The aim of the present study was to determine the effects of ulinastatin (UTI) on brain injury in rats subjected to cardiopulmonary resuscitation (CPR) following asphyxial cardiac arrest (CA) and identify the underlying mechanisms. In total, 100 healthy male Wistar rats were randomly divided into control and treatment groups (n=50). After 4 min of asphyxial CA, all the rats were immediately subjected to CPR. The treatment group animals were administered 15 mg/kg UTI at the onset of resuscitation. The mortality rate in the two groups was recorded at 24 h post-resuscitation. In addition, neurological function was evaluated at 24, 48 and 72 h post-resuscitation using a neurological deficit scale (NDS). Furthermore, the effects of UTI on the Toll-like receptor 4 (TLR4) signaling pathway in brain tissues were determined by assessing TLR4 mRNA expression, nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels at 1, 3, 6, 12, 24, 48 and 72 h post-resuscitation. After 24 h, the mortality rate significantly decreased in the treatment group when compared with the control animals . Additionally, an overt improvement was observed in the NDS score following UTI treatment when compared with the control (P<0.01). Finally, statistically significant decreases in the levels of TLR4 mRNA expression, NF-κB activity and TNF-α and IL-6 were observed in the treatment group at each time point (P<0.01). Therefore, UTI treatment at the onset of CPR significantly inhibits the TLR4 signaling pathway, thereby alleviating the inflammatory responses following resuscitation and improving neurological function. Statistical data show that ~1 million patients with cardiac arrest (CA) are recorded annually in the United States and Europe ,3. ThereOrgan injury following successful CPR from CA is considered to be associated with systemic inflammatory responses; levels of various cytokines and lipopolysaccharide (LPS) have been shown to be markedly elevated in patients that have been successfully resuscitated from CA ,5. LPS cUlinastatin (UTI), a urinary trypsin inhibitor extracted and purified from human urine, has been shown to possess anti-inflammatory properties, suppressing the infiltration of neutrophils and the release of elastinase and chemokines . In addiThe effects of UTI were investigated with regard to the mortality rate, TLR4 mRNA expression, NF-κB activity and the levels of TNF-α and IL-6. The aim of the present study was to determine whether UTI improved neurological function; thus, may be used in combination with CPR for the treatment of brain injury following CA.The study was approved by the Experimental Animal Protection and Ethics Committee of the Second Artillery General Hospital . In total, 100 male Wistar rats  were randomly assigned to control and treatment groups (n=50). After 4 min of asphyxial CA, all the rats were immediately subjected to CPR. The treatment group animals were administered 15 mg/kg UTI  at the onset of resuscitation .Asphyxial CA was induced as previously described –16. RatsBaseline physiological variables were recorded continuously for 10 min following surgery. CA was defined as a reduction in the mean arterial pressure (MAP) to a value of <10 mmHg after ventilation had been discontinued for ~3 min. After 1 min of CA, CPR was performed with continuous ventilation and external cardiac compressions at a rate of 200/min, along with an intravenous injection of 0.01 mg/kg epinephrine and 1 mmol/kg sodium bicarbonate. Successful CPR was defined as an achievement of ROSC, indicated by a rise in the MAP to a value of >50 mmHg. CPR was considered to have failed if no sign of ROSC was observed during the 5 min post-resuscitation.ad libitum.Following resuscitation, the rats were mechanically ventilated with an increased I/E ratio of 1/1.5 and treated with a continuous infusion of 8 ml/h lactated Ringer’s solution until the restoration of adequate spontaneous respiration. The rats were then weaned from the ventilator, extubated and observed for 10 min. This was followed by arteriovenous ligation and skin sutures if there were no abnormalities observed in the circulation and respiration. Following restoration in an oxygen tank for 30 min, the rats were finally returned to their respective cages with access to food and water The mortality rate in the two groups was recorded at 24 h post-resuscitation.MAP and HR were determined for all the animals during the first 60 min post-resuscitation. The time to CA (TCA) and time to ROSC (TROSC) were also recorded. The TCA was defined as the time from the initiation of ventilation to the onset of CA, while the TROSC was defined as the time from the start of resuscitation to ROSC.At 1, 3, 6, 12, 24, 48 and 72 h post-resuscitation, all the rats were decapitated, and their brains quickly removed and placed on ice for dissection. Following washing in ice-cold normal saline, the surface water on the organs was gently blotted off with filter paper. Left hemisphere brain tissues were stored in liquid nitrogen, until required for the analysis of TLR4 mRNA expression and NF-κB, TNF-α and IL-6 protein levels.A small sample of brain tissue (~50 mg) from the right hemisphere was extracted at 24 h post-resuscitation, as described previously. The brain tissues were immediately weighed on an electronic analytical balance, dried at 105°C for 24 h and weighed again. The BWC was calculated as follows: (wet weight - dry weight)/wet weight × 100%.Neurological function was evaluated in the rats at 24, 48 and 72 h post-resuscitation using a neurological deficit scale (NDS) scoring system , according to the manufacturer’s instructions. Briefly, fresh or frozen tissues (~100 mg) were homogenized in 1 ml TriPure reagent and submitted to centrifugation at 21,578 × g for 10 min at 4°C. Following the addition of 0.2 ml chloroform to the supernatants, the mixture was incubated at room temperature for 20 min with occasional stirring. The samples were then submitted to centrifugation again, as aforementioned. The resulting supernatants were mixed with 0.5 ml isopropanol, incubated for 5–10 min and centrifuged as aforementioned. The pellet containing total RNA was washed with 1 ml ethanol (75%) and centrifuged at 8,429 × g for 5 min at 4°C. The RNA pellet was air dried, dissolved in 3 ml diethylpyrocarbonate-treated water and the optical density (OD) values were measured at 260 and 280 nm on a Lambda 20 UV/Vis spectrophotometer . The RNA concentration was calculated as follows: RNA concentration (μg/ml) = OD10 copies/μl, respectively, and diluted with sterile water to final concentrations of 1×106, 1×105, 1×104 and 1×103 copies/μl. The qPCR mixture (25 μl) contained 1X PCR buffer, 4 mM MgCl2, 0.4 μM each primer, 0.4 μM TaqMan probe, 0.5 units AmpErase Uracil N-Glycosylase, 0.5 units AmpliTaq Gold DNA polymerase, 50 ng cDNA, double distilled water, 0.2 mM dUTP and 0.1 mM dATP, dGTP and dCTP.TLR4 gene expression was evaluated using quantitative polymerase chain reaction (qPCR). Plasmids of TLR4 and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were obtained at 3.67 and 2.67×10qPCR was conducted using an ABI Prism 7500 SDS RT-PCR system . The reaction conditions for the amplification of TLR4 and GAPDH were 40 cycles of 50°C for 2 min, 95°C for 2 min, 95°C for 15 sec and 60°C for 60 sec. The amplified products for TLR4 and GAPDH were 122 and 138 bp in size, respectively. The primers and fluorescent probes used for qPCR were as follows: TLR4 forward, 5′-TGAGAAACGAGCTGGTAAAGAATT-3′, reverse, 5′-GTGGAAGCCTTCCTGGATGATG-3′; and probe, 5′-AGTGCCCCGCTTTCAGCTTTGCCT-3′; GAPDH forward, 5′-CATGACCTTCCGTGTTCCTACC-3′, reverse, 5′-TAGCCCAGGATGCCCTTCAG-3′ and probe, 5′-CCTCAGACGCCTGCTTCACCACCT-3′.The threshold level was set at 10-times the standard deviation (SD) of the baseline fluorescence measured during PCR cycles 3–15. The threshold cycle (Ct) was defined as the cycle number at which fluorescence passed the fixed threshold, normalized against GAPDH. Standard curves of the Ct values  were constructed for TLR4 and GAPDH. The correlation coefficients of the standard curves for TLR4 and GAPDH were 0.986 and 0.971, respectively. The absolute copy numbers of TLR4 mRNA in each unknown sample were then calculated based on the Ct values. The GAPDH mRNA copy number in each sample was used to normalize TLR4 mRNA expression levels.Nucleoproteins were extracted from the brain tissues using a nucleoprotein extraction kit . Briefly, ~200 mg tissue samples were homogenized in 2 ml nuclear extraction buffer (NEB) A. The homogenates were then centrifuged at 599 × g for 30 sec, and the supernatants containing the complete nuclei were incubated on ice for 5 min. Following centrifugation at 3,746 × g for 10 min at 4°C, the nuclear precipitates (pellet) were resuspended in 150 μl NEB B and incubated for 30 min on ice. A final centrifugation at 29,371 × g for 1 min at 4°C yielded nucleoproteins in the supernatants, which were stored at −70°C until required for the assessment of protein concentrations using the Coomassie Brilliant Blue G-250 method .NF-κB activity levels in the nuclear extracts were detected by a specific high-sensitive enzyme-linked immunosorbent assay (ELISA), using a TransAM NF-κB detection kit (Active Motif), according to the manufacturer’s instructions. Antibodies were used at a 1:1,000 dilution in antibody binding buffer and incubations were performed at room temperature for 1 h. The reactions were visualized using 100 μl tetramethylbenzidine (TMB) chromogenic substrate for 10 min in the dark, and were stopped following the addition of 100 μl stop solution. Absorbance was measured immediately at 450 nm on a microplate reader.TNF-α and IL-6 levels were assessed using specific ELISA kits . Briefly, samples or standards  were added to the wells. Following incubation for 1 h at 37°C, and four washes in 350 μl washing solution, the plates were incubated in the presence of 100 μl biotin antibodies for 1 h at 37°C. Next, the plates were washed and incubated with 100 μl enzyme binding buffer for 30 min at 37°C. The reactions were visualized using 100 μl TMB chromogenic substrate for 15–20 min in the dark, and stopped following the addition of 100 μl stop solution. Absorbance was measured within 15 min at 450 nm on a microplate reader. Based on the measured OD values, the protein levels of TNF-α and IL-6 were determined using the corresponding standard curves.Statistical analyses were performed using SPSS statistical software version 12.0 . All the data are expressed as the mean ± SD. The Student’s t-test was used to compare differences between the two groups, where P<0.05 was considered to indicate a statistically significant difference, and P<0.01 indicated a higher degree of statistical significance.No statistically significant differences were observed in the baseline characteristics, including the body weight, HR, MAP and NDS, between the two groups (data not shown).As shown in Neurological data are summarized in According to the qPCR results , TLR4 mRAs shown in As shown in Patients that are successfully resuscitated from CA often experience neurological complications, including cognitive impairment and motor deficit, which potentially incapacitate the individuals and may result in mortality ,3. Thus,TLR4 is a pattern recognition receptor that predominantly recognizes endotoxins. A previous study demonstrated that TLR4 can bind to various exogenous ligands, including heat shock proteins, protein fragments from the extracellular matrix, hyaluronan, heparitin sulfate, vascular fibrin monomer-fibrinogen complex and phylaxin and elastase released from immune cells . The supOnce TLR4 is activated, the interaction of the TLR4 intracellular domains with the adapter protein, MyD88, promotes the expression of IL-1 receptor-associated kinase and TNF receptor-associated factor 6, which then upregulate NF-κB. Activated NF-κB further stimulates the expression of various inflammatory cytokines, including TNF-α, IL-1 and IL-6, resulting in inflammation . TNF-α aIn conclusion, UTI was found to alleviate brain injury following CPR by inhibiting the TLR4 signaling pathway and reducing the release of inflammatory cytokines; thus, exerting anti-inflammatory effects.


