1 with 0.1M NaOH solution and expressed as grams citric acid per liter of juice. The pH measurements were performed using a digital pH meter at 20°C. Total soluble solid content (SSC) was determined with a digital refractometer. Results were reported as°Brix at 20°C .rm), gross reproductive rate (GRR), net reproductive rate (R0), mean generation time (T), and finite rate of increase (λ) were analyzed according to the age-stage, two-sex life table using TWOSEX-MSChart (P < 0.05) (lx) and age-specific fecundity (mx) for both male and female  . The devd female . Additiokey test . CorrelaE. ceratoniae on various pomegranate cultivars is given in E. ceratoniae on the Shahvare-Danesefid cultivar was significantly shorter than on the other cultivars except for Malase-Yazdi. The longest pupal period was observed on the Esfahani-Daneghermez and Malase-Danesyah cultivars, whereas a shorter period was observed on the Toghe-Gardan cultivar. The shortest developmental time from egg to adult emergence was on the Shahvare-Danesefid cultivar (36.41 ± 0.31 days) and the longest was on the Esfahani-Daneghermez (41.80 ± 0.26 days) and Malase-Danesyah (41.46 ± 0.28 days) cultivars. The immature survival rate ranged from 0.68 ± 0.06 to 0.87 ± 0.05 with the lowest and highest survival rates observed on Esfahani-Daneghermez and Shahvare-Danesefid cultivars, respectively  of E. ceratoniae on different cultivars is shown in lx curve was more extended on this cultivar.The age-specific survival rate . Moreover, pupal weight showed significant variation as a function of the pomegranate cultivar tested . The highest values were observed on the Shahvare-Danesefid and Toghe-Gardan cultivars and the lowest ones were recorded on the Malase-Danesyah and Esfahani-Daneghermez cultivars  varied significantly as a function of the pomegranate cultivars used as food source. The TPOP was longest on the Esfahani-Daneghermez cultivar and shortest on Toghe-Gardan. Moreover, the oviposition period was the longest when E. ceratoniae females were reared on the Shahvare-Danesefid cultivar and the shortest when reared on the Esfahani-Daneghermez, Malase-Danesyah, and Malase-Yazdi cultivars. Total fecundity (mean number of eggs laid during the reproductive period) of E. ceratoniae varied significantly as a function of the 11 pomegranate cultivars tested; the highest values were observed on the Shahvare-Danesefid (82.74 ± 2.01 eggs) and Toghe-Gardan (70.46 ± 2.30 eggs) cultivars and the lowest was on Esfahani-Daneghermez (36.91 ± 1.35 eggs)  .mx) for E. ceratoniae reared on the pomegranate cultivars tested are shown in mx curve  was the greater on the Shahvare-Danesefid cultivar than the others.Age-specific fecundity rates  while those reared on Esfahani-Daneghermez showed the lowest GRR value. The net reproductive rate (R0) was the lowest on the Esfahani-Daneghermez cultivar and the highest on the Shahvare-Danesefid cultivar. The intrinsic rate of increase (rm) values ranged from 0.053 ± 0.004 to 0.090 ± 0.004 female progeny per female per day on the pomegranate cultivars under study  was similar to that observed for the intrinsic rate of increase while the mean generation time (T) of E. ceratoniae was shortest on the Toghe-Gardan cultivar . Also, the highest weight gain of larvae  was observed when larvae were fed on Shahvare-Danesefid  as well as efficiency of conversion of digested food (ECD)  compared with larvae fed on the other cultivars  was observed on the Esfahani-Daneghermez and Tabolarze-MehrMahi cultivars, while larvae reared on Gorche-Tafti had a lowest RCR. In addition, the highest value of relative growth rate (RGR)  was obtained for larvae reared on the Shahvare-Danesefid cultivar  was observed in those fed on the Shahvare-Danesefid cultivar . Similarly, the larvae fed on this cultivar showed the highest whole-body glycogen content . Larvae fed on the Shahvare-Danesefid and Tafti cultivars showed the highest lipid content  while the lowest value was observed for larvae reared on the Malase-Danesyah cultivar  compound compared with other cultivars. The cyanidin 3,5-diglucoside  was significantly higher in Esfahani-Daneghermez. Also, the highest value of delphinidin 3-glucoside , delphinidin 3,5-diglucoside , pelargonidin 3-glucoside  and pelargonidin 3,5-diglucoside  compounds were measured in Esfahani-Daneghermez  among the pomegranate cultivars. The carbohydrate content varied from 92.61 mg/mL in Esfahani-Daneghermez to 138.33 mg/mL in Shahvare-Danesefid cultivar . Furthermore, a higher content of condensed tannins was measured in Esfahani-Daneghermez and Shahvare-Daneghermez, whereas a lower content was detected in Shahvare-Danesefid and Toghe-Gardan cultivars   and total monomeric anthocyanin pigment (TMAC)  contents of different pomegranate cultivars. A higher value of TPC and TMAC was measured in Esfahani-Daneghermez cultivar and the lowest value of TMAC was detected in Shahvare-Danesefid and Tafti cultivars. The highest flavonoid content was measured in Esfahani-Daneghermez, whereas the lowest content was observed in the Shahvare-Danesefid and Toghe-Gardan cultivars   . There w 0.0001) .F = 8.30; df = 10, 32; P < 0.0001). Moreover, the highest pH value was observed in the Shahvare-Danesefid cultivar while the lowest value was in the Malase-Danesyah cultivar . The highest total titrable acidity value was obtained for the Malase-Danesyah cultivar, whereas the lowest value was seen for the Shahvare-Danesefid cultivar . The percentage of juice showed limited variation, ranging from 50.15 to 61.32 for the pomegranate cultivars under investigation . The soluble solids content was lowest in the Tabolarze-Mehrmahi cultivar  (Antioxidant activity (AA%) varied from 49.17 in Tafti to 60.13 (AA%) in the Esfahani-Daneghermez cultivar (= 0.040) .E. ceratoniae fed on different cultivars, on the one hand, and different biochemical traits of the various pomegranate cultivars tested, on the other hand; significant positive or negative correlations were observed  of pomegranate cultivars. Development time was positively correlated with major anthocyanin constituents, condensed tannins, total phenolic content, total monomeric anthocyanin pigment content, flavonoid, DPPH, and total titrable acidity of pomegranate cultivars, suggesting that pest development rate is negatively affected by respective metabolites and that these factors play a decisive role in the fitness of this insect. The longer development time of E. ceratoniae on Esfahani-Daneghermez cultivar may be due to higher levels of secondary metabolites n this cultivar. The range of larval and pupal periods of E. ceratoniae on tested cultivars is somewhat in agreement with the findings of In the present research, rbivores . Variatient time . The conE. ceratoniae in the current study was greater than that reported by E. ceratoniae. The reduced body weight of this pest on cultivars Malase-Danesyah and Esfahani-Daneghermez might be attributed to the lower carbohydrate content and higher contents of secondary metabolites in these cultivars.It has been reported that body weight is associated with the quality and quantity of food, which is one of the main biological indices of insect population dynamics . The lowE. ceratoniae fed on pomegranate cultivars were positively correlated with levels of the major anthocyanin constituents and secondary compounds, suggesting that phytochemical metabolites play a negative role in the growth of this insect. In this study, the range of male and female longevities of E. ceratoniae was close to those reported earlier for E. ceratoniae reared on three pomegranate cultivars, including Malase-danesyah, Gabri, and Shahvar . Sub-cluster A2 consisted of Esfahani-Daneghermez and Malase-Danesyah (most relatively unsuitable cultivars), and other cultivars were grouped in sub-cluster A1 based on their similarity in primary and secondary metabolite contents .In the present study, cluster analysis with respect to life table parameters, nutritional indices, and energy reserves of E. ceratoniae to feeding on various pomegranate cultivars, albeit with different degrees of success. Our results indicate that insects fed on the Shahvare-Danesefid cultivar displayed the fastest development and highest adult fecundity and longevity, immature survival rate and nutritional performance, apparently as a consequence of the higher primary metabolite and lower secondary metabolite contents of the host plant. Therefore, this cultivar was the most suitable (least resistant) cultivar for E. ceratoniae, among those tested here. Moreover, the Esfahani-Daneghermez cultivar was the least suitable (most resistant) pomegranate cultivar for E. ceratoniae, apparently as a consequence of the higher secondary metabolite contents of the host plant. Consequently, Esfahani-Daneghermez was designated as the most unsuitable cultivar among those tested in the present work; we recommend that it be grown in areas where damage by E. ceratoniae is typically high, with the view to protecting or at least delaying infestation of pomegranate by this pest.Results of the present study point to the apparent adaptability of All authors read and approved the manuscript. This research is a part of Ph.D. thesis of ZA. AG as supervisor and ZA proposed the research subject and wrote the manuscript. ZA conducted the experiments. MS, MH, AJ-N, and H-RA participated in the data analysis and acted as advisors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We demonstrate that IIH patients have a unique signature of androgen excess and provide evidence that androgens can modulate CSF secretion via the choroid plexus. These findings implicate androgen excess as a potential causal driver and therapeutic target in IIH.Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology, characterized by elevated intracranial pressure frequently manifesting with chronic headaches and visual loss. Similar to polycystic ovary syndrome (PCOS), IIH predominantly affects obese women of reproductive age. In this study, we comprehensively examined the systemic and cerebrospinal fluid (CSF) androgen metabolome in women with IIH in comparison with sex-, BMI-, and age-matched control groups with either simple obesity or PCOS . Women with IIH showed a pattern of androgen excess distinct to that observed in PCOS and simple obesity, with increased serum testosterone and increased CSF testosterone and androstenedione. Human choroid plexus expressed the androgen receptor, alongside the androgen-activating enzyme aldoketoreductase type 1C3. We show that in a rat choroid plexus cell line, testosterone significantly enhanced the activity of Na Women with idiopathic intracranial hypertension have a unique androgen excess signature that is associated with modulation of cerebrospinal fluid secretion dynamics. Idiopathic intracranial hypertension (IIH) is a chronic and disabling condition characterized by elevated intracranial pressure (ICP) . PatientIIH shares phenotypic characteristics with polycystic ovary syndrome (PCOS), in which androgen excess is a defining feature . Studiesex vivo investigations, comprehensively examining the androgen metabolome in serum, urine, and CSF in a large cohort of women with active IIH (papilledema and lumbar puncture pressure >25 cm CSF on the day of sample collection) compared with sex-, age-, and BMI-matched women with PCOS or simple obesity. We used state-of-the-art mass spectrometry–based analysis and employed cell models to investigate the functional role of androgens in CSF secretion.We aimed to delineate the androgen phenotype of women with active IIH and to explore the potential role of androgens in disease pathogenesis. To this end, we have performed complementary in vivo and n = 70), simple obesity (n = 40), and PCOS (n = 60) (P < 0.001). Conversely, the androgen precursor androstenedione (P < 0.001) and obesity (P < 0.05). In keeping with previous studies, serum androstenedione was higher in PCOS than in controls with obesity (P < 0.01) and IIH (P < 0.001). 11-Oxygenated androgens are adrenal derived and have recently been shown to be the major contributor to androgen excess in PCOS (P < 0.001) compared with controls with IIH and obesity.Age and BMI were not significantly different among the 3 groups comprising women with IIH ((n = 60) . We meas(n = 60) . Serum c(n = 60)  were sigenedione  was sign in PCOS ; therefo in PCOS  and 11KA in PCOS  were incP < 0.05).Systemic steroid metabolism in 24-hour urine samples was profiled using gas chromatography-mass spectrometry in the 3 study groups . The ratn = 9, demyelination n = 6, cerebrovascular disease n = 3, normal investigations n = 5, other n = 8) (P = 0.01); lean controls were significantly older (P < 0.001) than the IIH patients (https://doi.org/10.1172/jci.insight.125348DS1). CSF levels of the active androgen testosterone were significantly higher in IIH patients  but did not differ significantly between other groups, with CSF DHEAS highest in the obese control group and significantly greater in both lean and obese controls than in women with IIH ( < 0.0001). 11-Oxygenated androgens were detectable in CSF in both IIH patients and control women but did not differ significantly between the 2 groups. Serum androstenedione and testosterone both correlated significantly with CSF androstenedione and testosterone . Total amounts of serum and CSF androgens  were also positively correlated .We measured CSF androgen concentrations in 55 women with IIH, 19 healthy volunteer controls with obesity and no known comorbidities, and 31 lean controls (indications for lumbar puncture: headache r n = 8) . The obewith IIH  < 0.0001There were no significant correlations between serum or CSF androgen concentrations and BMI or markers of IIH disease activity  .n = 5) . These included 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), aldoketoreductase type 1 C3 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3), 5α-reductase type 1 (SRD5A1), aromatase (CYP19A1), 11β-hydroxylase (CYP11B1), aldoketoreductase type 1 D1 (AKR1D1), and the androgen receptor (AR)] , comparing expression levels to female human ovary, liver, and adrenal tissues (n = 3). Expression levels of the steroidogenic enzymes AKR1C3 (activation of androstenedione to testosterone), 17BHSD3, SRD5A1, and AR in the human choroid plexus samples were equivalent to or greater than expression levels observed in adrenal and ovarian tissue. Expression of SRD5A2, HSD3B2, CYP19A1, and AKR1D1 was negligible or undetectable in all samples. Furthermore, CYP11B1, which catalyzes the conversion of the classic androgen precursor androstenedione to 11-hydroxyandrostenedione, was also not expressed in human choroid plexus (HSD11B2 expression in human choroid plexus (IIH pathogenesis is speculated to involve either increased CSF secretion (predominantly at the choroid plexus) or reduced CSF-draining pathways . We haveor (AR)] , as welld plexus . This isAr  and the area under the curve of the ratio over 15 minutes , indicating increased Na+/K+-ATPase activity and by implication CSF production (Car2 and Car3), which establish the ion gradient to drive CSF secretion, were significantly upregulated (both P < 0.05) in testosterone-treated Z310 cells at 48 hours compared with vehicle-treated controls. Levels of Na+/K+-ATPase (Atp1a1) expression did not change.  expressed key steroidogenic enzymes and Ar . To deteAr  in Z310 ecretion . Testostoduction . Express change. .In the current study, we provide potentially novel insights into potential mechanisms relating to the pathogenesis of IIH. We highlight a distinct profile of androgen excess in women with IIH and demonstrate an impact of androgens on surrogate markers of CSF secretion in a cell model, providing evidence for a pathogenetic link between androgen excess and increased ICP in IIH.We show that women with IIH have a unique circulating androgen excess signature, with significantly higher active testosterone but lower concentrations of the androgen precursors DHEA and androstenedione than in women with PCOS or simple obesity. Previous studies of androgen metabolism in PCOS show complex contributions from both the classic  and 11-oAKR1C3 in human choroid plexus, indicating the presence of the enzymatic machinery required for activation of androstenedione to testosterone. Significant aromatization of androstenedione to estrone, or testosterone to estradiol, within the choroid plexus appears unlikely in view of the low levels of choroid plexus CYP19A1 expression and the well-recognized poor efficiency of this enzyme in converting androgens to estrogens . A further cohort of healthy control women was recruited from Adelaide and Meath Hospital, Dublin, Ireland. PCOS was diagnosed according to the Rotterdam European Society of Human Reproduction and Embryology 2004 criteria .The female lean control cohort for CSF evaluation consisted of patients with non-IIH neurological disease (after providing written informed consent) and a control cohort with obesity (with no known comorbidities) recruited solely for research evaluation through local advertising. Written informed consent was provided in all cases.g for 10 minutes and aliquoted. All samples were stored at –80°C and analyzed after a maximum of 1 freeze-thaw cycle.Exclusion criteria for the study were recent glucocorticoid therapy (within 3 months), pregnancy, recent oral contraceptive or antiandrogen use (within 3 months), hyperprolactinemia, and overt hyperglycemia. PCOS was excluded on clinical and biochemical assessment from the control cohorts. The cohorts were whole-group matched  for comparison unless otherwise stated. Participants attended the NIHR/Wellcome Trust Clinical Research Facility at University Hospital Birmingham after an overnight fast. Baseline anthropometric data were collected and serum samples were drawn for measurement of serum androgens. Precollected 24-hour urine samples for urinary steroid metabolite profiling were also provided by patients upon arrival to the research facility. In IIH patients, CSF samples were also collected by lumbar puncture (LP). CSF and blood samples were transported on ice before being promptly centrifuged at 226 x Visual assessments were recorded during the research visit. Best corrected visual acuity was measured with a Humphrey Visual Field analyzer using the Swedish Interactive Threshold Algorithm Standard 24-2 program and mean deviation was recorded. OCT  was acquired to record average and maximal retinal nerve fiber layer thickness. LP was performed after all visual assessments. LP pressure was evaluated with the participant breathing steadily in the lateral position, legs flexed 90° at the hip, with adequate time taken to ensure a stable reading. Headache disability was recorded using the HIT-6 . Quality+/K+-ATPase is a key rate-limiting step in driving CSF secretion  or acetazolamide  before recording ATP/ADP and applying 1 mM ouabain, the increase in Perceval fluorescence reflecting the portion of ATP consumed by Na+/K+-ATPase activity. A HEPES-bicarbonate buffer was used, containing the following in mM: 120 NaCl, 4.8 KCl, 24 NaHCO3, 0.5 Na2HPO4, 5 HEPES, 2.5 CaCl2, 1.2 MgCl2, and 25 D-glucose.Assays were conducted in Z310 cells , an immortalized cell line derived from primary rat choroid plexus epithelium transfected by simian virus 40 T antigen . Cells wmin was determined for each frame, where F is the intensity for the cell in that particular frame and Fmin is the lowest fluorescence intensity in the baseline period. This normalizes the change intensity to baseline, taking into account any quantitative difference in ATP/ADP ratio between cells or differences in Perceval protein expression. We used ouabain administration to initiate a change in ATP/ADP ratio or Δ .Images were acquired with MetaMorph (Molecular Devices). An image sequence was initially analyzed on ImageJ , where individual random cells are selected as a region of interest and motile cells are excluded from the analysis. Following this, the multimeasure function was selected to determine the mean intensity of individual cells at each frame. Subsequent to this the F/Ftio or Δ . This Δ t test (2-tailed), Mann-Whitney U test, or Kruskal-Wallis testing. P values < 0.05 were deemed significant.Statistical analysis was performed using SPSS Statistics . All comparisons were performed using Student’s . Ethical approval for CSF samples from patients with neurological disease was granted by Solihull Research Ethics Committee (04/Q2706/65). The Parkinson’s UK Brain Bank has ethical approval from the Wales Research Ethics Committee as a research tissue bank, which covers the use of tissue from the Brain Bank by researchers.For procedures involving PCOS and healthy controls, approval was granted by the South Birmingham (LREC5835), Edgbaston (12/WM/0206), and Adelaide and Meath Hospital Dublin (2006/10/02) Research Ethics Committees. Ethical approval for recruitment of IIH patients was granted by the Dudley (06/Q2702/64), Yorkshire and the Humber–Leeds West (13/YH/0366), and Black Country (14/WM/0011) local Research Ethics CommitteesAJS, MWO, and WA conceptualized and designed the clinical study. All authors were involved in aspects of study conduct as well as drafting and approving the final manuscript.


