The aim of this work is to investigate the effect of electroacupuncture (EA) on insulin sensitivity in high-fat diet (HFD) induced insulin resistance (IR) rats and to evaluate expression of AMPK/ACC signaling components. Thirty-two male Sprague-Dawley rats were randomized into control group, HFD group, HFD+Pi  group, and HFD+EA group. Acupuncture was subcutaneously applied to Zusanli (ST40) and Sanyinjiao (SP6). For Zusanli (ST40) and Sanyinjiao (SP6), needles were connected to an electroacupuncture (EA) apparatus. Fasting plasma glucose was measured by glucose oxidase method. Plasma fasting insulin (FINS) and adiponectin (ADP) were determined by ELISA. Triglyceride (TG) and cholesterol (TC) were determined by Gpo-pap. Proteins of adiponectin receptor 1 (adipoR1), AMP-activated Protein Kinase (AMPK), and acetyl-CoA carboxylase (ACC) were determined by Western blot, respectively. Compared with the control group, HFD group exhibits increased levels of FPG, FINS, and homeostatic model assessment of insulin resistance (HOMA-IR) and decreased level of ADP and insulin sensitivity index (ISI). These changes were reversed by both EA and pioglitazone. Proteins of adipoR1 and AMPK were decreased, while ACC were increased in HFD group compared to control group. Proteins of these molecules were restored back to normal levels upon EA and pioglitazone. EA can improve the insulin sensitivity of insulin resistance rats; the positive regulation of the AMPK/ACC pathway in the skeletal muscle may be a possible mechanism of EA in the treatment of IR. At the turn of this century 171 million individuals were estimated to have diabetes, and this is expected to increase to 366 million by 2030. AlthougObesity is associated with an increased risk of developing IR and T2DM. In obese individuals, adipose tissue modulates metabolism by releasing increased amounts of nonesterified fatty acids (NEFAs), glycerol, hormones—including leptin and adiponectin—and proinflammatory cytokines, and other factors that are involved in the development of insulin resistance . Previouα- (PPAR-α-) dependent manner [Increased NEFA delivery or decreased intracellular metabolism of fatty acids results in an increase in the intracellular content of fatty acid metabolites such as diacylglycerol, fatty acyl-coenzyme A, and ceramides, which, in turn, activate a serine/threonine kinase cascade leading to serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2), and a reduced ability of these molecules to activate PI3K, which is associated with altered rates of insulin-stimulated glucose uptake and transport , 14. By t manner . Thiazolt manner  and humat manner . Pioglitt manner . Pharmact manner . Eletroat manner . This tht manner , 21. TheTherefore, the goal of this study is to elucidate the EA effect on AMPK/ACC pathway proteins and further clarify the mechanism of EA in improving lipid metabolism in skeletal muscle of IR cases.Male Sprague-Dawley rats  were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (certificate number: SCXK (Guangzhou) 2013-0002). Animals were maintained in the Experimental Animal Center of Traditional Chinese Medicine Hospital of Guangdong Province. The animals were reared in separate cages in a room under controlled light, relative humidity (50%), and temperature (20±2°C) conditions. All animals had free access to basic feed and tap water. All animal experimental procedures were reviewed and approved by the Laboratory Animals Ethics Committee at Second Affiliated Hospital of Guangzhou University of Chinese Medicine Hospital (reference no. 2014016) and were conducted in accordance with local guidelines for animal welfare consistent with the National Research Council's ‘Guide for the Care and Use of Laboratory Animals' . All efforts were made to minimise the number of animals used and their suffering.According to a random number table, the rats were allocated to a negative control group  or a IR model group (n=24). Control group was fed with a regular diet (RD) consisting of 21% protein, 11% fat and 68% Carbohydrates. IR model group was fed a high fat diet (HFD) comprising 26.2% crude protein, 34.9% total fat, and 5% crude fiber. Twelve weeks later, the remaining 24 IR rats were subdivided into three groups according to a random number table (n=8 each): (1) an IR group remained untreated; (2) an IR + Pi group that received oral gavage of pioglitazone (10mg/kg/day); and (3) an IR + EA group that received EA only. Rats in the IR + EA group were physically restrained during EA and rats in the other three groups were restrained in the same way without receiving acupuncture to control for any effect of handling on study outcomes. Venous blood of rats was collected for fasting insulin, fasting plasma glucose, triglyceride, cholesterol, and HOMA-IR index measurement after treatment. All rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg) and were sacrificed by cervical dislocation for histopathological examination on the skeletal muscle. Skeletal muscles peeled off from quadriceps femoris were stored at −80°C for the following Western blotting.Fenglong) and SP6 (Sanyinjiao) [EA was administered every day to rats