The odds ratios (ORs) {95% confidence interval (CI)} of increasing serum bilirubin category were negatively associated with carotid IMT ≥1.0 mm and plaque in both genders. Compared to subjects with a serum bilirubin of Quartile-1, the multivariate-OR (95% CI) of carotid plaque was 0.25 (0.11–0.57) in the Quartile-4 male group, and 0.41 (0.21–0.78) in the Quartile-2 female group, 0.51 (0.26–0.98) in the Quartile-3 female group, and 0.46 (0.24–0.89) in the Quartile-4 female group. Our data demonstrated an independently negative association between serum bilirubin and carotid atherosclerosis in both genders.Serum bilirubin may have a beneficial role in preventing oxidative changes in atherosclerosis. Limited information is available on whether serum total bilirubin is an independent confounding factor for carotid atherosclerosis {for example, intima-media thickness (IMT), plaque} measured noninvasively by B-mode ultrasonography only among elderly persons. The study subjects were 325 men aged 79±8 (mean ± standard deviation) years and 509 women aged 81±8 years that were enrolled consecutively from patients aged ≥60 years in the medical department. Carotid IMT and plaque were derived via B-mode ultrasonography. Multiple linear regression analysis showed that in men age ( Serum bilirubin, a major intravascular product of heme catabolism, is an endogenous compound that can be toxic in infants under certain conditions like excessive production of bilirubin due to hemolysis The first report of a negative relationship between serum bilirubin levels and coronary artery disease was published as early as 1994 Carotid atherosclerosis {e.g., intima-media thickness (IMT), plaque} is an important and sensitive surrogate marker of CVD and can now be measured noninvasively by B-mode ultrasonography Firstly, this study investigated serum bilirubin levels and their relation to potential confounding factors such as hypertension, hyperglycemia and lipids. Secondly, this study investigated whether there is an independent association of serum bilirubin with a direct and early measure of carotid atherosclerosis by B-mode ultrasonography. To examine these two issues, cross-sectional data from elderly persons were used.2; total cholesterol (T-C) <100 mg/dL; and fasting plasma glucose (FPG) of <55 mg/dL or ≥400 mg/dL. Thus, 834  patients were enrolled in the study. The main causes of consultations of the patients were respiratory disorders including pneumonia in 98/107 (men/women) cases, metabolic and endocrine diseases in 51/72, stroke in 49/55, cardiovascular disease other than stroke in 29/65, digestive disorders in 35/37, neurological disorders other than stroke in 18/61, infection in 7/27, urinary or reproductive organ disorders in 3/26, neoplasm in 16/12, collagen or skin disorders in 5/10, mental disorders in 3/6, hematological disorders in 3/2, miscellaneous diseases in 8/29. All procedures were approved by the Ethics Committee of Seiyo Municipal Nomura Hospital, and written informed consent was obtained from each participants.Subjects for this investigation were recruited from among consecutive elderly patients aged ≥60 years that visited the internal medical department of Seiyo Municipal Nomura Hospital. Participants with serum total bilirubin ≥2.1 mg/dL or alanine transaminase (ALT) ≥80 IU/L or gamma glutamyl transpeptidase (GGT) ≥80 IU/L were excluded to avoid confounding factors due to the high possibility of potential Gilbert syndrome and hepatobiliary disease. We additionally excluded participants with severe cardio-renal failure or nutritional disorders that would affect blood pressure, lipid and glucose metabolisms were also excluded. Exclusion criteria were severe hypotension as defined by systolic blood pressure (SBP) <80 mmHg; renal failure with an estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m−1.154× Age−0.203×0.742  Information on demographic characteristics and confounding factors was collected using the medical records in all cases. Body mass index (BMI) was calculated by dividing weight (in kilograms) by the square of the height (in meters). Blood pressure was measured one time in the seated position by a nurse using a standard mercury sphygmomanometer or automatic manometer. Smoking status was defined as the number of cigarette packs per day multiplied by the number of years smoked (pack·year) irrespective of the difference between current and past smoking status: never, light (<20 pack ▪ year), moderate (20–39 pack ▪ year), and heavy (≥40 pack ▪ year). Blood samples were drawn after a 10 hour fast in the morning. T-C, triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), FPG, creatinine (enzymatic method), ALT, GGT, and serum total bilirubin were measured within 24 hours after admission. Low-density lipoprotein cholesterol (LDL-C) level was calculated by the Friedewald formula An ultrasonograph  equipped with a 7.5 MHz linear type B-mode probe was used by a specialist in ultrasonography to evaluate sclerotic lesions of the common carotid arteries on a day close to the day of blood biochemistry analysis (within 2 days). Patients were asked to assume a supine position, and the bilateral carotid arteries were observed obliquely from the anterior and posterior directions. We measured the thickness of the intima-media complex (IMT) on the far wall of the bilateral common carotid artery about 10 mm proximal to the bifurcation of the carotid artery  2 test for categorical data, respectively. Pearson's correlations were calculated in order to characterize the associations between various characteristics and carotid IMT. Forward stepwise multiple linear regression analysis  was used to evaluate the contribution of each confounding factor to carotid IMT. Subjects were divided into four groups based on sex-specific quartiles of serum bilirubin , and logistic regression analyses were used to test significant determinants of carotid IMT or plaque serving as the dichotomous outcome variable. A value of p<0.05 was considered significant.All values are expressed as the mean ± standard deviation (SD), unless otherwise specified, and in the cases of parameters with non-normal distribution , the data are shown as median (interquartile range) values. In all the analyses, parameters with non-normal distributions were used after log-transformation. Statistical analysis was performed using IBM SPSS Statistics Version 21 . The differences in means and prevalence among the groups were analyzed by Student's t-test for continuous data and χβ = -0.139), and prevalence of CVD (β = 0.133) were significantly and independently associated with carotid IMT, and in women age (β = 0.186), SBP (β = 0.104), DBP (β = -0.148), prevalence of antihypertensive medication (β = 0.126), FPG (β = 0.135), GGT (β = -0.104), serum bilirubin (β = -0.119), and prevalence of CVD (β = 0.103) were also independently associated with carotid IMT. In male model 2, age (β = 0.232) and smoking status (β = 0.155), SBP (β = 0.106), GGT (β = -0.136), and prevalence of CVD (β = 0.131) was significantly and independently associated with carotid IMT, while in female model 2, serum bilirubin (β = -0.107) as well as age (β = 0.168), prevalence of antihypertensive medication (β = 0.115), FPG (β = 0.134), GGT (β = -0.122), and prevalence of CVD (β = 0.093) was significantly and independently associated with carotid IMT.To find independently confounding factors for carotid IMT, in To examine any possible contribution of serum bilirubin to carotid atherosclerosis, we studied the relationship between CVD confounding factors, including serum bilirubin, and carotid IMT and plaque. This study showed that serum bilirubin was independently and negatively related to carotid atherosclerosis evaluated by ultrasonography. Increased carotid IMT occurred in parallel with the decline in serum bilirubin in women, and serum bilirubin quartiles are significantly and negatively associated with carotid IMT ≥1.0 mm including plaque and plaque, independent of other confounding factors in both genders. To our knowledge, this is the first study to indicate a negative relationship between serum bilirubin and carotid atherosclerosis among Japanese elderly persons.±0.08 mm versus 0.41±0.08 mm, p<0.001; 0.55±0.12 mm versus 0.40±0.07 mm, P = 0.002, in women and men, respectively) β = -0.34) P<0.001) There are few reports which studied the association between serum bilirubin and carotid atherosclerosis and they are all based on cross-sectional analysis. In healthy 111 men without manifested atherosclerosis, the mean IMT and age-adjusted odds ratio for carotid atherosclerosis (IMT >50% of controls) in hyperbilirubinemic subjects with Gilbert syndrome were significantly lower than that of controls The precise mechanisms that lead to a reduction in carotid atherosclerosis in individuals with mildly elevated serum bilirubin levels are not completely understood. Several studies have demonstrated a negative relationship between serum bilirubin and the presence of CVD. In addition, serum bilirubin correlated inversely with several known risk factors for CVD, such as smoking, LDL-C, diabetes, and obesity, and correlated directly with HDL-C However, interpreting our results has several limitations that must be considered. Firstly, based on its cross-sectional study design, the present findings are inherently limited in the ability to eliminate causal relationships between confounding factors and carotid atherosclerosis. Secondly, since all subjects were patients, we could not eliminate the possible effects of underlying diseases  and medications used for hypertension and dyslipidemia on the present findings. In our study, participants with serum total bilirubin ≥2.1 mg/dL or ALT ≥80 IU/L or GGT ≥80 IU/L were excluded, but individuals with Gilbert's syndrome and may be included in the Quartile-4  group. Thirdly, in this study our model could not account only for 11.3–15.7% of the variance in the IMT results. Secondary prevention interventions in obesity, hypertension, dyslipidemia and diabetes mellitus may be successful in reducing confounding factors, thus attenuating the observed association of confounding factors with disease. Fourth, it is generally known that elderly persons normally eat less  which leads inevitably to increased serum bilirubin levels The present study showed that a lower serum bilirubin is strongly associated with an increased prevalence of carotid atherosclerosis in both genders. The underlying mechanism behind this relationship is unclear, but it seems to be independent of traditional cardiovascular confounding factors such as age, BMI, smoking status, hypertension, dyslipidemia, uric acid, diabetes and liver enzymes. Serum bilirubin in addition to the consideration of present conventional confounding factors may be useful for individual risk assessment. Further prospective population-based studies are needed to investigate the mechanisms underlying this association.Figure S1Ultrasound images of common carotid artery.(XLSX)Click here for additional data file.Data S1Data of subjects.(PDF)Click here for additional data file.