R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods ( Förster resonance energy transfer (FRET) brings the sensitivity and speed of fluorescence spectroscopy to structure and distance determination . Since iThe major parameter that characterizes any FRET method is the Förster radius, the distance where the probability that the donor becomes deactivated by photon emission and external quenching is as likely as its deactivation by energy transfer to the acceptor . As donoEach donor is characterized by its natural fluorescence lifetime, the reciprocal decay rate in the absence of an acceptor. Depending on this lifetime, the donor can shorten its distance to the acceptor by Brownian motion, and, thereby, increase the probability of FRET events ,14. This6, with two donors of largely different radiative lifetimes, NAla and FTrp. The peptides were sufficiently long to avoid steric-hindrance effects and sufficiently short to lead to sizable FRET ble FRET . In addil-tryptophan required for peptide synthesis were prepared according to literature methods [3 M−1 cm−1 (FTrp) and 5.5 × 103 M−1 cm−1 (NAla) at 280 nm; they were adjusted to 10 µM in aerated solutions of varying ethylene glycol content, 25 °C, pH 5.0. The donor quantum yields were determined by comparison with N-acetyl-tryptophanamid  [Donor-acceptor and donor-only labeled peptides  were obt methods ,22. Othe pH 7.0) ,24,25. T pH 7.0) ,23.6-Dbo-NH2 and 5-F-Trp-(Gly-Ser)6-Dbo-NH2, and, in the absence of FRET, from the donor-only peptides, NAla-(Gly-Ser)6-NH2 and 5-F-Trp-(Gly-Ser)6-NH2. Upon excitation of NAla or FTrp at 280 nm and emission recording at 350 nm, the traces were analyzed by using the FLS920 instrument software  as described [Fluorescence lifetime decay traces in the presence of FRET were obtained from the donor-acceptor labeled peptides, NAla-(Gly-Ser)escribed  and by uDτrad) by plotting it as a function of Dτrad, or as a function of the product of the inverse viscosity times the inverse radiative lifetime or intrinsic decay rate (Dτrad ∝ η−1krad). Towards this goal, it will not be sufficient to only change the ethylene glycol content. To be convincing, we had to switch donors to achieve dramatically different radiative lifetimes that, together with the variation in viscosity, would allow us to cover two orders of magnitude changes in Dτrad. In the following, we outline the analysis that yielded the effective distances and the products of the inverse viscosities and radiative lifetimes.This study was designed to test whether the FDE depends symmetrically on the radiative donor lifetime and the end-to-end diffusion coefficient. The effective distance treated in the following depends only on the equilibrium distance distribution and the FDE; it decreases with increasing FDE and increases with decreasing FDE. As the end-to-end distance distributions of NAla- and FTrp-labeled peptides can be assumed to be identical, the effective distance changes only in dependence on FDE. Therefore, we can test whether FDE really depends only on the product of the diffusion coefficient and the radiative lifetime ((a) The effective distanceτDA, and of the donor-only peptides, τD, were converted into the corresponding decay rates, kD and kDA, according to Equation (1), which yielded the FRET rate constant, kFRET, according to Equation (2).The measured fluorescence lifetimes of the donor-acceptor-labeled peptides, At all ethylene glycol concentrations, the kinetic traces followed monoexponential time courses. In the case of the FTrp-labeled peptide at high ethylene glycol concentrations, biphasic fits seemed to be slightly more appropriate, and were used to calculate amplitude-weighted lifetimes, ith Ref. . The effThe Förster radius increases slightly with the ethylene glycol concentration because ethylene glycol increases the refractive index of the experimental solutions as well as the donor quantum yield of the experimental peptides. The experimental quantum yields were obtained as follows: The decay rate of the donor is the sum of the radiative and the non-radiative decay rate in the donor-only peptide (Equation (6)).D, is the ratio of the radiative decay rate, krad, and the decay rate, kD (Equation (7)). The values of the quantum yield and radiative decay rate in the absence of ethylene glycol have been reported [The donor quantum yield, Φreported .(7)ΦD=kn, of the experimental solution, which increases slightly with the ethylene glycol concentration (Equation (8)) [n0, equals 1.3328 under our conditions.The radiative decay rate depends on the refractive index, ion (8)) ,26. The Accordingly, the quantum yields were calculated from Equation (9):6 peptides in water, R0 = 9.6 Å; NAla/Dbo: R0 = 9.8 Å).How the Förster radii depend on the quantum yields and the refractive indices is captured in Equations (10) and (11). The Förster radii of the FTrp/Dbo and the NAla/Dbo donor/acceptor pair in the (GS)C, is calculated from Equation (11):The correction term, Accordingly, the Förster radii at a given ethylene glycol concentration were calculated from Equation (12):When the resulting R0, is also proportional to the sixth root of the orientation factor, κ2. The factor, κ2, captures that the dipole moments of the donor and acceptor can adopt various orientations towards each other and can vary between 0 (perpendicular vectors) and 4 (collinear vectors). The consequences of possibly misjudging the value of κ2 were recently discussed [The Förster radius, iscussed . The oriiscussed ; this isiscussed ,30, whiciscussed . Furtheriscussed . Thus, eiscussed  are met.(b) The product of radiative lifetime and diffusion coefficientτrad, (Equation (13)), and the diffusion coefficient, D; that is, on the product, Dτrad. We assumed that the diffusion coefficient is proportional to the reciprocal viscosity (Equation (14)). The radiative lifetimes have been determined as outlined above. If the effective distance depends symmetrically on the diffusion coefficient and the radiative lifetime (Equation (15)), it also depends symmetrically on the viscosity and radiative rate (Equation (16)), and vice versa. The relationship between the concentration of aqueous ethylene glycol solutions and their viscosity has been established in Ref. [xEG is the mole fraction of ethylene glycol.The assumption to be tested was that the FDE depends symmetrically on the radiative lifetime,  in Ref.  (Equatio(c) The end-to-end diffusion coefficient and distance probability distribution obtained from a global analysis based on the Haas-Steinberg equationN*, of chains with an excited donor in dependence of the donor-acceptor distance, N*/∂t, in donor-acceptor labeled chains to the sum of three terms accounting for the donor decay in the absence of FRET, for the donor decay caused by FRET, and for diffusion.The Haas-Steinberg equation ), first derived in reference , relatesN0*(r) or N0*, instantly after short-pulse donor excitation, mirrors the distance distribution in all peptide chains in the experimental sample because the probability of donor excitation does not depend on the distance of the acceptor. Thus, N0*(r), when normalized to ∫N0*(r) dr = 1, is identical to the probability distribution in the ground-state equilibrium ensemble of chains, i.e., N0*(r) = p(r). In the widely used skewed Gaussian distribution (Equation (19)), the meaningful parameters, a and b, determine the shape of the distribution, whereas c is a normalization constant determined by the condition that the integral of the probability density over all possible distances has to equal unity, ∫p(r) dr = 1.The initial distance distribution, kD, the Förster radius, R0, as well as the parameters describing chain conformation and dynamics, a, b, and D, are fed into the HSE—a linear partial differential equation, which can only be solved numerically, the HSE returns the fluorescence decay kinetics and, through further calculation, previously outlined in reference [Reff. As we determined the kD and R0 values for 48 donor/viscosity combinations, we could simultaneously analyze 48 equations, each with its own pair of kD and R0 values, with the diffusion coefficient, ηD, at viscosity, η, given by Equation (20)—where D and η0 are the values in water—and with the parameters of the distance distribution, a and b, whose values were kept constant in all 48 equations.In essence, when the photophysical parameters, the donor decay rate constant in absence of the acceptor, eference , the effa, b, and D were varied and the equations solved till the sum-of-squares difference between the computed and the experimental Reff values could not be further minimized. The result is illustrated in Figure 7 in the Results and Discussion section. This kind of optimization does not directly provide standard deviations of the parameters of interest. Therefore, we further tested the reliability of the obtained values by performing the same global optimization analysis on subsets that contained only a limited number (15 to 35) of data points, randomly chosen from the set of 48 experimental data points (200 optimizations). The computations were carried out in MATLAB (MathWorks).In the global optimization, 6 peptides occurs and how it is strongly influenced by ethylene glycol is illustrated in 6-Dbo-NH2, shows a fluorescence spectrum (red curve) with two regions of maximal intensity, one around 340 nm arising from NAla fluorescence and another at 440 nm due to Dbo fluorescence. Since Dbo in the absence of a donor cannot be optically excited at 280 nm [That FRET in the double labeled (GS)t 280 nm , it recet 280 nm .In τrad, of the NAla-only peptide  exceeds the radiative lifetime of the FTrp-only peptide (19.8 ns) by a factor of 13. While, in the presence of an acceptor, FRET shortens the donor lifetime –(16)). The viscosity of the experimental solutions increases exponentially with the ethylene glycol concentration 6-Dbo-NH2 (red) and in 5-F-Trp-(GS)6-Dbo-NH2 (blue) could now be obtained from Equation (5)  f.