in the IR + EA group only, seven days a week for 2 consecutive weeks. EA treatment was performed at classical acupuncture points, namely ST40 (yinjiao) . A pair All rats were fasted for 12 hours after the last acupuncture. On the second morning, blood samples from the orbital venous sinus were collected. Samples were measured for fasting insulin (FINS), fasting plasma glucose (FPG), triglyceride (TG), cholesterol (TC), and adiponectin (ADP). FPG was measured by glucose oxidase method. FINS and ADP were determined by ELISA method . TG and TC were determined by Gpo-pap method . All of above methods were performed according to the instructions from the manufacturer. Additionally, the following formula was used to calculate homeostasis model assessment-estimated insulin resistance (HOMA-IR)= FINS (IU/L) × FPG (mmol/L)/22.5. Insulin4% paraformaldehyde fixed skeletal muscle of quadriceps femoris samples (1mm ×1mm) of control, IR, IR + EA, IR + pioglitazone groups was collected and fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4) overnight at 4°C. After dehydration with a graded ethanol series, the sample was embedded in Epon812 and sectioned using a Leica EM UC6 ultramicrotome . Samples were viewed and photographed using Transmission Electron Microscopy (TEM) .Skeletal muscle were peeled off from quadriceps femoris and washed twice with ice-cold PBS (PH 7.4) and then homogenized with RIPA buffer  containing protease inhibitor cocktail . Tissue lysates were then centrifuged at 12,000 g for 5 min at 4°C. Protein concentration was determined using BCA , and 30 ug of total proteins was taken for 10% SDS polyacrylamide gel electrophoresis. The separated proteins were then transferred on to 0.2 pore polyvinylidene difluoride (PVDF) membranes  using tank transfer system . The nonspecific binding of antibodies was blocked with 5% nonfat dried milk in TBST buffer (0.1%Tween 20 in PBS). The membrane was then transferred to the hybrid bag and the first antibody, namely, adipoR1 , AMPK, ACC, and GAPDH , was added at 1:1000 dilution. The bag was then sealed and maintained at 4°C overnight. Subsequently, the second antibody  was added, and the membrane was incubated for 1 hour at 37°C and washed. After reacting with enhanced chemiluminescence (ECL) substrate for 1–3 min, the light signal was captured by Chemidoc gel imaging system , and the image was analyzed by Quantity One 1D Analysis Software .The statistical analysis was performed using the SPSS software, version 20.0 , and the data were expressed as the mean ± standard deviation. A one-way ANOVA was used after the normal distribution and homogeneity of variance were confirmed. For the nonnormally distributed data or for data with heterogeneous variance, a nonparametric test was used. The LSD method was applied for pairwise comparisons of the Western blot results. Statistical significance was set to p < 0.05 and high statistical significance was set to p < 0.01.p < 0.01). After EA and pioglitazone treatment, the levels of body weight, plasma FPG, FINS, TG, TC, and HOMA-IR in the HFD + Pi and HFD + EA group were significantly decreased, while the levels of ADP and ISI were significantly increased as compared with the HFD group . There were no significant differences observed between HFD+Pi and HFD+EA groups (p > 0.05).As shown in The quality of functional status of mitochondria in skeletal muscle was examined and evaluated by transmission electron microscope. Compared with the control group, many vacuoles with different sizes and subsarcolemmal accumulation of enlarged and abnormally shaped or swollen mitochondria were observed in HFD group. One of the hallmarks for the disordered lipid metabolism of skeletal muscle is the high population of damaged or swollen mitochondria and disordered fiber arrangement or dysfunctional fiber structure in skeletal muscle. The normal arrangement of mitochondria and apparently less vacuoles were observed in skeletal muscle from HFD + Pi and HFD + EA groups .p < 0.05, p < 0.01). The HFD + Pi and HFD + EA group showed increased expression of both adipoR1 and AMPK and decreased expression of ACC compared with those of the HFD group . No significant differences were obtained between the HFD + Pi and HFD + EA group (p > 0.05).This study demonstrated that EA intervention improved insulin resistance in HFD rats. Insulin sensitivity as measured by HOMA-IR index and ISI got worse in HFD rats after 12 weeks of HFD. This was consistent with previous reports that HFD rats developed insulin resistance and had higher plasma free fatty acids (FFAs) and triglycerides . In the It is believed that EA may be used to prevent diabetes and metabolic syndrome since it can ameliorate insulin sensitivity. However, the underlying mechanism of action remains uncertain. Obesity is commonly associated with T2DM, coronary artery disease, and hypertension, and the coexistence of these diseases has been termed the metabolic syndrome . InsulinIt is known that the skeletal muscle is one of the most susceptible areas to insulin resistance. Adiponectin receptor 1 (adipoR1) is abundantly expressed in skeletal muscle which serve as receptor for