Objective. To examine risk taking behaviours associated with alcohol consumption amongst UK undergraduate students. Design and Methods. A cross-sectional web survey was used to assess attitudes and health behaviours. The survey included the Alcohol Use Disorders Identification Test (AUDIT). Students were also asked about why they drank alcohol; about their preferred alcoholic beverage; and if they had experienced any consequences associated with drinking alcohol as well as questions relating to sexual risk taking, drug use, and smoking. Results. 2779  students completed some part of the survey. Of these, 98% (n = 2711) completed the AUDIT. Of the 92% that drank 66%  were categorised as being AUDIT positive. 8% (n = 224) were categorised as probably alcohol dependent. Higher AUDIT scores were significantly associated with negative consequences such as unplanned sexual activity, physical injuries, and arguments. Other risk taking behaviours such as drug use and smoking were also found to be positively correlated with higher AUDIT scores; drug use; and smoking. Conclusions. The results from this study provide insight into students' alcohol consumption and associated risk taking. University policies need to protect students' overall health and wellbeing to ensure academic potential is maximised. University life brings with it the introduction to various social activities and new socialisation groups; this exposes students to various situations in which risk taking behaviour is deemed socially acceptable . StudentRisky drinking is a major public health problem and often equates to risk taking behaviour . The impForty-five percent of men and 46% of women aged 16–24 in the general population report binge drinking (drinking more than twice the recommended daily amounts of 3-4 units per day for men or 2-3 units for women) . Risky dThe objective of this study was to examine risk taking behaviours associated with alcohol consumption amongst UK undergraduate university students and to discuss the findings in relation to the WHO Healthy University Framework.A cross-sectional web survey, adapted from the School Health Education Unit's (SHEU) Health Related Behaviour Questionnaire, was emailed to all undergraduate students registered with a university email account in one university in Northern England to assess attitudes and health behaviours . A prizeThis present study relates to the questions in the survey regarding alcohol, mental health, sexual health, smoking, and drug misuse. Demographic data was also collected on age, gender, ethnicity, living arrangements, type of course studied, and year of study.Research suggests standardised alcohol screening tools, such as the Alcohol Use Disorders Identification Test (AUDIT) , are a hIn relation to mental health, questions were asked about whether students had experienced emotional or psychological problems  that they felt interfered with their life; responses were never; yes more than 12 months ago; and yes within the last 12 months . MoreoveQuestions were asked about whether contraception had been used on the last occasion that students had had sexual intercourse; whether emergency contraception had been used in the last 12 months; and whether the student or their partner had had a sexually transmitted infection in the last 12 months. Students were asked if they had ever smoked or currently smoked .Questions related to drug use were based on suggestions from the local drug alcohol team. Students were asked to indicate whether they had ever used drugs; used them once or twice; used them regularly in the past but not now; currently used them less than once a week; and used them weekly or more often. Less than once a week and using weekly or more often were classified as current use. Cannabis, speed/amphetamine, cocaine, crack, acid/LSD, magic mushrooms, ecstasy, aerosol/glue/solvents, poppers, heroin, body-building steroids; nonprescribed sedatives or tranquilisers, nonprescribed antidepressants, and ketamine were included on the list of drugs as well as space to add others.The protocol for the study was reviewed by the local Primary Care Trust (PCT) research and development department and granted approval as an evaluation; therefore ethics was not applicable whilst the lead author was working there as part of her Public Health Training. Governance mechanisms were established through approval from the PCTs senior management group and a multidisciplinary steering group provided an overview and scrutiny role as a substitute for an approval by an ethics committee.The web survey was piloted with members of the steering group and 30 representatives from the student union and amended based on feedback received. The survey was changed to include the ability to skip questions that students did not want to answer. The results from the pilot were not included in the final study. The pilot also indicated that students thought an incentive for completing the survey would be useful and therefore a prize draw for an iPad was included in the study. The survey was reviewed by senior managers of the university prior to dissemination.The survey was emailed to all students registered on campus' email addresses. The survey was live for three weeks from the end of April to the beginning of May 2012, just before exams began. An introductory email from the University Academic Registrar highlighted the purpose of the survey and emphasised that all responses would remain anonymous and confidential and that a final report would be collated at a group level. Contact information was provided at the end of the survey for local support services (including university support services) in case any sensitive issues had been raised whilst completing the questionnaire. One email reminder was sent midway through the three-week time period.U test  in the case of two groups or the Kruskal-Wallis analysis of variance by ranks  in the case of more than two groups.The questionnaire data was analysed using SPSS v.18. All responses were weighted by age, gender, and ethnicity to make the findings representative of the university population at the time of the survey. Statistically significant results were identified by using the 1% level, instead of the conventional 5%, due to large numbers of tests being run on the data. As AUDIT scores in the sample were positively skewed, nonparametric tests were used to look for statistically significant differences, an approach used in similar studies . Differen = 1806) of respondents were female and 84%  White British. The age profile of the survey respondents consisted of 5% (n = 145) 18 year olds, 20% (n = 550) 19 year olds, 23% (n = 649) 20 year olds, 23% (n = 624) 21 year olds, 23% (n = 647) 22–24 year olds, and 6% (n = 164) 25+ year olds. Eleven percent (n = 277) of those that answered the question reported current smoking .Out of possible 14,814 undergraduate students registered with the university in 2011/12, 2779 (19%) completed the survey. Sixty-five percent  stated they were abstinent (7% (n = 97) males, 9% (n = 124) females). Of those that drank (scored more than zero on the AUDIT) the mean AUDIT score was 10.70; SD 6.16; range 1–39 . Of those that said they drank 66%  were categorised as being AUDIT positive (score of 8+) (71% (n = 902) males, 61% (n = 741) females). Of those that drank, 8% (n = 224) of the overall sample were categorised as probably alcohol dependent (11% (n = 148) males, 6% (n = 76) females)  students completed the AUDIT. Eight percent  .Z = −4.70, P < 0.0005), “to feel confident” (“strongly agree/agree” 11.17 (SD 6.19); “strongly disagree/agree” 8.67 (SD 5.58); Z = −6.21, P < 0.0005); “to get drunk” (“strongly agree/agree” 11.10 (SD 6.08); “strongly disagree/agree” 6.48 (SD 4.58); Z = −9.34, P < 0.0005); “to look cool” (“strongly agree/agree” 9.72 (SD 6.29); “strongly disagree/agree” 11.68 (SD = 5.79); Z = −7.92, P < 0.0005), and “because friends do” (“strongly agree/agree” 10.45 (SD 6.23); “strongly disagree/agree” 11.72 (SD 5.97); Z = −3.99, P < 0.0005).There were significant differences in mean AUDIT scores relating to the reasons why students drink alcohol  “to feeln = 221) a range of overlapping reasons were stated for this. Forty-five percent (n = 98) said that they did not drink for religious beliefs (53% (n = 50) of males and 39% (n = 48) of females), 36% (n = 78) said that they did not drink for lifestyle reasons such as fitness (44% (n = 42) of males and 29% (n = 36) of females), 47% (n = 102) did not like the taste of alcohol (37% (n = 35) of males and 55% (n = 67) of females), 2% (n = 5) had previous problems with alcohol and were now abstinent , and 8% (n = 18) have experienced family problems through drinking and have chosen to be abstinent as a result (8% (n = 8) of males and 8% (n = 10) of females).Of those who stated they were abstinent .Males had statistically significant higher AUDIT scores than females ; females 9.80 (SD 5.79); Z = −3.59, P < 0.0005), 20 year olds ; female 9.83 (SD 5.83); Z = −4.88, P < 0.0005), and 21 year olds ; female 9.09 (SD 5.05); Z = −3.73, P < 0.0005). When looking at gender by ethnicity AUDIT scores were significantly different amongst those classified as “White” ; female 10.18 (SD 5.75); Z = −7.73, P < 0.0005).When looking at gender by age group, males had statistically significant higher mean AUDIT scores in some ages: 18 year olds ; female 11.06 (SD 5.84); χ2 = 133.01, df = 5, and P < 0.0005). Significant differences in age started between 19 and 20 year olds  and for all age groups thereafter. There were also significant differences in mean AUDIT scores by ethnicity (“White” 11.12 (SD 6.03); “Chinese” 4.55 (SD 4.63); “other” 8.19 (SD 6.17); χ2 = 142.86, df = 2, and P < 0.0005).There were significant differences in mean AUDIT scores between students of different ages (18s mean 12.46 (SD 6.52); 19s mean 11.86 (SD 6.59); 20s mean 10.84 (SD 5.95); 21s mean 10.21 (SD 5.63); 22–24s mean 9.03 (SD 5.54); 25+ mean 6.93 (SD 4.55); χ2 = 53.33, df = 3, and P < 0.0005). However, there were no significant differences in mean AUDIT score between students in their first and second years of study  whilst significant differences existed between second, third, and fourth year (or over) of study .There was a significant overall difference in mean AUDIT scores of year of study with scores reducing as students' progress through university (first year 11.58 (SD 6.54); second year 10.76 (SD 6.08); third year 10.07 (SD 5.64); fourth year (or over) 8.54 (SD 5.29); χ2 = 106.07, df = 4, and P < 0.0005). There were significant differences in mean AUDIT scores between all combinations of residence except for students living in “parental/family home” and “own home”  and “own home” and “other” .There was a significant difference in mean AUDIT scores of where students were currently living ; “halls” 12.00 (SD 6.57); “privately rented” 10.43 (SD 5.89); “own home” 7.53 (SD 5.73); “other” 5.17 (SD 4.97); Results showed significant differences between the mean AUDIT scores of students who had and had not experienced various negative consequences as a result of drinking. In all but one of the consequences listed, the mean AUDIT score was significantly higher in those students who had experienced the specific consequence. The only consequence not significant was that of “police caution or arrest for assault/affray” .Z score, were “your alcohol use” (“often/most days” 19.44 (SD 6.25); “never/rarely/sometimes” 9.93 (SD 5.52); Z = −18.09, P < 0.0005), “your smoking” (“often/most days” 15.97 (SD 7.01); “never/rarely/sometimes” 10.49 (SD 6.02); Z = −7.94, P < 0.0005), “sex” (“often/most days” 13.24 (SD 6.93); “never/rarely/sometimes” 10.41 (SD 6.00); Z = −6.52, P < 0.0005), and “sexually transmitted infections” (“often/most days” 15.49 (SD 7.27); “never/rarely/sometimes” 10.56 (SD 6.06); Z = −6.10, P < 0.0005)  .Z = −25.64, P < 0.0005). There were also significant differences in mean AUDIT scores of students who “strongly disagreed/disagreed” and “strongly agreed/agreed” that the amount they drank at present was harmful to their studies (“strongly disagreed/disagreed” 9.20 (SD 5.15); “strongly agreed/agreed” 18.14 (SD 6.21); Z = −20.52, P < 0.0005).There were significant differences in mean AUDIT scores of students who “strongly disagreed/disagreed” and “strongly agreed/agreed” that the amount they drank at present was harmful to their health (“strongly disagreed/disagreed” 8.03 (SD 4.64); “strongly agreed/agreed” 16.32 (SD 5.99); n = 219) currently use any of the drugs listed in the survey . Of those that drank (scored more than zero on the AUDIT) (n = 219) the mean AUDIT score was 15.91, SD = 6.24, range 2–32 . There were significant