ηkrad, of viscosity, η, and inverse radiative lifetime, krad (ηkrad)−1, is directly proportional to D𝜏rad (Equations (13)–(16)). To ease the understanding of ηkrad, and, equivalently, D𝜏rad, which influences the FDE. With a decrasing FDE (6 model peptides seemed to be appropriate as their dimensions emerged to be a good match to the Förster radii (ca. 10 Å) of the sdFRET methods. The remarkable increase of the effective distance emphasizes that any FRET-based study and inferrence on peptide structure could be gravely incorrect if a possible FDE is simply ignored.With the combined data in hand, it is possible to retrieve the relationship between the effective distance and the product, me, krad , top. Thsing FDE , the effDτrad or ηkrad symmetry has important implications. In ηkrad. The dotted line marks the simultaneous switch from high to low viscosity and from a short to a long donor lifetime. If the coadditive has no impact on peptide dimensions, this course is continuous (case (2)). If the intrachain distances contract upon addition of the coadditive, they should expand again when the experimental series goes from highest to lowest ethylene glycol content (case (1)). If the intrachain distances expand upon addition of the coadditive, they should contract again when the experimental series starts again at 0% ethylene glycol (case (3)). In this study, we clearly observed a type-2 course. Even though ethylene glycol changes not only the viscosity and the refractive index of the solutions, but also the dielectric permittivity and other properties, it has, even at very high concentrations, no impact on the peptide dimensions.The How a coadditive influences peptide dimensions is a topic of interest to the protein folding community and in the field of natively unfolded proteins or intrinsically disordered peptides ,35. The 2/ns in the absence of ethylene glycol, and a skewed Gaussian distance distribution, p(r) = cr2∙exp(−a(r − b)2, with a = 1.27 × 10−3 Å−2 and b = −25.3 Å ), as described in the Material and Methods section. The result of the global optimization is shown in  −25.3 Å b at all 2/ns [2/ns [2/ns, reported in reference [16 peptides, is only slightly larger than our value of 55 Å2/ns for the (GS)6 peptides that we studied here. At short chain lengths, the diffusion coefficient is expected to grow with an increasing length of the probe-intermittent chain [End-to-end and site-to-site diffusion coefficients in linear polymers, mostly in peptides and proteins, have been published previously ,39,40,41ns [2/ns . Most ofns [2/ns ,36,39,40ns [2/ns . To dissns [2/ns . Haas etns [2/ns . Other pns [2/ns , or the ns [2/ns . We chalns [2/ns ,36. Howeeference  and measIn an outlook, we would like to emphasize that future applications of sdFRET are not limited to low-molecular weight polymers or short peptides. In investigations of protein folding processes, the ability to detect when two labeled chain sites approach each other closely can become crucial ,45,46,47D𝜏rad symmetry can be employed to conclude on the impact of viscogenic coadditives on peptide chain dimensions. Measurements in the presence of a viscogen can even provide absolute parameters of chain dynamics and dimensions.Through a two orders of magnitude variation in parameter range, we have shown that the FRET diffusion enhancement depends symmetrically on the diffusion coefficient and the radiative donor lifetime. That this relationship holds has been expected on the grounds of theoretical considerations , however


Patients with chronic obstructive pulmonary disease (COPD) have increased risk of cardiovascular events.This study evaluated the association between high-sensitivity cardiac troponin I concentration and cardiovascular events in patients with COPD and heightened cardiovascular risk.In a double-blind randomized controlled trial, 16,485 patients with COPD and cardiovascular disease or risk factors were randomized to once daily inhaled placebo, fluticasone furoate (100 μg), vilanterol (25 μg), or their combination. Plasma high-sensitivity cardiac troponin I concentrations were measured in a subgroup of 1,599 patients. Outcomes were on-treatment cardiovascular events and COPD exacerbations over a median of 18 months, and cardiovascular death over a median of 27 months.Baseline plasma cardiac troponin I concentrations were above the limit of detection (1.2 ng/l) in 1,542 (96%) patients. Concentrations were unaffected by inhaled therapies at 3 months (p > 0.05). Compared with the lowest quintile (cardiac troponin <2.3 ng/l), patients in the highest quintile (≥7.7 ng/l) were at greater risk of cardiovascular events  and cardiovascular death  after adjustment for risk factors. By contrast, there were no differences in exacerbations between quintiles .NCT01313676)In patients with COPD and heightened cardiovascular risk, plasma cardiac troponin I concentrations are a specific and major indicator of future cardiovascular events and cardiovascular death. Inhaled therapies did not affect cardiac troponin I concentrations consistent with their neutral effect on mortality and cardiovascular outcomes. (Study to Evaluate the Effect of Fluticasone Furoate/Vilanterol on Survival in Subjects With Chronic Obstructive Pulmonary Disease [SUMMIT];  Cardiovascular (CV) disease, including ischemic heart disease and stroke, accounts for 1 in 4 deaths globally and is increasing in prevalence Notwithstanding prior major advances, future clinical trial conduct is hampered by several important and increasing challenges. It is well recognized that clinical trial participants represent a relatively low-risk subset of the real-world patient population. Consequently, modest event rates necessitate large and costly trials in order to demonstrate treatment efficacy. Conversely, this low event rate creates the potential for researchers to fail to recognize CV harms related to new medications before their clinical approval An ideal CV biomarker needs to be a sensitive as well as a specific indicator of CV risk. High-sensitivity cardiac troponin I is such a potential suitable candidate. Plasma concentrations can be reliably quantified in most apparently healthy individuals, and numerous studies have demonstrated clear associations between elevated plasma troponin concentrations and CV events in both primary and secondary prevention populations SUMMIT  1) ≥50% and ≤70% of the predicted value, a ratio of post-bronchodilator FEV1 to forced vital capacity ≤0.70, and a score ≥2 on the modified Medical Research Council dyspnea scale. Patients were additionally required to have a history, or be at increased risk, of CV disease. CV disease was defined as coronary artery disease, peripheral arterial disease, prior stroke or myocardial infarction, or diabetes mellitus with target organ disease. Increased CV risk was defined as being ≥60 years and receiving medications for ≥2 of the following: hypercholesterolemia, hypertension, diabetes mellitus, or peripheral vascular disease.The prospective, multicenter, international randomized controlled SUMMIT trial sought to determine whether treatment with an inhaled LABA in combination with an ICS versus either component, could improve clinical outcomes in patients with moderate COPD and increased CV risk compared with placebo. Details regarding study design have been previously published Although prior ICS and LABA treatments were discontinued before study entry, other COPD medications were permitted during the trial. Participants were then allocated equally to 1 of 4 randomized treatments: placebo, fluticasone furoate (100 μg), vilanterol (25 μg), or their combination  inhaled once daily as a dry powder. A total of 16,485 patients were enrolled and included in the final intention-to-treat efficacy population.In addition to the primary endpoint of all-cause mortality by intention-to-treat analysis, the secondary CV endpoint was time to first-on-treatment CV event comprising CV death, myocardial infarction, stroke, unstable angina, and transient ischemic attack STAT high-sensitive cardiac troponin I assay , which has a limit of detection of 1.2 ng/l, coefficient of variation <10% at 4.7 ng/l, and sex-specific 99th percentile upper reference limits of 16 and 34 ng/l in women and men, respectively Venous blood samples were obtained before randomization and at 3 months. Blood was processed and plasma stored at −80°C until analyzed. As previously described, before analysis, samples were thawed and underwent centrifugation twice  according to the manufacturer’s instructions to ensure homogeneity A value of 0.5 ng/l was imputed for patients without reportable troponin values. Cardiac troponin I concentrations were log-transformed before statistical modeling, and results transformed back to the original scale. To determine which patient characteristics were associated with baseline cardiac troponin I, regression modeling was performed. The final model was achieved using backwards selection where to remain in the model all variables needed to have p < 0.10. To test whether ICS or LABA therapy affected cardiac troponin I values at 3 months, an analysis of covariance was performed adjusting for baseline cardiac troponin