adiponectin. AdipoR1 expression in the skeletal muscle of T2DM patients has been reported to be decreased . Our resAMPK represents an energy sensor and metabolic regulator that responds to hormone and nutrition status in vivo and exerts a regulatory effect in the hypothalamus and multiple peripheral tissues . ActivatAlthough the present study has shown that electroacupuncture alters expression of AMPK/ACC signaling pathway related molecules and enhances insulin sensitivity in a rat model of IR, there are some details which are expected to improve. First, phosphorylated-AMPK and phosphorylated-ACC will be measured to demonstrate better about the activation of the AMPK/ACC signaling pathway in the next study. Second, specific inhibition of AMPK can be used as a negative control to explain the effect of electroacupuncture on AMPK biological function more objectively. Finally, the acupuncture protocol can be changed to every other day and last for 4 weeks, which can ensure the acupuncture stimulation and be convenient for both patients and acupuncturists to follow.The present study suggests that EA increase insulin sensitivity via modulating secretion of adiponectin, as well as disordered lipid metabolism in rat model of IR; and the expression level of adipoR1 protein in skeletal muscle is upregulated. These improvements in IR were observed associated with significant reduction of fat accumulation in skeletal muscle along with activation of the AMPK/ACC signaling pathway. In conclusion, EA is evidenced to be able to prevent disordered lipid metabolism and to ameliorate insulin sensitivity. Therefore, EA may represent a promising therapeutic strategy for diseases with obese related to insulin resistance.


Background: Studies have shown that the serum total bilirubin (TBil) is associated with the mortality of the general population and of hemodialysis patients. However, few studies have examined the associations of the direct bilirubin (DBil) and indirect bilirubin (IBil) with the mortality of peritoneal dialysis (PD) patients.Methods: This was a retrospective cohort study. Clinical and laboratory data were collected from 740 PD patients. The primary endpoint was 5-year all-cause mortality. Survival analysis was performed using the Kaplan–Meier method with the log-rank test. The mortality hazard ratio was evaluated using Cox regression models.Results: Among the 740 PD patients, the mean age was 49.9 ± 15.0 years, 54.9% were men, and 20.3% had diabetes. During the median follow-up period of 28 months , 178 patients died. Kaplan–Meier analysis revealed that all-cause mortality was higher in the patients in the higher TBil group than in the lower TBil group  and in patients in the higher IBil group than in the lower IBil group . Multivariate analysis showed that compared with the lower TBil group, the 5-year mortality risk was higher in the higher TBil group . Similarly, there was a 56% higher risk of 5-year mortality in the higher IBil group than in the lower IBil group . However, no such associations were observed between the DBil and the mortality risk.Conclusions: The baseline serum TBil and IBil levels were significantly associated with 5-year all-cause mortality among PD patients. At present, more than 272 000 ESRD patients receive PD worldwide, which accounts for approximately 11% of dialysis patients ,4.Bilirubin, as the end-product of heme metabolism, has been discovered to have antioxidant properties , whetherCurrent evidence indicates that mildly elevated bilirubin is associated with protection from cardiovascular disease and from all-cause mortality in adults . MoreoveTherefore, this study aimed to evaluate the relationship of the levels of the three types of serum bilirubin  with the 5-year all-cause mortality of PD patients.2.2.1.All incident patients who used PD as their first renal replacement treatment modality from the PD Center of the First Affiliated Hospital, Nanchang University, Jiangxi, China between 1 November 2005 and 28 February 2017 were screened. Patients who were aged ≥18-years-old at the start of PD and survived for at least 90 days from their initial PD therapy were enrolled. Patients who were catheterized in other hospitals shifted from permanent HD or failed renal transplantation therapy were excluded. Patients who met the following criteria were also excluded: (1) patients with hepatitis B virus or hepatitis C virus; and (2) patients with serum bilirubin levels higher than the upper limit of the normal range, who did not have a known baseline bilirubin level, or who showed abnormal liver enzyme tests  > 40 IU/L or aspartate aminotransferase (AST) > 40 IU/L). The study was conducted in compliance with the ethical principles of the Declaration of Helsinki [The primary endpoint of this study was 5-year all-cause mortality. All patients were followed up until the cessation of PD, death, or 31 May 2017. Baseline demographic data were collected, including age, sex, the primary cause of ESRD, the presence of diabetes and CVD. Clinical and biochemical data were collected at the initiation of PD, including blood pressure, estimated glomerular filtration rate (eGFR), hemoglobin, serum albumin, blood urea nitrogen, total cholesterol (CHOL), triglyceride (TG), ALT, AST, TBil, DBil, serum creatinine (SCr), blood uric acid, serum phosphorus (P), serum calcium (Ca), intact parathyroid hormone (iPTH), and total urea clearance (tKt/V). The IBil level was calculated using the following formula: IBil  =  TBil − DBil. All baseline data were obtained during the first 1–3 months of PD. Baseline residual renal function was assessed by eGFR using the Chronic Kidney Disease Epidemiology Collaboration creatinine equation .2.2.n (%). Differences between groups were assessed using chi-square or Student’s t tests or the Kruskal–Wallis test. Survival analysis between the groups was performed using the Kaplan–Meier method with the log-rank test. The mortality hazard ratio was evaluated using Cox proportional hazards models. Sensitivity analyses by excluding patients with follow-up periods less than 1 year were performed to assess the robustness of the results. The censored data included those of patients who switched to HD, who underwent renal transplantation, who transferred to another center, who were lost to follow-up, or who were still at our PD center on 31 May 2017. Covariates with p < .1 in the univariate Cox analyses or that were thought to be clinically significant were used for multivariate Cox proportional hazards regression analysis. The results are expressed as the hazard ratios (HR) and 95% confidence interval (95% CI). All descriptive and multivariate analyses were conducted using SPSS version 21.0 . A value of p < .05 was considered statistically significant.We calculated the area under the receiver operating characteristic (ROC) curve and selected the optimal cut-off value with the maximum sum of specificity and sensitivity, and we divided the patients into two groups according to the cut-off values of the three types of serum bilirubin levels: the lower bilirubin group  and higher bilirubin group . Numeric parameters are presented as the mean ± standard deviation and categorized data are presented as the number and percentage 3.3.1.n  =  1011) were recruited and monitored at our hospital. Out of 1011 patients, 34 patients were excluded based on our exclusion criteria, as follows: 3 patients were under 18 years of age, 2 patients were transitioned from a failed renal transplantation, 8 patients were transitioned from permanent HD, and 21 patients received PD for less than 3 months. The remaining 977 patients were enrolled. Of the 977 patients, patients were excluded for the following reasons: 2 patients with serum bilirubin levels greater than the upper limit of the normal range or who did not have baseline serum bilirubin measurements; 83 patients with ALT > 40 IU/L or AST > 40 IU/L; and 152 patients with hepatitis B virus or hepatitis C virus. Finally, 740 PD patients were enrolled in the study (All PD patients (he study . As show3.2.As shown in 3.3.p = .017) (p = .026) (p = .018. The corresponding HR (95% CI) for IBil was 1.442 (1.044–1.991), p = .026. After adjusting for age, sex, diabetes, cardiovascular disease, hypertension, eGFR, albumin level, and hemoglobin, the mortality rate was higher in the higher TBil group than in the lower TBil group . Similarly, there was a 56% higher risk of 5-year mortality in the higher IBil group than in the lower IBil group . However, no such associations were observed between the DBil and mortality risk. Moreover, the sensitivity analysis showed the same results after excluding patients with less than 1-year follow-up period . By the end of the study, 41 (5.5%) patients underwent renal transplantation, 123 (16.6%) patients were shifted to HD, 2 (0.3%) patients were transferred to other PD centers, 20 (2.7%) patients were lost to follow-up and the remaining 376 (50.8%) patients were still being followed at our PD center. Of the 178 deaths, 96 (53.9%) were caused by CVD, 16 (9.0%) were caused by infectious disease, 2 (1.1%) were caused by malignancy, 13 (7.3%) were caused by cachexia, 16 (9.0%) were caused by other reasons and 35 (19.7%) were due to unknown causes. The correlation between 5-year all-cause mortality and bilirubin levels was evaluated by using Kaplan–Meier methods and the results are shown in  = .017)  and in p = .026) . Further4.In this retrospective cohort study, we investigated the relationship of the levels of the three types of serum bilirubin  with the 5-year all-cause mortality of PD patients. We found that the TBil and IBil levels were significantly associated with 5-year all-cause mortality among PD patients. In addition, we observed a significantly higher mortality rate in PD patients with higher levels of serum TBil or IBil than in patients with lower levels of serum TBil and IBil. However, no such associations were observed between the DBil and mortality risk. These results were confirmed in sensitivity analyses after excluding patients with follow-up of less than 1 year. Besides, we also found that chronic glomerulonephritis was the main cause leading to ESRD and CVD was the main cause of death among PD patients, which were consistent with other previous studies ,20.Studies have indicated