differences between the mean AUDIT scores of student drinkers who did and did not currently use drugs .Nine percent of students (n = 163) currently use cannabis . Of those that drank (n = 163) the mean AUDIT score was 15.71, SD = 6.38, range 2–32 . There were significant differences between the mean AUDIT scores of student drinkers who did and did not currently use cannabis .Six percent of students .Students who smoked had significantly higher mean AUDIT scores than nonsmokers (“smoker” 14.17 (SD 6.60); “nonsmoker” 10.28 (SD 5.93); Z = −4.67, P < 0.0005). There were also significant differences in mean AUDIT scores of students who specifically used emergency contraception and those who did not (“did use” 13.05 (SD 6.06); “did not use” 10.76 (SD 5.80); Z = −7.20, P < 0.0005) and significant differences also existed between mean AUDIT scores of students who had contracted a sexually transmitted infection in the last year ; “not had an STI” 11.04 (SD 5.87); Z = −4.99, P < 0.0005).Students who did not use any sort of contraception (or used the morning after pill) had significantly higher AUDIT scores than those who used some form of contraception the last time they had sex (“did not use” 12.50 (SD 6.58); “did use” 10.50 (SD 6.08); This cross-sectional survey of risk taking behaviours associated with alcohol consumption amongst undergraduate students found, as other studies have, that students most likely consume alcohol at increasing risk levels or above are male, 18–20 year olds of a White British ethnicity, and living in halls in their first year of study , 27. ThiThe insight gained into some of the consequences experienced by students when drinking alcohol is of importance as it can affect students' general health and wellbeing. The significantly increased chances of being arrested for antisocial behaviour, having arguments with friends/family, having a physical injury , and having unplanned sex make the student population who drink at higher levels more vulnerable to negative risky experiences which could have impacts on their chosen career paths and longer term health and wellbeing. This finding has also been found amongst students in previous research , 16.aspires to create a learning environment and organisational culture that enhances the health, wellbeing and sustainability of its community and enables people to achieve their full potential” [So, the question is how these results can be used by universities to work towards changing behaviours. With more than 2.3 million students in the UK and approximately 370,000 staff, the university setting is an ideal context to embed health and wellbeing programmes and policies . The Wortential” . It provtential” . The restential” . RecommeThe results of this present study provide insight into student drinking and positively correlated wider risk taking behaviours, which can inform university policies to protect students' health and wellbeing. The study university has considered the research results and has started to approach alcohol in a holistic and proactive manner. A multidisciplinary alcohol working group has been established to update the university alcohol policy in relation to staff and students. Numerous operational procedures have also changed. Since 2013 student enrolment documents have included core messages about the negative impact of alcohol and evidence based top tips to reduce these negative consequences. Also since 2013, Freshers' week was organised differently to ensure all promotional T-shirts did not advertise alcohol and that local pubs and clubs did not promote alcohol offers on campus grounds. The university offered increased financial assistance to societies on the basis that they did not take up sponsorship from external companies . Discussions are taking place to use the study's finding in relation to the demographic profile of students drinking at the highest levels to develop a social marketing campaign to raise awareness of the impact risky alcohol consumption can have.As with any study there are limitations. The survey response of 19% was low compared to other published electronic surveys  and is tFinally, universities do not sit in isolation but are an integral part of the city within which they are based. The Healthy University Framework provides an opportunity to develop a multidisciplinary task group to review how a university can approach student alcohol consumption. Partnership working with local authority alcohol action groups can have an impact on pubs and clubs targeting students and promoting binge drinking. The study published here provides a case study example of how to gain an understanding of students' attitudes and behaviours towards alcohol and how a university can reduce the wider negative impact caused by risky consumption.


Stereotactic body radiotherapy (SBRT) for non-small cell lung cancer (NSCLC) is primarily a treatment option for medically inoperable patients, who are often elderly. However, few studies report the effects of SBRT in elderly patients. Thus, we retrospectively analyzed clinical outcomes and feasibility following treatment of very elderly patients (age ≥ 85 years) with stage Ι NSCLC and younger patients (age < 85 years) with SBRT in our institution.From January 2006 to December 2012, 81 patients  with stage Ι NSCLC received SBRT. Prescription doses of 48 Gy were delivered in 4 fractions over 2 weeks or doses of 60 Gy were delivered in 10 fractions over 3 weeks.Local control was achieved in 91.8% of all patients at 3 years , and the 3-year overall survival (OS) rate was 69.4% . OS rates were significantly shorter for the very elderly group than for the younger group, with a 3-year cause-specific survival (CSS) rate of 77.9%  and a 3-year progression-free survival (PFS) rate of 59.5% . Multivariate analysis revealed a significant correlation between T stage and OS. Grades 2 and 3 radiation pneumonitis (RP) occurred in 7 (8.6%) and 2 (2.5%) patients, respectively. Among patients of very elderly and younger groups, grade 2 RP occurred in 4 (20%) and 3 (4.9%) patients, and grade 3 occurred in 2 (10%) and 0 (0%) patients, respectively. No grade 4 or 5 toxicity was observed, RP was significantly more severe among very elderly patients.SBRT for stage Ι NSCLC was well tolerated and feasible in very elderly patients. The efficacy of SBRT was comparable to that achieved in younger patients, although very elderly patients experienced significantly more severe RP. Although this study cohort included only 20 very elderly patients, the present data suggest that decreasing volumes of normal lung tissues exposed to ≥ 20 Gy and mean lung doses reduces the risk of RP in very elderly patients. The present data warrant studies of larger very elderly cohorts. Numbers of elderly patients with non-small cell lung cancer (NSCLC) are currently increasing . HoweverEligibility criteria were as follows: (1) identification of T1N0M0 or T2aN0M0 (stage Ι) primary lung cancer according to the Union for International Cancer Control in the 7th lung cancer TNM classification and staging system using computed tomography (CT) of the chest and upper abdomen, bone scintigraphy, and brain magnetic resonance imaging, (2) confirmation of NSCLC from histology or clinical information such as increased maximum standardized uptake valued (SUVmax) on 18-fluoro-deoxyglucose-positron emission tomography (FDG-PET), tumor enlargement on CT images, or elevated tumor marker levels during the observation period, (3) predominantly peripheral localization of the tumor, and (4) arterial oxygen pressure of ≥ 60 mmHg and predicted postoperative forced expiratory volume of ≥ 700 ml at 1 s. These respiratory criteria are identical to those prescribed by the Japan Clinical Oncology Group 0403 . PatientFrom January 2006 to December 2012, 81 patients with stage Ι NSCLC received SBRT. Table SBRT was performed with 6 MV X-rays using a CLINAC C21EX linear accelerator  or a Novalis Tx linear accelerator . A CT simulator and a 3D radiotherapy planning system  were used to plan treatments for all patients. A BlueBAG system  was used to immobilize patients. To maintain tumor positions during irradiation, patients were instructed in the self-controlled breath-hold technique using an Abches  respiratory monitoring system ,21.Follow-up after SBRT comprised CT scans at 1 month, and then at 3-month intervals for the first 2 years. Thereafter, follow-up CT scans were performed every 4 months. Local recurrence was diagnosed according to pathological confirmation, high uptake on FDG-PET (SUVmax ≥ 8) ,23, enlat test, ordinal quantitative variables were compared using Mann–Whitney U test, and qualitative variables were compared using chi-squared test with Fisher’s exact test. The Kaplan–Meier method was used to calculate local control (LC), overall survival (OS), cause-specific survival (CSS), and progression-free survival (PFS) rates, and group comparisons were made using the log-rank test. The Cox proportional hazard model was used to identify predictors of OS in both univariate and multivariate analyses. Multivariate analyses were performed for variables with probability (p) values of < 0.20 in univariate analysis, and differences were considered significant when p < 0.05. All statistical analyses were performed using StatView software version 5.0 .Continuous quantitative variables were compared using Student’s p = 0.002). Numbers of patients who were capable of self-controlled breath-holding during irradiation were significantly fewer in the very elderly group than in the younger group (p = 0.02). CTV were similar in each group, whereas PTV, V20 , and mean lung doses (MLD) were slightly but insignificantly greater in the very elderly group than in the younger group. No other significant differences were found between the groups.Table p = 0.0306; Figure Among all 81 patients, 1-, 2-, and 3-year LC rates were 97.4%, 95.9%, and 91.8%, respectively, and did not differ significantly between very elderly and younger patient groups , whereas CTV, PTV, V20, and MLD were not. However, V20 and MLD showed predictive tendencies for the severity of RP, with p values of 0.054 and 0.052, respectively  and 2 2.5%) patients, respectively. In the very elderly and younger groups, grade 2 RP was observed in 4 (20%) and 3 (10%) patients, and grade 3 RP was observed in 2 (10%) and 0 (0%) patients, respectively. No patients suffered from grade 4 or 5 toxicity. However, RP was more severe in the very elderly group than in the younger group . Thus, very elderly patients tended to die of causes other than NSCLC.Prognostic factors for OS that were identified in univariate analysis included age, T stage (T1a vs. others), and CTV. However, in subsequent multivariate analyses, only T stage was a significant prognostic factor for OS, indicating that tumor size is a stronger prognostic factor than age. In agreement, Palma et al.  showed tToxicity of SBRT is primarily related to RP. However, whereas grades 2 and 3 RP reportedly occur in 4.6%–13.8% and 0%–20% of patients, respectively ,2,30,31,3 of the rib were significant prognostic factors for rib fracture [Rib fracture was observed on follow-up CT in 18.5% of all patients (25% in the very elderly group and 16.3% in the younger group). Only two (2.4%) patients in the younger group complained of chest pain, and no patients exhibited symptoms of grade 3 or more. All elderly patients with rib fractures were asymptomatic, and no significant differences in rib fracture rates were found between the groups. In a study by Nambu et al. , rib frafracture -43, HoweSBRT for very elderly patients (age ≥ 85 years) with stage Ι NSCLC was well tolerated and feasible, with comparable efficacy to that observed in younger patients. Although very elderly patients suffered significantly more severe RP than younger patients, SBRT decreased V20 and MLD and reduced the risk of RP in very elderly patients. Although only 20 very elderly patients with NSCLC were included, the present analyses indicate that SBRT is curative, and warrant future studies with larger patient numbers.The authors declare that they have no competing interests.All authors read and approved the final manuscript. SH contributed to the study design, data collection, and analysis, and drafted the manuscript. HT contributed to the study design, data collection and analysis, and approved the final version of the manuscript. YK participated in data analysis and interpretation. YO performed clinical evaluations of patients at follow-up and collected data. HH contributed to study data abstraction, critical reviewed and improved intellectual content, and approved the final version of the manuscript.