I, age, sex, prior myocardial infarction and hypertension.Patients were grouped into quintiles based on their baseline cardiac troponin I concentrations. To explore the effect of baseline cardiac troponin I quintile on each of the study endpoints , analysis of time-to-first event was performed using Cox proportional hazards regression modeling, adjusted for age, sex, study therapy, and CV risk factors of prior myocardial infarction and hypertension. In a sensitivity analysis, we also included statin therapy and C-reactive protein concentrations as model covariates. Cardiac troponin I was also examined as a continuous variable where the best fitting model was selected from a variety of polynomial or logarithmic models using 2-term fractional polynomials Previous reports have identified that adverse CV outcomes are associated with plasma troponin I concentrations ≥5 ng/l NCT01313676).Scientific oversight of the trial was provided by a steering committee composed of academic experts and employees from GlaxoSmithKline, who were collectively responsible for the study design and analysis, and for the review and interpretation of the data. This study is registered with ClinicalTrials.gov  and above the sex-specific 99th percentile  in 42 participants (2.6%). The median cardiac troponin I concentration was 4.0 ng/l (interquartile range [IQR]: 2.6 to 6.7 ng/l).The patient characteristics in the biomarker substudy population were broadly similar to the overall SUMMIT population, except that those in the biomarker population were more likely to be female, have a higher body mass index, have fewer previous COPD exacerbations, and have differences in CV history and CV therapy . Partici1 was associated with lower cardiac troponin I concentrations at baseline.A number of patient characteristics were associated with baseline plasma cardiac troponin I concentration . In a muIn the biomarker substudy, during a median on-treatment follow-up of 1.5 (IQR: 0.8 to 2.5) years, there were 74 patients (4.6%) with a composite CV event, and 587 (36.7%) patients with moderate or severe exacerbations of COPD. During a median on- and post-treatment follow-up of 2.3 (IQR: 1.6 to 3.1) years, there were 25 CV deaths.Compared with the lowest quintile, participants in the highest quintile were at greater risk of experiencing a CV composite event . This diPlasma cardiac troponin I concentrations at 3 months were unchanged from baseline  . There wOf the 1,258 patients with baseline and 3-month measurements, 673 (53%) had cardiac troponin concentrations <5 ng/l on both occasions. Compared with this group, patients who had a plasma troponin ≥5 ng/l at either time point had increased rates of the composite CV endpoint  and a markedly increased risk of CV death  . By contWe have identified a strong association between plasma high-sensitivity cardiac troponin I concentration and CV outcomes in patients with COPD at heightened CV risk. Importantly, this relationship was specific for CV events, particularly CV death, with no demonstrable association with the risk of COPD exacerbations despite the apparent association with baseline pulmonary function. Possible mechanisms underpinning this relationship have been described previously and may include increased inflammation, systemic hypoxia, or right ventricular strain associated with pulmonary hypertension Our findings highlight the potential use of high-sensitivity cardiac troponin concentration as a surrogate biomarker endpoint in early-phase clinical trials of CV interventions. Our findings also have important clinical implications. Recognizing the risk associated with increased troponin concentrations might encourage clinicians to address CV risk due to lifestyle choices and make patients more likely to engage with these recommendations. Similarly, improved risk stratification may facilitate more appropriate targeting of preventive medications This study has a number of notable strengths that distinguish it from previous reports on the use of plasma cardiac troponin within the outpatient setting. First, trial participants comprised a broad spectrum of risk, including primary and secondary prevention populations. Second, as a substudy within the context of a large international randomized trial, we ensured comprehensive follow-up and rigorous adjudication of clinical events. Third, the troponin assay chosen for this analysis is both widely available and analytically robust with <5% of samples below the limit of detection. Finally, the availability of paired plasma samples pre-treatment and after 3 months of therapy allowed assessment of any potential relationship between treatment-related changes in plasma troponin concentration and modification of clinical risk.Chronic obstructive pulmonary disease is an important risk factor for the onset of CV disease. Although there is a clear correlation between both these conditions and established predisposing factors such as age and smoking history, it appears that the chronic inflammatory milieu that exists in patients with COPD provides additional proatherosclerotic impetus Within this study, the troponin concentration that determined the upper quintile was 7.7 ng/l. Compared with the lowest quintile (<2.3 ng/l), the upper quintile was associated with a greater than 3-fold increased risk of all CV events and a 20-fold increased risk of CV death. The application of a troponin concentration threshold of ≥5 ng/l also robustly dichotomized individuals into high- and low-risk groups. This cutpoint of risk is remarkably consistent with previous descriptions Our analyses are nonrandomized comparisons, and there is a risk of bias if factors correlated with both troponin and the outcome were not included in our analysis. Due to the additional requirement for specimen collection and storage at baseline and 3 months, this substudy contained only 1,599 (10%) of the total number of participants included in the primary SUMMIT analysis. The baseline characteristics and clinical outcomes appear broadly consistent with the full study analysis although there were some differences in key characteristics such as sex, body mass index, and use of concomitant therapies. In the absence of electrocardiography, we were unable to adjust for the presence of arrhythmia, conduction defects, or left ventricular hypertrophy. Despite employing a robust and precise assay, the average troponin concentrations were low and similar to the concentration where total imprecision of the assay is 10%. This reinforces the need for physicians and trialists to be aware of the analytical characteristics of the locally available troponin assay before implementing cardiac troponin monitoring in clinical practice. Although analytical variation is modest, even at low concentrations PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: In patients with COPD, plasma concentrations of cardiac troponin I are associated with future fatal and nonfatal CV events but not with exacerbations of COPD. Long-acting beta-agonists or ICS, alone or in combination, do not reduce plasma troponin I concentrations or CV events.TRANSLATIONAL OUTLOOK: Serial measurements of high-sensitivity cardiac troponin I could act in future studies as a surrogate market of CV status in patients with COPD and potentially assess the impact of treatment before clinical events occur.In patients with combined respiratory and CV diseases, high-sensitivity plasma cardiac troponin I concentration is a prognostic marker that is specific to CV, but not respiratory, events. Plasma troponin I concentrations were not modified by the inhaled therapies for COPD investigated in this trial; a finding concordant with the primary SUMMIT findings. As such, high-sensitivity cardiac troponin I represents a plausible surrogate indicator of the CV consequences of novel medical therapies and interventions.


Iberian primitive breeds exhibit a remarkable phenotypic diversity over a very limited geographical space. While genomic data are accumulating for most commercial cattle, it is still lacking for these primitive breeds. Whole genome data is key to understand the consequences of historic breed formation and the putative role of earlier admixture events in the observed diversity patterns.ST values ranging from 4 to 16% and have levels of nucleotide diversity similar or larger than those of their European counterparts, namely Jersey and Holstein. All eight breeds display significant gene flow or admixture from African taurine cattle and include mtDNA and Y-chromosome haplotypes from multiple origins. Furthermore, we detected a very low differentiation of chromosome X relative to autosomes within all analyzed taurine breeds, potentially reflecting male-biased gene flow.We sequenced 48 genomes belonging to eight Iberian native breeds and found that the individual breeds are genetically very distinct with FOur results show that an overall complex history of admixture resulted in unexpectedly high levels of genomic diversity for breeds with seemingly limited geographic ranges that are distantly located from the main domestication center for taurine cattle in the Near East. This is likely to result from a combination of trading traditions and breeding practices in Mediterranean countries. We also found that the levels of differentiation of autosomes vs sex chromosomes across all studied taurine and indicine breeds are likely to have been affected by widespread breeding practices associated with male-biased gene flow.The online version of this article (10.1186/s12864-019-5685-2) contains supplementary material, which is