that bilirubin is not merely an end product of heme degradation but is also an effective antioxidant that effectively scavenges peroxyl radicals and suppresses the oxidation of lipids and lipoproteins ,7,21,22;In addition, Chen et al.  and TemmIn addition to the TBil, we also investigated the effects of the DBil and IBil levels on the all-cause mortality of PD patients. To the best of our knowledge, no studies have reported the association of IBil with mortality in PD patients until now. Only a very limited number of studies have found that high serum IBil levels were independent protective factors for the development of diabetic nephropathy and CKD in patients, but there was no significant association among these factors in the general population ,15,29. IThe IBil is extremely poorly water-soluble and it is strongly bound to albumin in plasma ; in contThe present study had some limitations. First, this was a retrospective study, which can only reveal associations but not causality. Second, we only had information regarding all-cause mortality, but we did not know the exact cause of death of the PD patients; however, based on clinical experience, most dialysis patients die from cardiovascular events. Third, in this study, we only examined the baseline data, without considering the changes that occurred during the follow-up period. Fourth, because of the limited sample size, the potential risk factors were not all adjusted for in this cohort study. Hence, the effects of residual confounding factors could not be completely eliminated. Our future studies will address these issues.5.Our findings suggest that the TBil and IBil levels were significantly associated with 5-year all-cause mortality among PD patients. However, no such associations were observed between the DBil and mortality risk.


A controversy exists about the potential effect of childhood varicella vaccination on Herpes Zoster (HZ) incidence. Mathematical models projected temporary HZ incidence increase after vaccine introduction that was not confirmed by real-world evidence. These models assume that absence of contacts with infected children would prevent exogenous boosting of Varicella-Zoster-Virus (VZV) immunity and they do not include an endogenous VZV immunity-boosting mechanism following asymptomatic VZV reactivation. This study aims to explore the effect of various assumptions on exogenous and endogenous VZV immunity-boosting on HZ incidence in the general population after introduction of routine childhood varicella vaccination.An age-structured dynamic transmission model was adapted and fitted to the seroprevalence of varicella in France in absence of vaccination using the empirical contact matrix. A two-dose childhood varicella vaccination schedule was introduced at 12 and 18 months. Vaccine efficacy was assumed at 65%/95% (dose 1/dose 2), and coverage at 90%/80% (dose 1/dose 2). Exogenous boosting intensity was based on assumptions regarding HZ-immunity duration, age-dependent boosting effect, and HZ reactivation rates fitted to observed HZ incidence. Endogenous boosting was the same as pre-vaccination exogenous boosting but constant over time, whilst exogenous boosting depended on the force of infection. Five scenarios were tested with different weightings of exogenous (Exo) - endogenous (Endo) boosting: 100%Exo–0%Endo, 75%Exo–25%Endo, 50%Exo–50%Endo, 25%Exo–75%Endo, 0%Exo–100%Endo.HZ incidence before varicella vaccination, all ages combined, was estimated at 3.96 per 1000 person-years; it decreased by 64% by year 80 post vaccine introduction, for all boosting assumptions. The 100%Exo-0%Endo boosting scenario, predicted an increase in HZ incidence for the first 21 years post vaccine introduction with a maximum increase of 3.7% (4.1/1000) at year 9. However, with 0%Exo-100%Endo boosting scenario an immediate HZ decline was projected. The maximum HZ incidence increases at 10, 3, and 2 years post vaccination were 1.8% (75%Exo-25%Endo), 0.8% (50%Exo-50%Endo) and 0.2% (25%Exo-75%Endo), respectively.Assuming modest levels of endogenous boosting, the increase in HZ incidence following childhood varicella vaccination was smaller and lasted for a shorter period compared with 100%Exo-0%Endo boosting assumption. Endogenous boosting mechanism could partly explain the divergence between previous HZ-incidence projections and real-world evidence.The online version of this article (10.1186/s12879-019-3759-z) contains supplementary material, which is available to authorized users. Varicella (chickenpox) is a highly contagious infectious disease with a peak incidence among preschool and school-aged children. It is caused by the varicella-zoster virus (VZV) , 2. AfteVaricella is considered as a self-limiting disease which annually infects a large number of people, mostly children, almost equal to the size of the annual birth cohort in temperate regions. The disease can lead to serious complications in 3% of cases under 15 years of age and 6% of cases above 15 years of age in France, including secondary bacterial infections of skin and lungs, sepsis, aseptic meningitis, encephalitis, and Reye syndrome , 6. SeveThe disease burden and the