Amyloid precursor protein knockout mice (APP-KO) have impaired differentiation of amacrine and horizontal cells. APP is part of a gene family and its paralogue amyloid precursor-like protein 2 (APLP2) has both shared as well as distinct expression patterns to APP, including in the retina. Given the impact of APP in the retina we investigated how APLP2 expression affected the retina using APLP2 knockout mice (APLP2-KO).Using histology, morphometric analysis with noninvasive imaging technique and electron microscopy, we showed that APLP2-KO retina displayed abnormal formation of the outer synaptic layer, accompanied with greatly impaired photoreceptor ribbon synapses in adults. Moreover, APLP2-KO displayed a significant decease in ON-bipolar, rod bipolar and type 2 OFF-cone bipolar cells . Reduction of the number of bipolar cells was accompanied with disrupted dendrites, reduced expression of metabotropic glutamate receptor 6 at the dendritic tips and alteration of axon terminals in the OFF laminae of the inner plexiform layer. In contrast, the APP-KO photoreceptor ribbon synapses and bipolar cells were intact. The APLP2-KO retina displayed numerous phenotypic similarities with the congenital stationary night blindness, a non-progressive retinal degeneration disease characterized by the loss of night vision. The pathological phenotypes in the APLP2-KO mouse correlated to altered transcription of genes involved in pre- and postsynatic structure/function, including CACNA1F, GRM6, TRMP1 and Gα0, and a normal scotopic a-wave electroretinogram amplitude, markedly reduced scotopic electroretinogram b-wave and modestly reduced photopic cone response. This confirmed the impaired function of the photoreceptor ribbon synapses and retinal bipolar cells, as is also observed in congenital stationary night blindness. Since congenital stationary night blindness present at birth, we extended our analysis to retinal differentiation and showed impaired differentiation of different bipolar cell subtypes and an altered temporal sequence of development from OFF to ON laminae in the inner plexiform layer. This was associated with the altered expression patterns of bipolar cell generation and differentiation factors, including MATH3, CHX10, VSX1 and OTX2.These findings demonstrate that APLP2 couples retina development and synaptic genes and present the first evidence that APLP2 expression may be linked to synaptic disease.The online version of this article (doi:10.1186/s13041-016-0245-z) contains supplementary material, which is available to authorized users. The amyloid precursor protein (APP) is known to play, together with its proteolytic fragments, numerous and varied roles in cell physiology and pathology, including in Alzheimer disease –4. The ao and GRP179 [As part of the CNS, the neural retina is arranged in well-defined laminar structures. Photoreceptors (rods and cones) transmit visual information to ganglion cells via bipolar cells. In the “classical” rod pathway, rods feed into rod bipolar cells that provide input to both the ON and OFF pathway via AII/glycinergic amacrine cells. The rod bipolar cell light-evoked response is initiated by a decrease in glutamate occupancy of mGluR6, which decreases its G-protein activity culminating in the opening of the TRPM1 channel –12. The d GRP179  and othed GRP179 , 19. Thed GRP179 . The OPLGRM6, TRPM1, GRP179, NYX and LRIT3 [CACNA1F were identified in patients with iCNSB [CACNA1F encodes the α1F subunit of the L-type voltage-gated Ca2+ channel, Cav1.4, and is located at the photoreceptor ribbon synapse. Abnormal synapses in the outer nuclear layer (ONL) detected by a noninvasive imaging technique using optical coherence tomography (OCT) have been observed in some cases of iCSNB [CABP4, encoding a Ca2+-binding protein located in photoreceptor synaptic terminals and associated with Cav1.4, lead to autosomal recessive iCSNB [CABP4 mutations have recently been shown to display high hyperopia [CABP4 and the Cacna1f I745T mutation lead to similar ERG alterations, visual impairments and an improper maturation of the synapse architecture [CSNB encompasses a group of genetically and clinically heterogeneous retinal disorders that primarily affect night vision. The non-progressive night vision loss is present at birth and there is no cure for CSNB. The prevalence of CSNB is approximately 1 in 10,000 . CSNB isnd LRIT3 –33. Mutath iCNSB , 35. CACof iCSNB , while tof iCSNB . Mutatiove iCSNB . Most ofyperopia , 40. A myperopia  and theyyperopia . In mousitecture , 43, indBHLHB4, MATH5, or PRDM8, which encode three transcription factors required for either maturation of rod bipolar cells or differentiation of several bipolar cell subtypes, may be candidate genes for CSNB [Although different genes responsible for the pathogenic mechanisms of CSNB have been identified, further studies are needed to clarify the molecular mechanisms of the disease. For CSNB patients in whom the genetic causes are still to be discovered, mutations are likely to be found in genes that function in photoreceptor pre- and postsynaptic processes that affect retinal transmission. It was suggested that for CSNB , 45. Recfor CSNB , reinforfor CSNB . In thisGiven the role of APLP2 in synapse function in adult brain and peripheral nervous system (PNS) , 48, andDevelopmental expression of APLP2 was examined by immunohistochemistry on WT neural retinas. During postnatal development, retinal ganglion cells (RGCs) and amacrine cells are the first cells to differentiate and form the IPL Fig. . Then, hUsing a panel of antibodies against specific markers of retinal cell subtypes, we showed that APLP2 colocalized with RGCs, amacrine (starburst and AII), rod bipolar and horizontal cells in adult retina , histology and transmission electron microscopy (TEM) in young and adult mice. The SD-OCT images showed an identical laminar organization in the inner and outer retina in young APLP2-KO and WT Fig.  mice sinThe thicknesses of the total neural retina, inner retina and outer retina were not significantly different between young and adult APLP2-KO , a b-wave with a subnormal amplitude was observed in young APLP2-KO  reduced in APLP2-KO (866 ± 191 μV) compared to WT (1218 ± 115 μV) with no alteration in half saturation luminance (K) or the slope of the b-wave sensitivity curve (n) . With increasing flash intensities, the b-wave amplitude increased incrementally in both age groups but the maximal cone b-wave amplitude was significantly (p < 0.05) reduced in adult (97.1 ± 8.9 μV) compared to young WT (138.5 ± 8.5 μV)  in APLP2-KO (100.3 ± 7.8 μV) compared to WT (138.5 ± 8.5 μV)  Fig. , consistμV) Fig. , indicatμV) Fig. , indicatv1.4-KO mice show altered localization in the OPL of presynaptic proteins, including RIBEYE and PSD95, and impaired distribution of PNA-labeled cone pedicles [Capedicles . Furtherpedicles  and has We next used TEM (16 K - 41 K x magnification) to monitor the photoreceptor terminals at the ultrastructure level. In the WT OPL region, the cone pedicles contained multiple triads composed of two lateral horizontal processes flanking a central bipolar cell dendrite Fig. . The maj13, a specific marker for ON bipolar cells (rod bipolar and cone bipolar cells (types 5a and 5b and types 6–9)) stained three rows of ON bipolar cell somas in the WT INL  in the INL  (MATH3 and BHLHB5) and homeodomain (HD)  factors involved in the generation or differentiation of bipolar cells –67. UsinSince rod bipolar cell dendrites contact photoreceptor terminals in the OPL by PN10, we investigated PKCα immunostaining of bipolar cell dendrites at this developmental stage. In WT, PKCα-labeled rod bipolar cells displayed their typical elongated cell somas in the INL and developed dendrites . At PN10, WT staining for VGLUT1 was preferentially located in the OFF sublamina where the bipolar cell terminals were located Fig. . In PN10Activation of microglial cells occurs during postnatal development to phagocytose inappropriate synapses to form a regular array in the two retinal plexiform layers . Given tv1.4 . Two main components of the soluble NSF attachment protein receptors, SNAP25 and syntaxin-1, were also upregulated. As were genes coding for two principal calcium sensors on synaptic vesicles, synaptotagmin 1 and Munc13-1 (2- to 3-fold). Genes coding for the presynaptic proteins: synapsin 2, PSD-95, synaptobrevin 2/VAMP2 and Munc18 had similar expression levels between WT and APLP2-KO.Although CSNB is a synapse pathology, the transcriptional consequences on genes encoding pre- and postsynaptic proteins of gene mutations responsible of CSNB remain unknown. Therefore, we hypothesized that deleting APLP2 may affect a transcriptional network essential for retinal synapse structure/function. To gain insights into the molecular mechanisms underlying synaptic defects in APLP2-KO, we analyzed mRNA expression of 12 proteins specifically involved in synaptic organization and function Fig. . In adulOα. In the dark, mGluR6 is constantly stimulated by glutamate and activates Goα which closes the TRPM1 channel, and PKCα accelerates and terminates glutamate-driven signal transduction. With the exception of TRPM1, which was significantly upregulated (9-fold), the other components of the ON bipolar cell signalling including mGluR6, GOα and PKCα were all significantly down regulated in APLP2-KO  mice have been described –98. Eye In vivo assessment of mice retinal thickness was performed on anesthetized animals using spectral domain optical coherence tomography (SD-OCT)  adapted for small animal eyes. Temporal, nasal, and superior quadrants of the retina were analyzed, using the optic nerve head and the retina