available to authorized users. Bos taurus) in the Iberian Peninsula alone  [10, 12reconquista of the 11th–13th centuries AD [Currently, there are two broad groups of cattle breeds, those under intensive management with strong specialization in dairy or meat phenotypes , and the so-called “primitive” breeds, traditional cattle with a low dependence from external inputs that make use of naturally available food resources. Iberian native cattle are found in diverse agro-ecological systems including coastal, mountain, and lowland arid environments Fig. a. Inheriuries AD , 15 and uries AD , as welluries AD , and a suries AD . This hiuries AD , 20. ThiTo uncover genome-wide patterns of diversity associated with the formation of primitive cattle breeds, we sequenced the genomes of 48 individuals belonging to eight breeds of native Iberian cattle Fig. a. Their B. taurus) and zebu (B. indicus) cattle genomes from Europe, Africa and Asia [B. primigenius) [We confirm that there is a clear genetic distinction between Iberian cattle breeds. In addition, we demonstrate that breed management and associated demographic processes had profound effects on genomic diversity and resulted in unusual patterns of genetic differentiation for autosomes vs sex chromosomes. We further describe genome-wide diversity and introgression in Iberian breeds in relation to 60 previously published taurine  . We confB. mutus) [The 48 Iberian cattle genomes and the previously published shotgun resequencing data from 60 additional individuals including taurine and indicine cattle (Table . mutus) . DetailsK populations is set to two . In general, we observed high levels of differentiation (average 9%), even when admixture has occurred, which precludes the use of Iberian cattle as a single evolutionary unit. Consistent with their higher heterogeneity, the breed pair Arouquesa/Mertolenga had a low FST value of 0.06. The highest FST corresponded to the pairwise comparison of Mirandesa and Alentejana (FST = 0.16) and the lowest FST values were obtained for Preta vs Mertolenga (FST = 0.04)  Table .Table 2FST values between Iberian breeds and other taurine cattle ranged from 12 to 33%, partially overlapping the divergence values observed for comparisons within Iberian breeds ; Brava has historically been reproductively isolated from other breeds living in semi-feral conditions for the main purpose of its use in bullfights [The observed genetic homogeneity within Iberian cattle and the high differentiation of Mirandesa and Brava are expected to result from genetic drift due to drastic demographic changes: in the 1970s, Mirandesa was raised in a vast area of the Portuguese territory with over 200,000 animals  and sincllfights . PCs 3 aK = 7  caused by breeding practices, observed in an overall higher Tajima’s  relative X Fig. , with siHere we sequenced whole genomes of locally adapted Iberian cattle (for which genomic resources were lacking), and compared them to commercial cattle to uncover genomic patterns associated with the different breeding contexts. Our analyses confirm that these breeds are genetically very distinct and show high levels of genetic variation unlike what would be expected given their limited geographical distribution. Also, Iberian cattle retain much of the original paternal and maternal diversity, which appears to derive from the dispersal of a heterogeneous population since the Neolithic along the Mediterranean route with strong influences from North African taurine cattle, rather than from recent admixture with transboundary commercial cattle. This may have significant impact on the resilience of Iberian cattle to foreseen environmental changes. Not only these breeds produce high-quality certified meat products under local extensive conditions, as they can provide the source for genetic material to improve breeds with depleted genetic diversity, i.e. transboundary commercial cattle. Furthermore, we show that the complex processes underlying the formation of taurine breeds in general had profound effects on genomic diversity and resulted in unusual patterns of genetic differentiation for autosomes vs. sex chromosomes. Our results indicate that genetic differentiation measured using chromosome X might be more representative of the native populations of domesticated cattle, and that comparisons between breeds using autosomal data might be misleading without an appropriate demographic model.Bos taurus), Ogaden (Bos indicus), Boran (Bos indicus) and Kenana (Bos indicus) [Information regarding the breeds and the type of genetic data used to investigate genome diversity and genetic relationships is summarized in Table indicus)  (Bioprojindicus)    (Englandindicus) , 355,330indicus) , and 946indicus)  were usehttps://www.diagenode.com/en/p/bioruptor-pico-sonication-device), genomic libraries were prepared using the TruSeq DNA PCR-free Library Preparation Kit  according to the manufacturer’s protocols. Whole-genome paired-end resequencing data was obtained by pooling 16 samples in each lane and using an Illumina HiSeq1500 instrument with 2 × 100 bp reads.Genomic DNA was extracted using a modified salting-out precipitation method  according to the manufacturer’s recommendations. We prepared equimolar DNA concentrations for all animals before library construction using Nanodrop™ 2000 (Thermo Scientific) and Qubit™ Fluorometer  measurements. Following DNA fragmentation by sonication using a program specific for 550 bp inserts  [http://broadinstitute.github.io/picard/) and local realignment around indels was done with GATK [The 48 samples were sequenced to between 1.4X and 2.3X depth of coverage   was doneith GATK .Sequencing error rates were determined in ANGSD (version 0.917)  using a NGSadmix version 32  was usedA principal component analysis using the same SNP set for the Iberian breeds was done with PCAngsd  which esTreemix (version 1.13)  was usedThe software RAxML  version Bos mutus) as an outgroup. All samples were mapped to the yak outgroup genome assembly [To determine the pattern of excess shared derived alleles between taxa, indicative of introgression, we estimated D-statistics using the wild yak  , 49 to eAdditional file 1:Note S1. Brief description of the Iberian native cattle breeds sampled in our study. Table S1. Iberian breeds databases. Table S2. Individual sample information. The Y-chromosome haplogroups in bold were determined in this study (n.a.: not applicable). Table S3. FST values between taurine breeds. The highest value is shown in bold and the lowest in italic. Figure S1. Average error rate per sample. Figure S2. Population structure plots determined by NGSadmix; each individual is represented by a stacked column for 3, 4, 6 and 8 proportions. Other K values are shown in Fig. Figure S3. Principal component analysis done with PCAngsd . Figure S5. D-statistics determined as in [Figure S6. Approximately-maximum-likelihood phylogeny of cattle Y-chromosome sequences  determined in FastTree [Figure S7. FST per chromosome for all pairwise comparisons within Iberian cattle and between Iberian cattle and the African taurine N’Dama and the African indicine Ogaden. The proportion of shared variation oscillates throughout the genome, reaching extreme values for chromosome 21 (the largest between Iberian and African taurine cattle) and sex chromosome X . (DOCX 1909 kb)FastTree . Labels


PRNP. Interestingly, we observed enhanced levels of CSF-MDH1 in all genetic prion disease patients compared to neurological controls (without neurodegeneration). While E200K and V210I carriers showed highest levels of MDH1 with diagnostic discrimination from controls of 0.87 and 0.85 area under the curve (AUC), FFI and GSS patients exhibited only moderately higher CSF-MDH1 levels than controls. An impact of the PRNP codon 129 methionine/valine (MV) genotype on the amount of MDH1 could be excluded. A correlation study of MDH1 levels with other neurodegenerative marker proteins revealed a significant positive correlation between CSF-MDH1 concentration with total tau (tau) but not with 14-3-3 in E200K, as well as in V210I patients. In conclusion, our study indicated the potential use of MDH1 as marker for gCJD patients which may complement the current panel of diagnostic biomarkers.The exploration of accurate diagnostic markers for differential diagnosis of neurodegenerative diseases is an ongoing topic. A previous study on cerebrospinal fluid (CSF)-mitochondrial malate dehydrogenase 1 (MDH1) in sporadic Creutzfeldt–Jakob disease (sCJD) patients revealed a highly significant upregulation of MDH1. Here, we measured the CSF levels of MDH1 via enzyme-linked immunosorbent assay in a cohort of rare genetic prion disease cases, such as genetic CJD (gCJD) cases, exhibiting the E200K, V210I, P102L (Gerstmann–Sträussler–Scheinker syndrome (GSS)), or D178N ) mutations in the  Res) in brain tissue. They can occur as sporadic, iatrogenic, or hereditary forms. Genetic prion diseases represent between 10 and 15% of all prion disease cases. More than 50 different mutations in the open reading frame of PRNP have already been described [Transmissible spongiform encephalopathies (TSE) belong to the group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein resistant  [In addition to the P (PrPC) , as wellP (PrPC) ,4,5,6,7.Currently, the number and the accuracy of diagnostic test systems for prion diseases had been improved; however, autopsies are still required for a confirmation.All biomarkers which are currently available, such as 14-3-3, total tau (tau), S-100 calcium-binding protein B, alpha-synuclein or