viral transmission of VZV are reduced by the routine use of varicella vaccine in children. Several live attenuated varicella vaccines have been developed, with well-established efficacy and safety profiles , 7, 8. IThe potential negative effects of routine childhood varicella vaccination (RVV) on HZ incidence in older population originate from the “exogenous boosting theory”, which postulates that individuals susceptible to HZ coming into contact with VZV-infected children could maintain their cell-mediated immunity (CMI), thereby reducing the risk of reactivation and developing HZ –19. The The alternative hypothesis of endogenous boosting suggests that internal factors (e. g. stress) can cause asymptomatic VZV reactivation and boost CMI, thus preventing HZ , 37, 38.In this study, we aim to explore the impact of childhood varicella vaccination on HZ incidence through scenarios with different relative weighting of both exogenous and endogenous boosting mechanisms. This may be of importance when modelling the public health impact of RVV on HZ.The model we used for this study is an adaptation of the model developed by Ouwens et al.  which weBriefly, the model structure Fig.  combinesThe progression from state R to state Sz; then from state Sz to state Iz (as a result of VZV-reactivation) depends on three key parameters: (1) immunity duration against HZ after a varicella infection, (2) the risk of boosting event as a function of age when susceptible to zoster, and (3) VZV reactivation rates.For the first parameter, the duration of immunity 1/δ) against HZ is unknown. Therefore, fixed values were assumed and tested in sensitivity analyses: 10 years in the base case and values of 2 and 20 years in the scenario analysis. Regarding the second parameter on the risk of exogenous boosting, the same values are used as in Brisson et al.  with a p/δ againsIncidence was presented following the French age groups of preschool and school children, young adults, working adults, and older-age population: < 1 year, 1–4 years, 5–9 years, 10–14 years, 15–24 years, 25–44 years, 45–64 years, and ≥ 65 years. The lack of population-level data on endogenous boosting does not allow a full calibration of these parameters. Therefore, the constant age-dependent e(a) was assumed to be the same as the exogenous boosting before vaccination (pre-vaccination equilibrium). This level of boosting was derived by Brisson et al. from results of a trial on the live-attenuated HZ vaccine efficacy , 44.Five different relative weights of endogenous and exogenous boosting effects were tested as different scenarios in the model  so that the total force of boosting combining both exogenous and endogenous effects was assumed to be the same before vaccine introduction in all scenarios. In absence of data on the boosting effect, we set the total force of boosting to what was assumed by Brisson et al.,  i.e. 75%The model output was the evolution of HZ incidence over time after childhood varicella vaccine introduction in each scenario.Tables Vaccine efficacy was obtained from the clinical trials and assumed to be 65% for the first, and 95% for the second dose . VaccineHZ incidence was well reproduced by the mathematical model for each age group and all boosting- effect assumptions. Before varicella vaccine introduction, the overall population HZ incidence was estimated at 3.96 per 1000 individuals, increasing with age Table . This deFor the other 3 scenarios , higher proportions of endogenous boosting led to lower temporary increase in HZ incidence of 1.8, 0.8 and 0.2% and for shorter duration of increase of 10, 3, 2 years respectively Table .In addition, higher proportions of endogenous boosting lead to shorter time to maximum temporary increase in HZ incidence: 6 years for 25%Exo–75%Endo, 8 years for 50%Exo–50%Endo, 9 years for 75%Exo–25%Endo.The impact of introducing an endogenous boosting effect on HZ incidence in the general population was less important with the 2-year VZV immunity scenario, while a substantial difference was observed for 20 years of VZV immunity Table .Specifically, for 100% exogenous boosting, with the scenario of 2-year VZV immunity in comparison to the base-case scenario (10 years), the temporary initial increase in HZ incidence was projected to be smaller (1.3% versus 3.7%) and shorter (3 years versus 21 years). Thus, the estimated impact of increased endogenous boosting effect was smaller in the 2-year immunity scenario than in the base case.When 20-year VZV immunity was assumed and with 100% exogenous boosting, the temporary initial increase in HZ incidence was projected to be higher (5.7% versus 3.7%) and to last longer (33 years versus 21 years) than in the base-case scenario. On the other hand, introducing 25% of endogenous boosting had a larger impact, with a lower maximum HZ incidence increase  and a shorter duration of HZ incidence compared to pre-vaccination levels (− 10 years from 33 to 23 years).The maximum estimated increases in HZ incidence compared to the pre-vaccination rate in the base- case scenario were in the group 25–44 years of age Fig. , althougAdditional file A small number of studies have