vessels as landmarks. Each 2-dimensional B-scan recorded at 30° field of view consisted of 1536 A-scans with an optical resolution reaching 3.5 μm. Thickness of full retina, inner retina, and ONL were quantified using software provided by Heidelberg Engineering (Heidelberg Eye Explorer Version 1.6.2.0).Retina cross-sections were mounted and stained with toluidine blue. Images of whole retina cross-sections were obtained using Leica leitz Aristoplan microscope equipped with DFC 480 camera (Leica) and LAS 3.7 version software. Thickness of the different nuclear and synaptic layers were determined using Visilog 6.4 version software (Noesis).Retinal expression of APLP2 during embryonic and postnatal development was investigated by immunohistochemistry as previously described . Serial Identification and visualization of specific cells and cellular structures in the retina was performed by immunofluorescence microscopy as previously described . PrimaryAfter dissection, retinas were fixed in 2.5 % glutaraldehyde cacodylate buffer  and then fixed in 1 % osmium tetroxyde in cacodylate buffer  and progressively dehydrated in graduated ethanol solution, then in propylene oxide. Semi-thin sections (1 μm) were cut with an ultra microtome (Reichert OmU2) and stained with toluidine blue. Ultra-thin sections (80 nm) were contrasted by uranyl acetate, and analyzed with a transmission electron microscope (Philips CM10).g at 4 °C. Protein concentrations were determined using a Bio-Rad kit. Retina lysates were mixed with 3× Laemmli buffer and heated for 5 min at 95 °C. They were then resolved by SDS-PAGE (10 % or 15 % polyacrylamide gels), electroblotted onto polyvinylidene difluoride (PVDF) membrane , and probed with antibodies directed against Chx10, Islet1, VGLUT1, RIBEYE mGluR6 and actin , and centrifuged for 10 min at 10,000 Ct value for each sample recorded at the end of the reaction. The average and standard deviation of the three Ct values were calculated. Gene expression levels were normalized to β-actin for each retinal tissue sample and calculated relative to WT retinal tissue with the following equation: relative expression = 2Ct ─ WT sample ΔCt)─(APLP2 sample Δ where ΔCt = mean Ct(target) − mean Ct(β-actin).Mouse retina tissue was exposed to TRIZOL reagent  according to the manufacturer’s instructions, and SuperScript II Reverse Transcriptase (Invitrogen) was used to reverse transcribe 1 μg of mRNA. Amplification reaction assays contained 1× SYBR Green PCR Mastermix (Applied Biosystems). All real-time PCR oligonucleotide primers had previously been validated experimentally by qPCR, agarose gel analysis, sequencing, and blast. The PCR primer sequences are available on request. A hot start at 95 °C for 5 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min with the 7300 SDS thermal cycler (Applied Biosystems). Controls with no reverse transcriptase were run for each assay to confirm the absence of genomic DNA contamination. Control qRT-PCRs were performed without cDNA templates. The standard curve method  was used for relative quantification of gene expression. For each experiment, each individual sample was run in triplicate and the 2 and neutral density filter were used to attenuate luminance. Flash stimuli were given through an integrative sphere in order to uniformly illuminate all retina. A single ERG was recorded for seventeen increasing luminance  via Ag/AgCl electrodes. The signal was amplified  then processed and stored in a computer. For each ERG, a- and b-wave amplitude and latency were calculated. The mean values are plotted against the flash stimulation luminance. The software program  was used to fit the b-wave sensitivity curves to calculate the saturated b-wave amplitude (Bmax), the half-saturation luminance (K); and the slope of the curve in its linear part (n). For photopic conditions, mice were light-adapted 10 min at 30 cd/s m2 and then the retinal activity recorded in response to single flashes ranging from 10−2 to 30 cds/m2, in the same illumination conditions. For each stimulus intensity, 10 to 15 traces were averaged. Amplitude of the a-wave was defined as the difference between the baseline level at the time of stimulation and the peak of the a-wave. Amplitude of the b-wave was defined as the difference between the peak of the b-wave and the peak of the a-wave (or the baseline level when the a-wave was not detectable). Amplitudes are expressed in μV.Animals were dark adapted overnight. Under dim-red-light, they were anesthetized with a mixture of ketamine/xylazine and their pupils dilated with 1 drop of atropine. A photostimulator (Type PS 33) was used to generate ERG. The highest flash stimulation was 11.8 cd.s.mAll data are presented as mean ± s.e.m. Statistical significance in pairwise comparisons was assessed using two-tailed t-tests with unequal variance.In conclusion, our findings demonstrated the important and specific role of APLP2 in the retina. Our results demonstrate the non-redundant actions of APLP2 and APP in the central nervous system that are mediated through different effects on specific neurons and on ribbon synaptic networks during late embryonic and early postnatal development. Most importantly and of clinical relevance, there are striking similarities to be found between the phenotypic features of bipolar cell dendrites and photoreceptor ribbon synapses, and vision alteration in APLP2-KO mice and congenital stationary night blindness. APLP2 may be a bridge between retina development and synaptic genes involved in the pathogenic events in CSNB. Our studies have identified APLP2 as a new molecule that warrants investigation for its involvement in a range of eye pathologies.v1.4, calcium channel, voltage-dependent, L type, alpha 1 F subunit; cGMP, cyclic guanosine monophosphate; Chx10, Ceh-10 homeodomain-containing homolog; CNS, central nervous system; CSNB, congenital stationary night blindness; DSCAM, down syndrome cell adhesion molecule; ERG, electroretinogram; GCL, ganglion cell layer; Gγ13, G protein gamma 13 subunit; HD, homeodomain; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; Islet1, insulin related protein 1; L1CAM, L1 cell adhesion molecule; LRIT3, Leucine-rich repeat, immunoglobulin-like and transmembrane domains 3; MATH5/ATOH7, atonal BHLH transcription factor 7; mGluR6, metabotropic glutamate receptor 6; mRNA, messenger ribonucleic acid; NBL, neuroblastic layer; NFL, nerve fiber layer; NYX, nyctalopin; OCT, optical coherence tomography; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; OTX2, orthodenticle homeobox 2; PKCα, protein kinase C alpha; PN, postnatal day; PNA, peanut agglutinin; PNS, peripheral nervous system; PP2A, Protein phosphatase 2A; PRDM8, RIZ homology domain containing 8; PSD95, postsynaptic density protein 95; RAC1, Ras-related C3 botulinum substrate 1; RGC, retinal ganglion cells; RhoA, Rho family GTPase; RP2h, Retinitis Pigmentosa 2 homolog; RPE, retinal pigment epithelium; RT-PCR, reverse transcription polymerase chain reaction; SD-OCT, spectral domain optical coherence tomography; SNAP25, synaptosomal-associated protein 25; SynCAM, synaptic cell adhesion molecule; TEM, transmission electron microscopy; TRPM1, transient receptor potential cation channel subfamily M member 1; VGLUT1, vesicular glutamate transporter 1; VSX1, visual system homeobox 1; WT, wild-type; XRLS, X-linked RetinoschisisAPLP2, amyloid precursor-like protein 2; APP, amyloid precursor protein; BHLH, basic helix-loop-helix; BHLHB4, basic helix-loop-helix domain containing, class B 4; C1q, complement 1 q; CABP4, calcium binding protein 4; CACNA1F/Ca


Chlamydia gallinacea is known to occur in domestic poultry and other birds. Its potential as an avian pathogen and zoonotic agent is under investigation. The whole-genome sequence of its type strain, 08-1274/3, consists of a 1,059,583-bp chromosome with 914 protein-coding sequences (CDSs) and a plasmid (p1274) comprising 7,619 bp with 9 CDSs.The recently introduced bacterial species  Chlamydia gallinacea is an obligate intracellular bacterium classified under the single genus Chlamydia  were identified through mapping to the Gallus gallus genome using Segemehl  and Gallus gallus mitochondria, respectively. Thus, the assembly resulted in scaffold 1 , scaffold 2 , and scaffold 3 , all representing the chromosome, and scaffold 4  representing plasmid p1274. To close the gaps, primer sites were selected in flanking scaffold regions. The primers were used in PCR to generate DNA fragments of 600 to 800 bp (gap 1) and 1,300 to 1,500 bp (gap 2), which were sent to Eurofins Genomics  for Sanger sequencing. Alignment of Sanger sequences to scaffolds 1 to 3 using BLAST and MAFFT (In the present work, the data set was subjected to Segemehl . The remSegemehl  with k-mnd MAFFT  eventualThe complete chromosomal sequence comprises 1,059,583 bp. Provisional annotations using Prokka  revealedC. gallinacea. It can serve as a reference genome for future studies.This is the first report of a completely assembled genome sequence of C. gallinacea type strain 08-1274/3 and its plasmid p1274 have been deposited in NCBI GenBank under accession numbers CP015840 and CP015841, respectively.The updated whole-genome sequence data of