neuron specific enolase, and the recently established in vitro protein misfolding amplification assays, real-time quaking-induced conversion (RT-QuIC), have a good accuracy for sporadic CJD (sCJD) diagnosis ,16,17,18Previous studies of our group revealed a significant upregulation of mitochondrial malate dehydrogenase 1 (MDH1), an enzyme that reversibly catalyzes the oxidation of malate to oxaloacetate  , in sCJDPRNP codon 129 MV polymorphism and estimated the association of MDH1 level with surrogate marker proteins, such as tau and 14-3-3.These promising observations encouraged us to determine the levels of MDH1 in cerebrospinal fluid (CSF) of different types of genetic prion disease patients , GSS, and FFI). We calculated the diagnostic sensitivity of MDH1, analyzed the influence of We retrospectively analyzed 205 CSF samples obtained from patients recruited at the surveillance units from different participant centers (1) Clinical Dementia Center and the National Reference Center for CJD Surveillance at the University Medical Center, Goettingen, Germany, (2) Neurochemistry Laboratory, Neurology Department of Coimbra University Hospital, Coimbra, Portugal, (3) Alzheimer’s Disease and Other Cognitive Disorders Unit, Hospital Clinic, Barcelona, Spain, (4) National Centre of Microbiology-Carlos III Institute of Health, Madrid, Spain, (5) National Registry of CJD and related disorders at Istituto Superiore di Sanità, Rome, Italy, and (6) Institute of Neurology, Medical University of Vienna, Austria. The patients were classified according to established diagnostic criteria .Our study was done in accordance with the Declaration of Helsinki and with informed written consent provided by all patients or by their next of kin in the case of cognitive impairment. Our study was approved by local ethic committees Goettingen (No. 24/8/12) and from ethical committees of other participating centers.n = 45), with PRNP mutation V210I-gCJD cases (n = 46), D178N-M-FFI (n = 36), P102L-GSS (n = 7), and neurological controls (NC) (n = 71) (The cohort of patients in this study consisted of E200K-gCJD cases ((n = 71) . NC groug  at room temperature (~22 °C), aliquoted in 1 mL portions and stored at −80 °C. Blood-stained CSF samples were excluded from the study. The analysis of the codon 129 MV genotype of PRNP was performed after isolation of genomic DNA from blood samples according to standard methods [CSF samples were centrifuged for 10 min at 720×  methods .Levels of MDH1 were determined in CSF by a commercially available enzyme-linked immunosorbent assay (ELISA) kit , following the manufacturer’s instructions. Briefly, levels of MDH1 were measured in arbitrary units (AU) per mL. The detection range of the assay spans from 9.38 to 600 AU/mL. To be in this range, CSF samples were measured in a dilution of 1:100. Colorimetric signal was analyzed at 450 nm with a 1420 Multilabel Counter Victor 2  .We followed a slightly modified immunoblot protocol as described previously ,26. A poAs a secondary antibody we used a polyclonal horseradish peroxidase-conjugated antibody  (diluted 1:7500).The CSF levels of protein tau were measured by a commercially available ELISA kit from INNOTEST hTAU-Ag  with a detection range of total tau (phosphorylated and non-phosphorylated) between 50 and 2500 pg/mL (detection limit: 34 pg/mL). Briefly, before antibody incubations, each sample (75 µl) was diluted 1:1 in sample diluent. The colorimetric reaction was measured at 450 nm with a 1420 Multilabel Counter Victor 2  (PerkinElmer).p-Values below 0.05 are considered as significant.Statistical data analysis was conducted by the use of the statistic software GraphPad Prism 6.01 . For comparison between more than two groups, we used the Kruskal-Wallis test followed by Dunn’s post-hoc analysis. All correlation studies were computed by using the non-parametric Spearman’s correlation test (two-tailed) with a confidence interval of 95%. In our study, we measured the MDH1 levels in CSF in different types of genetic prion diseases by using a commercial MDH-ELISA kit. Firstly, we excluded a potential sex-influence on MDH1 amount .p < 0.001 ***) (p < 0.001)  A. The co< 0.001) A.When we performed a receiver operating characteristic curve analysis for each group, the diagnostic accuracy to discriminate gCJD (E200K and V210I) from controls was 0.87 area under de curve (AUC), 95% Cl = 0.81–0.94, sensitivity 84% for E200K and 0.85 AUC, 95% CI = 0.77–0.92, sensitivity 80% for V210I B,C. In cPRNP codon 129 MV polymorphism on MDH1-levels in CSF, we stratified our genetic prion disease cohort according to the PRNP codon 129 genotype in gCJD . In none of the groups we observed changes in the CSF-MDH1 levels depending on the codon 129 MV genotype, suggesting that this polymorphism has no impact on CSF-MDH1 concentration (To study the influence of the ntration A–C.To explore a putative association between CSF-MDH1 levels and neurodegenerative biomarker proteins for prion diseases (tau and 14-3-3), we performed a correlation study.r = 0.39; p < 0.01 **) and V210I  cases but not in FFI cases  or as negative (14-3-3 band is not visible). The comparison of MDH1 levels from 14-3-3 positive and negative patients revealed for E200K, V210I, and FFI cases no significant association between MDH1 and 14-3-3 B,D,F.Spearman’s correlation test (two-tailed) with a confidence interval of 95%. A p-value < 0.001 is considered as highly significant (***), <0.01 as very significant (**), <0.05 as significant (*), and ≥0.05 as not significant (ns).All correlation studies were computed by using the non-parametric PRNP point mutations, such as V210I or E200K, are associated with variable disease onsets  and penetrance [PRNP mutation [Some netrance ,28. The mutation ,28,29. Imutation , highligA previously conducted two-dimensional proteomic study on CJD brain homogenates and CSF, as well as a recent retrospective study, indicated a specific regulation of MDH1 in sCJD patients compared to controls ,21,22.These interesting observations encouraged us to investigate a potential regulation of MDH1 levels in CSF of genetic prion disease patients in comparison to a neurodegenerative control cohort. Moreover, we were also interested in comparing MDH1 levels with different genetic prion disease groups, such a gCJD E200K, V210I, FFI, and GSS.Meso Scale Discovery (MSD) technology, revealed a diagnostic accuracy for gCJD E200K of 0.96 AUC and for FFI of 0.75 AUC [Our data indicate an enhancement of MDH1 amount as a consequence of rapid neurodegeneration in CSF of genetic prion disease patients compared to controls. Within the genetic prion disease groups, gCJD E200K and V210I carriers showed the highest MDH1 levels in CSF with AUC value of 0.87 and 0.85 followed by GSS  and FFI cases , which revealed a dependency of MDH1 levels on the type of genetic prion disease. The diagnostic accuracy of MDH1 in gCJD is comparable to sCJD (specificity of 85% and a sensitivity of 83%) . In the 0.75 AUC  which isA limitation of this analysis is the small number of GSS cases that did not allow proper statistics. Therefore, it was not possible to calculate the diagnostic accuracy for this group.PRNP codon 129 MV polymorphism (MM and MV) on MDH1 levels in genetic prion disease patients, we did not find any difference on MDH1 concentrations for any genotype. Our results are in agreement with a previous study [When we analyzed the impact of the us study , in whicus study ,36,37.Correlation studies of MDH1 levels with other neurodegenerative marker proteins, such as tau or 14-3-3 revealed a significant correlation only between MDH1 and tau in gCJD (E200K and V210I) but not for FFI, which most probably depend on the low upregulation of MDH1 in this group. As opposed to previous observations on sCJD patients, in our present study on genetic prion diseases, we observed no correlation between MDH1 and 14-3-3. This disagreement may be explained by the detection method for 14-3-3 . UnfortuFor a further validation of CSF-MDH1 as a protein marker for genetic prion disease diagnosis.MDH1 levels should be determined in a larger patient cohort, which might be challenging due to the rareness of genetic prion disease cases worldwide.In the present study we determined the levels of CSF-MDH1 in a cohort of genetic prion disease patients. Our data indicated an upregulation of MDH1 levels in gCJD (E200K and V210I) patients (with a sensitivity of 80–84%) recommending MDH1 either alone or in combination with other tests as a potential biomarker for gCJD  diagnosis.


Dear editor,10.1051/sicotj/2016008 (PMID: 27163103). The objective of the study was to assess the TTTG distance in 100 MRI scans of skeletally matured Indian knees.We were interested in reading “Patellar instability in Indian population: relevance of tibial tuberosity and trochlear groove distance” by Sourabh Kulkarni et al. published in the esteemed Sicot Journal Published online on 2016 Mar 25. DOI: The authors defined TT–TG distance by measuring the distance between two parallel vertical lines, one passing through the apex of tibial tuberosity and the other through the apex of the trochlear groove . The tecFurthermore, the author uses the center of the patellar tendon as a reference point for tibial tuberosity to obtain the TT–TG distance. This in fact is PT–TG (patellar tendon–trochlear groove) distance which is found to be different from the osseous TT–TG distance. Wilcox et al.  in his sTo conclude, the methodology used in the study needs to be verified and the TT–TG distance measured should be ideally referred to as PT–TG distance.