attempted to model the effect of childhood varicella vaccination on the incidence of HZ in the population in the presence of endogenous boosting and its relative weight with exogenous boosting. Our analysis shows that even with a limited fraction of endogenous boosting, a substantial reduction occurs in the duration and magnitude of temporary initial HZ incidence increase after introduction of vaccination relative to pre-vaccination levels.The current insight from the model means that relative weighting of exogenous and endogenous boosting effects may have an important and varied impact on VZV immunity and predicted HZ burden in the population.Previous modelling studies projected that RVV would increase HZ incidence in the general population above pre-vaccination levels for about 20 to 50 years after the introduction of childhood vaccination under assumption of reduced exogenous boosting , 43, 46,Using varicella and HZ data from the Netherlands, Marinelli et al. , compareMoreover, complex interactions between exogenous and endogenous boosting mechanisms as well as other factors may exist, and the effect on HZ incidence may not be solely influenced by reactivation rates and exogenous boosting, as assumed in dynamic transmission models.Introduction of HZ vaccination in older adults , 50, aloOur study has limitations. The various assumptions , and the uncertainties around these assumptions can have a significant impact on model-based projections.Some key parameters of the model like vaccination coverage, detection rate of HZ, demography and age-structured contact pattern are based on current knowledge and held constant over the analysis period. These are common hypotheses for population models in high-income countries. Also, due to the compartmental structure of the model, as opposed to individual-based models, heterogeneity in the immune-system status of individuals of the same age is not taken into account. Differences in HZ risk have been reported for different groups in the population e.g., higher risk in female, lower risk for black individuals . Our modIn a VZV dynamic transmission model, assumptions on relative weighting of exogenous and endogenous boosting effects may have an important and varied impact on the predicted HZ burden following introduction of childhood varicella vaccination. The HZ burden in the general population is projected to decrease for all boosting scenarios, but a temporary increase in HZ precedes this effect when exogenous boosting alone is considered. This predicted increase in HZ is markedly reduced even when endogenous boosting is assumed to have a small weight. Endogenous boosting could therefore partly explain divergence between real-world evidence on HZ burden in countries utilizing childhood varicella vaccination and model projections based on exogenous-only boosting assumption. A safe and effective Varicella vaccine seems to offer the greatest promise controlling even long-term issues caused by this virus.Additional file 1:Highlights / Patient Focus summarizes the content of the study in a form that could be shared with patients by healthcare professionals. (DOCX 13 kb)


However, how plant lineages responded to the cooling has remained unknown until now. Between Mesocyparis sinica from the early Paleocene of Jiayin, Heilongjiang, northeastern China. By integrating lines of evidence from phylogeny and comparative morphology of Mesocyparis, we found that during ca.70–60 Ma, the size of seed cone of Mesocyparis more than doubled, probably driven by the cooling during the K-Pg transition, which might be an effective adaptation for seed dispersal by animals. More importantly, we discovered that the northern limit of this genus, as well as those of two other arboreal taxa Metasequoia Miki ex Hu et Cheng (gymnosperm) and Nordenskioldia Heer (angiosperm), migrated ca.4–5° southward in paleolatitude during this time interval.Here we report a new species, Mesocyparis and have driven the migration of plants southwards.Our results suggest that the cooling during the K-Pg transition may have been responsible for the increase in size of the seed cone of The online version of this article (10.1186/s12870-019-1980-y) contains supplementary material, which is available to authorized users. Metasequoia around the world since the Late Cretaceous has shown the knowledge of the distributional changes of this genus across the K-Pg transition [The cooling from the Maastrichtian to the Paleocene that occurred during the Cretaceous-Paleogene (K-Pg) transition ca. 70–60 Ma), has been recorded by land plant leaf data  and mari0 Ma, hasansition . NeverthMesocyparis McIver & Basinger (Cupressaceae) was distributed in eastern Asia and western North America from the Late Cretaceous to the Paleocene, ca. 70–60 Ma [Mesocyparis fossils from Northeast China, together with the other four species of this genus in the Northern Hemisphere, provide us with an opportunity to evaluate adaptions within the genus.The extinct genus 70–60 Ma –22. HereOrder: Coniferales.Family: Cupressaceae.Subfamily: Cupressoideae.Genus:Mesocyparis McIver et Basinger.Species:Mesocyparis sinica