After 1, 3, 8, or 24 weeks, arteries were isolated for in vitro study. High flow (HF) induced outward hypertrophic remodeling in WKY rats after 1 week and persisted until 24 weeks without change in wall to lumen ratio. In SHRs, diameter increase was delayed, occurring only after 3 weeks. Nevertheless, it was reduced at 8 weeks and no longer significant after 24 weeks. In parallel, media cross-section area increased more with time in SHRs than in WKY rats and this was associated with increased contractility and oxidative stress with decreased NO-dependent relaxation. Low flow induced progressive inward remodeling until 24 weeks in both strains with excessive hypertrophy in SHRs. Thus, a chronic increase in flow induced transitory diameter expansion and long-lasting hypertrophy in SHRs. This could contribute to the higher susceptibility of hypertensive subjects to ischemic diseases.Hypertension is a major risk factor for cardiovascular disorders. As flow-mediated outward remodeling has a key role in postischemic revascularization, we investigated this remodeling in mesenteric resistance arteries of normotensive (WKY) and spontaneously hypertensive rats (SHRs) aged 3 to 9 months. Sequential ligation of mesenteric resistance arteries allowed modifying blood flow  Arterial hypertension is a major public health problem concern worldwide. This insidious disease that causes few if any symptoms or warning signs is nevertheless an important risk factor for myocardial infarction, stroke, renal failure, and peripheral arterial disease. Chronic increase in blood pressure induces a structural vascular remodeling associated with endothelial dysfunction and increased vascular tone in resistance arteries , 2. HypeShear stress exerted by blood flow at the surface of vascular endothelium produces vasorelaxation and in the long term an outward arterial remodeling. Indeed, a chronic increase in blood flow induces arterial diameter expansion normalizing shear stress, while the associated compensatory hypertrophy normalizes wall strain , 6. Such2, and endothelin-1 [In previous studies, we have shown that nitric oxide (NO) is essential to mesenteric resistance arteries remodeling induced by a chronic increase in blood flow , 14. Thethelin-1 , 19. Thethelin-1 , and NO thelin-1 , 20.2O2 level in SHRs arteries together with a high NO concentration that cannot be further elevated following chronically increased blood flow [High flow-mediated remodeling, when measured after 1 week, is reduced in young (10 weeks old) SHRs as compared to age-matched WKY rats . Neverthood flow . Althougood flow  and smalood flow , oxidatiood flow . Neverthood flow  and old ood flow , high flood flow . NAD(P)Hood flow .In this study, we investigated the evolution and the nature of flow-mediated remodeling in the context of hypertension and aging. To address this issue, we conducted functional and biochemical studies on mesenteric resistance arteries chronically submitted to high blood flow for 1 week up to 6 months in 3-month-old WKY rats and SHRs to evaluate the parallel evolution of flow-mediated remodeling, arterial contractility, and endothelium-mediated relaxation.Forty-eight 10-week-old male WKY rats and 48 SHRs  were anaesthetised  and pretreated with buprenorphine . A loop of intestine was then exposed and local mesenteric artery blood flow was surgically reduced as previously described , 28. Brin = 12 rats per group) and after 1, 3, 8, or 24 weeks, they were anesthetized for blood pressure measurement in the carotid artery [Rats were divided in 4 groups . The protocol was approved by the regional ethical committee (Protocol CEEA PdL number 2008.10).3; 3.7, KCl; 1.2 KH2PO4; 1.2, MgSO4; 11, glucose; 1.6, CaCl2; and 5, N-2-hydroxy-ethylpiperazine-N-2ethylsonic acid, pH 7.4, PO2 160 mmHg, and PCO2 37 mmHg. Mesenteric arteries were dissected free of fat and connective tissue using a dissection microscope. From each HF, LF, and NF artery, one segment was quickly frozen in liquid N2, one was used to determine the pressure-diameter relationship, and one was used for a pharmacological study.The mesenteric vascular bed was removed and kept in ice-cold physiological solution of the following composition (mM): 130, NaCl; 15, NaHCO2+-free PSS containing EGTA (2 mmol/L) and sodium nitroprusside . Pressure was controlled by a servo-perfusion system  and increased by steps from 10 to 150 mmHg. Diameter changes were continuously measured and recorded .One segment was then cannulated at both ends in a video-monitored perfusion system  as previously described . BrieflyAt the end of each experiment, pressure was set at 75 mmHg, and the arteries were fixed in a 10% buffered formaldehyde solution, as previously described  and storμm thickness) were obtained from the fixed arterial segments and stained with orcein. External diameter, lumen diameter, and media thickness were determined after images acquisition  and analyzed using the Histolab software  for cross-sectional area (CSA) calculation as previously described [Sections (7 escribed .μm diameter) were inserted in the lumen of the arteries and connected to a force transducer and a micrometer, respectively. Arteries were bathed in the PSS described above [μmol/L). Acetylcholine- (Ach 1 μmol/L) induced relaxation was then obtained after phenylephrine-induced preconstriction  in the presence or in the absence of the NO synthesis blocker L-NAME (100 μmol/L) or of superoxide dismutase (120 U/mL) plus catalase (80 U/mL) [The last arterial segment (2 mm long) was mounted on a wire-myograph  as previously described . Brieflyed above . A wall ed above  and vess80 U/mL) .μg total protein from each sample) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with the primary antibody , washed (3 times for 15 min), and incubated with horseradish peroxidase-conjugated secondary antibody (Amersham) for 90 min at room temperature. The proteins were visualized using the ECL-Plus Chemiluminescence Kit (Amersham) and bands intensity was quantified by densitometry using Image J software. The results were normalized to beta-actin immunoreactivity [Frozen arterial segments were pulverized in liquid nitrogen. The powders were resuspended in ice-cold lysis buffer: 150 mM NaCl, 1% IgepalCa630, 0,5% Na deoxycholate, 0,1% SDS, proteases inhibitors cocktail . Vessel extracts were incubated in this buffer on ice for 15 min and then centrifuged . The detergent soluble supernatant fractions were retained, and protein concentration in samples was equalized by using a Micro BCA Protein Assay Kit (Pierce) . Proteinactivity .t-test) to compare pressure-diameter curves in the different groups. In the other set of experiments, means were compared by unpaired Student's t-test. P values less than 0.05 were considered to be significant.Results were expressed as means ± standard error (SEM). Significance of the differences between groups was determined by analysis of variance (two-way ANOVA for consecutive measurements followed by the Bonferroni n = 12 per group).Mean arterial blood pressure was not significantly affected by the ligations in either strain. In the 4 studied groups  mean arterial pressure was 146 ± 6, 162 ± 7, 173 ± 9, and 170 ± 8 mmHg in SHRs and 92 ± 5, 95 ± 6, 94 ± 7, and 95 ± 7 mmHg in WKY rats  and from 326 ± 38 to 350 ± 37 μL/min in SHRs (n = 5 per group). In HF arteries blood flow ranged from 673 ± 52 to 724 ± 79 μL/min in WKY rats (n = 5 or 6 per group) and from 654 ± 45 to 688 ± 70 μL/min in SHRs . In LF arteries, blood flow ranged from 110 ± 21 to 54 ± 16 μL/min in WKY rats  and from 93 ± 18 to 50 ± 14 μL/min in SHRs .Blood flow , measureP < 0.01). Remodeling decreased then after 8 weeks  and after 24 weeks .In isolated mesenteric resistance arteries, stepwise increases in pressure induced a rise in diameter Figures . ArteriaOutward remodeling in HF arteries was significantly lower in SHRs than in WKY rats after 1 week and after 24 weeks, whereas it was higher after 3 weeks and equivalent after 8 weeks. Inward remodeling of LF arteries was equivalent in SHRs and in WKY rats and developed progressively with time .Media cross-section area (CSA) was higher in SHR compared to WKY rats arteries as previously described . Chronicμmol/L) was higher than in NF vessels . Nevertheless, contractility rose progressively from 1 week to 24 weeks in HF arteries from SHRs, whereas the contraction was stable in HF arteries from WKY rat from 3 to 24 weeks. The contraction induced by depolarizing potassium concentration (KCl 80 mM) followed a similar pattern with the exception that it was higher in LF arteries in SHR compared to WKY rats (Phenylephrine- (PE-) induced contraction  was highWKY rats .μmol/L) was lower in HF and NF arteries from SHR than in equivalent arteries from WKY rats  induces endothelium-mediated dilation, which is impaired in SHRs due to excessive vasoconstrictor agents  production Indeed, p67phox and gp91phox expression levels were higher in SHRs than in WKY rats. This may lead to excessive oxidative stress and consequently to hypertrophy and hypercontractility as previously described in various vascular territories in hypertension –43. Our  oxidase . In the  oxidase . A possirtension , 45 as wrtension , metabolrtension , 47, andrtension , 49.Finally, in arteries exposed to a reduced flow, intraluminal diameter decreased continuously over the 6-month duration of the protocol without reaching a plateau and without difference between SHRs and WKY rats. Low flow-mediated inward remodeling is the consequence of unopposed constrictor tone as flow-mediated EDRF release is chronically reduced , 27, 50.To conclude we found a biphasic evolution of high flow-mediated outward remodeling of mesenteric resistance arteries in SHRs. Nevertheless, remodeling evolved rather negatively over time in SHRs with a progressive worsening of the arterial function and structure, whereas in WKY rats outward remodeling persisted over time with an improved endothelium-mediated relaxation. Such a reduced response to a chronic increase in blood flow could provide a possible explanation for the deleterious outcome of ischemic disorders in hypertensive patients.


Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder  anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection.Outer membrane protein C of  Edwardsiella tarda is a Gram-negative pathogen and the etiological agent of edwardsiellosis, which affects a wide range of marine and freshwater fish [ter fish ,2,3, cauter fish ,5. The cter fish . Vaccineter fish , and theEscherichia coli and Salmonella typhi had showed that OmpC is a major cell surface antigen, and the expression of OmpC during the infection period and its capacity of exhibiting heterologous epitopes on the cell surface make it an attractive candidate antigen in vaccine development [E. coli, when in form of subunit vaccine, could induce the production of specific serum antibodies and produce highly protective effects against E. coli infection and cross-protection against Shigella strains [Salmonella revealed better protection against virulent Salmonella challenge when compared with formalin-killed, whole-cell bacterial vaccine in a bird model [Shigella flexneri 3a OmpC epitope was recognized by human umbilical cord sera and associated with protective activity, mice immunized with OmpC retained long-lasting protection against a lethal dose of both homologous and heterologous strains of the pathogens [Aeromonas hydrophila and S. enterica serovar Typhi, has also been reported to have the potential in vaccine development [E. tarda is a pathogen bacteria that threatens fish, reptiles, amphibians, and humans [E. tarda.Outer membrane protein C is a porin that resides in the outer membrane of Gram-negative bacteria. Studies in human pathogen elopment ,9,10,11. strains . It was rd model ,14. Moreathogens . Additioelopment . E. tardd humans , and gred humans , no commE. tarda was amplified and recombinantly expressed. To test the immunogenicity of OmpC, outer membrane proteins of E. tarda were extracted and analyzed by Western blot using flounder  anti-rOmpC and anti-E. tarda antibodies. Meanwhile, the immune response of flounder after being injected with rOmpC was investigated, including the production of specific serum antibodies, the proliferation of sIg+ lymphocytes and the expression of immune-related genes. Moreover, the immune protective effects were examined by challenging with E. tarda in the flounder model.In the present study, OmpC of E. coli BL21 (DE3) with pET-32a system . Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) revealed that OmpC was successfully expressed after IPTG induction with a distinct band representing about 60 kDa  when in form of recombinant protein could produce immune-protective effects in flounder against edwardsiellosis [E. tarda infection [To test the immunogenicity of OmpC, the OMPs of iellosis ,21. OmpCiellosis ,23,24. Anfection ,26. ThesE. coli and formulated with certain adjuvants, could induce the production of specific serum antibodies and produce highly protective effects [E. tarda challenge. All of these results suggested that OmpC was a protective antigen and 2 mg/mL rOmpC was a safe dose. Nevertheless, only one concentration was set in the present study, which might not be the most optimal concentration, so we also believed that a further research was needed to optimize the injection dose by setting a series of concentrations. Meanwhile, the levels of sIg+ lymphocytes and specific antibodies induced by rOmpC were also examined. The results showed that rOmpC could induce the proliferation of sIg+ lymphocytes and the peak levels in blood, spleen, and pronephros occurred at week 5, week 4 and week 5 after immunization. In accordance with the change variation of sIg+ lymphocytes, the specific antibodies increased and reached the peak level at week 5 after immunization. These results may indicate that rOmpC could induce a strong humoral immune response.Subunit vaccines are safe and have negligible adverse effects, several researchers have confirmed that subunit vaccines, when in form of recombinant proteins purified from  effects ,27. In tMHCI and MHCII genes responsible for encoding cell-surface glycoproteins, which will bind foreign peptides and present self- and non-self peptide to T-cells’ receptors [MHCIIα and CD4-1 genes significantly up-regulated after immunization, while MHCIα and CD8α were not induced. Though E. tarda was an intracellular pathogen [IL-1β, TNF-α, and IL-6, were also induced by rOmpC. These cytokines play an important role in activating the pro-inflammatory cytokine cascade, in activating the functions of macrophages, and also in activation of the adaptive immune system [TLR2 and TLR5M genes were not induced after vaccination with rOmpC, suggesting that the TLR2 and TLR5 pathways were not activated.It was reported that eceptors ,29. It weceptors , and foreceptors . In our pathogen , OmpC whe system . TLRs are system , howeverE. tarda EIB202 (Genbank No. CP001135.1), the gene sequence encoding OmpC was obtained and the accession number was ETAE3470. Specific primers were designed to amplify the open reading frame (ORF) of OmpC excluding the region coding for its signal peptide. The primers used were all listed in E. coli BL21 (DE3). The transformant was cultured in LB medium to a mid-logarithmic phase and induced by adding isopropyl β-d-1-thiogalactopyranoside. His-tagged rOmpC was purified using His Trap™ HP Ni-Agarose  followed by the manufacturer’s instruction. The purified protein was dialyzed for 24 h against phosphate-buffered saline (PBS) and treated with Triton X-114 to remove endotoxin [Based on the genome sequence of E. tarda mixed with complete Freund’s adjuvant. The flounder were boosted with the same amount of rOmpC or inactivated E. tarda mixed with incomplete Freund’s adjuvant at two weeks after the initial immunization. The fish were sacrificed at two weeks post the last boost, and blood was collected from the caudal veins of the fish. The blood was placed at 4 °C for overnight to allow clotting, and sera were obtained by centrifugation at 3000× g for 10 min [Ten healthy flounder weights of 750 ± 50 g were purchased from local farm and divided randomly into two groups and were immunized via intraperitoneal injection with rOmpC or inactivated r 10 min , and theE. tarda were extracted by the method as described [E. tarda serum or the serum of healthy flounder for 1 h at 37 °C, then washed three times with PBS containing 0.05% Tween-20 (PBST), and then incubated with mAb 2D8 for 1 h at 37 °C, which was a monoclonal antibody against flounder IgM previously produced by our laboratory [2, pH 9.5) containing nitroblue tetrazolium  and 5-bromo-4-chloro-3-indolyphosphate  for 5 min and stopped by washing with distilled water. The immune-reactive proteins were excised from polyacrylamide gels and analyzed by an ABI5800 matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) assay system .The OMPs of escribed . The extboratory , then waTwo hundred healthy flounder with body weights of 30 ± 5 g were obtained from a fish farm in Rizhao City of Shandong province, China. The fish were maintained in tanks containing aerated, sand-filtered seawater at 21 ± 0.5 °C for one week prior to processing. Flounder were equally divided into two groups. For rOmpC vaccination, the concentration of rOmpC was adjusted to 2 mg/mL, and one group was intraperitoneally injected with 100 μL rOmpC mixed with complete Freund’s adjuvant at equal volume. The other one was intraperitoneally injected with 100 μL PBS as the control group.g for 10 min and stored at −20 °C until usage. For qRT-PCR, the liver, spleen, and head kidney were randomly collected from three fish in each group at 6 h, 12 h, 24 h (1 day), 48 h (2 days), 72 h (3 days), 96 h (4 days), 5 days, 7 days, 14 days, 21 days and 28 days after immunization. Tissues were placed in a sample protector  and stored at −80 °C until usage. Before manipulations, the fish were anaesthetized or over-anaesthetized with MS-222 as before.The serum and the lymphocytes in blood, spleen, and pronephros were randomly sampled from three fish in each group before immunization and at week 1, 2, 3, 4, 5 and 6 after immunization. For serum isolation, blood was collected from the caudal veins and allowed to clot overnight at 4 °C. The serum was obtained by centrifugation at 3000× 6 cells/mL, then incubated with mAb 2D8 (1:3000 diluted in PBS) for 1 h at 37 °C. Subsequently, cells were washed three times with PBS containing 5% (v/v) Newborn Calf Serum, then incubated with goat-anti-mouse Ig-FITC  for 1 h at 37 °C, and washed again. After that, the cell suspensions were analyzed with an Accuri C6 cytometer .The lymphocytes in blood, spleen, and pronephros of vaccinated flounder were isolated according to the technique as described in our previous study . The lymE. tarda (108 CFU/mL) at 4 °C. The wells were washed three times with PBST and then blocked with 3% BSA in PBS for 1 h at 37 °C. After washing, the serum (1:100 diluted in PBS) sampled from vaccinated fish at different times were added 100 μL per well and incubated for 2 h at 37 °C. Following washing, 100 μL mAb 2D8 (1:3000 diluted in PBS) was added for serum antibody detection. After incubation at 37 °C for 1 h and washing, 100 μL goat-anti-mouse Ig-alkaline phosphatase conjugate (Sigma) diluted 1:5000 in PBS was added and incubated for 1 h at 37 °C. After the last washing, 100 μL 0.1% (w/v) p-nitrophenyl phosphate  in 50 mM carbonate-bicarbonate buffer (pH 9.8) containing 0.5 mM MgCl2 was added to each well and incubated at room temperature for 30 min in the dark. The reaction was stopped by adding 50 μL per well of 2 M NaOH and absorbance was measured with an automatic ELISA reader  at 405 nm.Wells of flat-bottom microplates  were coated overnight with 100 μL/well of ® 480 II Real-Time System . Each assay was performed in triplicate with 18S gene as the internal control. All dates were analyzed relative to the 18S gene by the 2Ct−∆∆ method, then the difference in the vaccinated and control groups were employed to assess changes in the expression of genes. The primers for TLR2, TLR5M, MHCIα, MHCIIα, CD4-1, CD8α, IL-1β, IL-6, IFN-γ, TNF-α and 18S were listed in Total RNA was extracted from the liver, spleen, and head kidney of vaccinated fish using trizol reagent according to the manufactures’ instruction and measured by a Nanodrop 8000 Spectrophotometer . Single-strand cDNA was synthesized from 2 μg total RNA using PrimeScript™ RT-PCR Kit  according to the manufactures’ instruction. qRT-PCR was carried out using SYBR GreenI Master  in a LightCyclerE. tarda used for challenge was cultured in LB media at 30 °C for 24 h. The fish was intraperitoneally injected with a dose of 100 μL per fish containing 1 × 107 CFU live E. tarda. Mortalities were monitored over a period of 15 days after the challenge, and relative percentage of survival rate (RPS) was calculated as previously described [Forty vaccinated fish were randomly selected from each vaccinated group for challenge test at week 6 after immunization. The escribed .p < 0.05.The statistical analysis was performed using Statistical Product and Service Solution (SPSS) software , differences were analyzed with one-way analysis of variance (ANOVA) and the results were expressed as mean ± SEM. In all cases, the significance level was defined as E. tarda is an immunogenic surface protein, which could induce strong innate immune response and humoral immune response in flounder and finally evoke highly protective effects against E. tarda challenge when in the form of a recombinant protein. This indicates that OmpC is a promising vaccine candidate against E. tarda infection.In conclusion, OmpC of