Sepsis is the primary cause of death from infection. We wanted to improve the outcome of sepsis by stimulating innate immunity in combination with modulating the severity of inflammatory responses in rats.  Sepsis was induced by the injection of feces suspension (control). A 5-day course of G-CSF treatment was given before the septic insult (G-CSF). The inflammatory response was decreased using various doses of the LPS-blocking peptide LBPK95A . Survival rates were observed. Bacterial clearance, neutrophil infiltration, tissue damage, and the induction of hepatic and systemic inflammatory responses were determined 2 h and 12 h after the septic insult. α and IL-6 levels. Irrespective of dose, the blockade of the LPS-LBP interaction was associated with low systemic cytokine levels and delayed increases in hepatic TNF-α and IL-6 mRNA expression. The delayed increase in cytokines was associated with the phosphorylation of STAT3 and AKT.  High-dose LBPK95A (100% Combi) reduced the survival rate to 10%, whereas low-dose LBPK95A (10% and 1% Combi) increased the survival rates to 50% and 80%, respectively. The survival rates inversely correlated with multiorgan damage as indicated by the serum levels of ALT and urea. G-CSF treatment increased the white blood cell counts, hepatic neutrophil infiltration, and bacterial clearance in the liver, lung, and blood. The blockade of the LPS-LBP interaction decreased neutrophil infiltration, led to increased white blood cell count, and decreased hepatic neutrophil infiltration, irrespective of dose. However, bacterial clearance improved in the 1% and 10% Combi groups but worsened in the 100% Combi group. G-CSF increased TNF- Our results revealed that increasing innate immunity by G-CSF pretreatment and decreasing inflammatory responses using LBPK95A improved the survival rates in a rat sepsis model and could be a novel strategy to treat sepsis. Sepsis is defined as the overwhelming reaction to the invasion of microorganisms and their components. The organism mounts an innate immune response to eliminate pathogens. Sepsis is frequently associated with increased blood levels of endotoxin . EndotoxDespite years of research, optimal strategies that specifically target the aggressive immune response that characterizes sepsis are not yet available . ModulatGranulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is released after infection and increases the number and function of polymorphonuclear neutrophils (PMNs) . In the In previous experiments, we observed that the injection of G-CSF leads to an increase in lipopolysaccharide binding protein (LBP) expression . LBP is Taken together, one reason for the observed conflicting results of G-CSF-treatment could be an inappropriate balance of the putatively beneficial effect of LBP-mediated bacterial clearance and the detrimental effect of LPS-sensitization in the course of sepsis development. Therefore, we hypothesized that the dynamic balance between LBP-mediated LPS-sensitization and bacterial clearance was decisive for the therapeutic success of G-CSF-induced modulation of innate immunity in sepsis.Male inbred Lewis rats  were used in this study. All animals were housed under standard animal care conditions and had access to water and rat chow ad libitum. Male rats were used to avoid hormonal fluctuations. The permission for animal experiments was given by the “Thüringer Landesamt für Verbraucherschutz” (AZ: 2226840402026/13).μg/kg).Animals were allowed to adapt to laboratory conditions for at least seven days. All procedures were carried out according to German Animal Welfare Legislation and were performed under inhalation anesthesia with 3% isoflurane  complemented by the injection of the analgesic drug Torbugesic Vet  for 5 days before septic insult. The inflammatory response was blocked by interfering with the LPS-LBP interaction using an LBP inhibitory peptide, LBPK95A , by intraperitoneal injection [n = 3). The rats were sacrificed 24 h after injection, and the serum ALT was measured. To modulate the severity of inflammatory response, three doses—5 mg/kg (100% Combi group), 0.5 mg/kg (10% Combi group), and 0.05 mg/kg (1% Combi group) of LBPK95A—were selected and were injected simultaneously with the septic insult after G-CSF pretreatment. According to the previous results [To induce sepsis, rats were injected intraperitoneally with human stool suspension (control group). Innate immunity was enhanced by pretreating rats with G-CSF . In the kinetic experiment, the rats were sacrificed at 2 h (n = 6) and 12 h (n = 6). Tissue injury, bacterial clearance, neutrophil infiltration, activation of signal pathways, and induction of local and systemic inflammatory responses at 2 h and 12 h after septic insult were analyzed.For survival analysis, rat activity was monitored every 3 h up to 72 hours  into the right lower quadrant of the abdomen with a 21-gauge cannula.A sepsis model mimicking the complexity of the clinical situation was used. Therefore, peritoneal contamination and infection (PCI) was performed in rats, using a human feces suspension comprised of several bacteria, induced as described by Gonnert et al. : 3 μl/g At 2 h and 12 h after septic insult, the rats were sacrificed under 3% isoflurane anesthesia. The ascites were collected, and the volume was recorded. Blood was taken from the vena cava for blood count, liver enzyme, and cytokine analysis. The liver and lung were sampled for histological evaluation. Liver tissue samples were stored in −80°C until use in gene and protein expression studies.μm) were cut and stained with hematoxylin-eosin (HE). Slides were digitalized using a virtual slide scanner . Histological evaluation was performed according to a standardized semiquantitative scoring system [Liver and lung tissue was fixed in 4.5% buffered formalin for at least 24 h. Paraffin embedding was performed, and sections .α and IL-6 levels, commercially available enzyme-linked immunosorbent assay kits were used . The procedures were performed according to the manufacturer's suggestions. Measurements of the ELISA were performed in 96-well polystyrene plates using an SLT Spectra ELISA plate reader at 450 nm.For the analysis of serum TNF-Fifteen micrograms of total liver lysate protein or serum were loaded per well and were separated on 12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blotting and staining with rabbit anti-phospho-AKT (Ser 473) , rabbit anti-AKT , rabbit anti-phospho-Stat3 (Tyr 705) , rabbit anti-STAT3 , orrabbit anti-caspase 3 . Signals were detected with Lumi-Light Western blot substrate  and exposure to X-ray film .μg) was reverse transcribed using the First-Strand cDNA synthesis kit . Real-time PCR was carried out in an M3000P QPCR System , using the Brilliant probe-based QPCR Master Mix kit , probes , and primers: IL-6: CCTGGAGTTTGTGAAGAACAACT, GGAAGTTGGGGTAGGAAGGA, and probe #106; TNF-α: TGAACTTCGGGGTGATCG, GGGCTTGTCACTCGAGTTTT, and probe #63; hypoxanthine guanine phosphoribosyltransferase (HPRT): GACCGGTTCTGTCATGTCG, ACCTGGTTCATCATCACTAATCAC, and probe #95. The reaction consisted of 50 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 50°C, and extension for 30 s at 72°C. A standard curve was generated using a serial dilution of pooled sample of all single cDNAs. Expression of target genes was normalized using HPRT.RNA was isolated from liver tissues using the RNeasy kit , and the procedure was performed according to the manufacturer's instructions. The concentration of RNA was quantified by the relation of absorbance at 260 nm to absorbance at 280 nm. Total RNA  to detect aerobic bacteria. Plates were incubated for 24 h at 37°C, and colonies were counted.The bacterial loads in tissues and blood were determined to investigate bacterial clearance under various treatment conditions to reach a better understanding of the delicate balance between enhancing the innate immune response and attenuating the inflammatory response. Lung tissue, liver tissue, and blood were examined for bacterial load. The tissues were homogenized in 3 ml NaCl for 1 minute before dilution to 5 ml with brain heart infusion (BHI) broth . For blood preparation, EDTA blood was used. Three concentrations  were prepared of the working solution using BHI. Ten t-test in case of normal distribution of the data. If data were not normally distributed, the Mann–Whitney rank sum test was employed to compare sets of data in different animal groups. Data were expressed as the mean ± SD. p < 0.05 was considered statistically significant in this experiment.All values were expressed as the mean ± SD. All statistical calculations were performed using SigmaStat . The 72 h survival results were shown in a Kaplan-Meier curve, created using SigmaPlot 12. Groups of animals were compared employing the Student Previous experiments revealed that G-CSF pretreatment improved the survival rate from 36% to 56% . The surAs shown in Organ damage is one of the leading causes of mortality of sepsis. The liver and the lung are particularly affected in most cases. Thus, we investigated the histomorphological alterations in liver and lung in the various groups to assess organ damage. The extent of liver and lung damage matched the survival data. As shown in The release of liver enzymes also reflected the severity of hepatocellular injury. As shown in To evaluate the function of innate immunity, levels of circulating WBC and hepatic neutrophil infiltration were assessed. G-CSF pretreatment improved neutrophil promotion and upregulation of LBP expression, as reported previously . CountinBacterial clearance was assessed by tissue and blood culture . G-CSF pIn this kinetic experiment, we measured serum levels of proinflammatory cytokines  that are highly elevated in sepsis Figures . Serum THepatic expression of TNF-a and IL-6 mRNA was assessed to explore the induction of local inflammatory responses Figures . G-CSF pThe mRNA expression of inflammatory cytokines was associated with phosphorylation of STAT3 and AKT . The phoThis study was designed to explore whether the dynamic balance between LBP-induced bacterial clearance and LBP-mediated LPS-sensitization was decisive for the therapeutic success of G-CSF-induced modulation of innate immunity in sepsis. We showed in immune-stimulated G-CSF-pretreated animals that blocking the LBP/LPS interaction with a low dose of blocking peptide (0.05 mg/kg) simultaneously with the feces suspension for sepsis induction was associated with a high survival rate. By contrast, increasing the dose of the blocking peptide in immune-stimulated animals to modulate the inflammatory response was associated with decreased survival rate, increased local neutrophil infiltration, and increased tissue damage.G-CSF is needed for bacterial clearance. In vivo, monocytes and macrophages are major sources of G-CSF, particularly upon activation by pathogens, e.g., endotoxin and infection . BacteriHowever, conflicting effects of G-CSF were observed in septic patients and animal models. During the past few decades, there have been a variety of studies investigating the effect of G-CSF in patients with sepsis. However, consistent results have not been reported and no individual study has established whether G-CSF brings clinically important benefits for septic patients. Recently, Bo et al. demonstrated in a meta-analysis  that G-COur results may help to explain the conflicting results observed in clinical trials. The conflicting effects of G-CSF could be explained by the dynamic balance between innate immunity and the inflammatory response. The dynamic balance reflects the beneficial effects of G-CSF regarding bacterial clearance in relation to the detrimental adverse effects. In addition to upregulation of neutrophils, G-CSF leads to an increase in LBP .LBP upregulation is associated with sensitization to LPS . LBP is Many strategies have been developed to decrease circulating endotoxin and to minimize inflammatory responses . Direct α and IL-6, reduced cytokine-induced neutrophil chemoattractant, reduced macrophage inflammatory protein production, and attenuated recruitment of polymorphonuclear leukocytes to the site of infection. However, depletion of LBP caused sensitization to E. coli infection, as LBP−/− mice showed increased mortality, decreasing bacterial clearance and severe organ damage. This shows that LBP is necessary for bacterial elimination but is also detrimental due to sensitization to LPS. Araña et al. showed for the first time that interfering with the interaction between LPS and LBP using an LBP inhibitory peptide (LBPK95A) decreased inflammatory injury to animals [LBP blockade could be considered a novel strategy in modulation of inflammatory response in sepsis. Neutralization of LBP was accomplished by inhibition of the transfer of LBP-LPS complexes to the TLR4 complex to protect from induction of the inflammatory response . Knapp e animals . Taken tThe beneficial effect of immune therapy can be attenuated by tissue damage caused by inflammation. In our study, the severity of inflammatory response was successfully downregulated using various doses of LBPK95A. However, it appears that a delicate balance between induction of innate immunity and blockade of inflammation is needed. These observations suggest that combining augmentation of innate immunity together with moderate blockade of the inflammatory response could be a novel strategy to treat sepsis.