Y. M. Cui, W. Wang, D. K. Ferguson, J. Yang et Y. F. Wang, sp. nov.Diagnosis: Seed cones ovate to orbicular, borne in opposite pairs , specimen no. WY0801 . The full fossil description and a morphological comparison with the other four species in Mesocyparis are provided in supplementary text of the electronic supplementary material.Here we treat the Wuyun fossils as a new species based on a detailed morphological comparison, which shows two or more significant morphological differences between the Wuyun fossils and other Mesocyparis is a member of the Cupressoideae and clusters with Hesperocyparis, Callitropsis–Xanthocyparis, and Juniperus–Cupressus (BS = 51%) , and the three Asian species, M. beringiana, M. rosanovii and M. sinica, formed another . Mesocyparis sinica is sister to M. rosanovii (BS = 62%).Our family-wide phylogenetic analysis indicates that 1%) Fig. . Mesocypsus BS = 1%  seed cones became larger , 2) the umbo shape on the scale changed from erect  is apparent in modern Cupressus, one of the nearest living relatives of extinct Mesocyparis  feed on intact seed cones and we envisage similar feeding strategies existed in early birds during the K-Pg transition. The increasing size of the seed cone might be an adaptive mechanism for seed dispersal . An animal’s preference for larger seed cones might select and even amplify the increasing size of seed cones in Mesocyparis. Evidence has shown that the rapid radiation and diversification of birds [Specific morphological features can be linked to a specific biological function, and may allow us to speculate about the reason behind of birds , 26 and of birds  that, ocIn general, the geographical distribution of plants at high latitudes is strongly constrained by the terrestrial thermal regime, particularly the mean coldest monthly temperature (MCMT) and/or the mean annual temperature (MAT) , 29. ThuMesocyparis south by ca. 4° N from the Maastrichtian to the Paleocene  and Nordenskioldia (Trochodendraceae). Metasequoia’s northern limit moved south by ca. 4 °   could haMesocyparis sinica from the early Paleocene of Jiayin, Heilongjiang, northeastern China. By integrating lines of evidence from phylogeny and the comparative morphology of Mesocyparis, we found that during ca. 70–60 Ma, the size of the seed cone of Mesocyparis increased by ca. 130%, probably driven by the cooling during the K-Pg transition, which might be an effective adaptation for seed dispersal by animals. Meanwhile, our analyses indicate that the northern limits of this genus, as well as those of two other arboreal taxa Metasequoia (gymnosperm) and Nordenskioldia (angiosperm), have migrated ca. 4–5° southwards in paleolatitude during this interval, in association with the cooling. This is the first report to link plant evolutionary and ecological responses to the climate cooling during the K-Pg transition. This study not only enriches our understanding of how specific plant taxa responded to the climate changes during the K-Pg transition, but also provides a reversed case study for the scientific community to speculate about plant responses and distributional changes under global warming today.We report a new fossil species, Mesocyparis sinica described here were collected from the upper part of the Wuyun Formation, at Wuyun coalmine   as the polar-radius of the ellipsoid and seed cone width (scw) as the two equatorial-radii. The estimated volume (V) of Mesocyparis is expressed by the following ellipsoid volume equation:scl = median value of seed cone length, scw = median value of seed cone width. The scls of five species in Mesocyparis are 3.9 mm (M. umbonata), 3 mm (M. beringiana), 4 mm , 4.25 mm (M. rosanovii) and 4.8 mm (M. sinica), while their scws are 2.95 mm (M. umbonata), 3 mm (M. beringiana), 4 mm , 3.5 mm (M. rosanovii) and 4.55 mm (M. sinica). Based on the volume equation, the estimated volumes are 5.657 mm3 (M. umbonata), 4.5 mm3 (M. beringiana), 10.667 mm3 , 8.677 mm3 (M. rosanovii) and 16.562 mm3 (M. sinica). Thus, we can estimate that the average volumes of Mesocyparis in the Maastrichtian and the Paleocene were 5.079 mm3 and 11.969 mm3 respectively, so the increase in volume of Mesocyparis from the Maastrichtian to the Paleocene was about 136%.Here we use a volume formula of an ellipsoid to estimate the average cone size of each species in Mesocyparis, we calculated the potential relationship between the seed cone sizes of extant Cupressus and temperatures in their habitats. We examined 45 samples of five Cupressus species  naturally occurring in China from PE Herbarium, which have female cones and entire information on locality and elevation. The diameters of the female cones were measured to represent their seed cone sizes. Mean annual temperature (MAT) used the climate data set of New et al. [To explore the influence of climate change on the morphology of seed cones in w et al.  and thenMesocyparis [Metasequoia  [Nordenskioldia  [The coordinates of paleolatitude and paleolongitude were compiled from previous reports of ocyparis –22, Metanosperm)  and Nordiosperm)  in the liosperm) .Additional file 1:Table S1. Morphological characters matrix including both extant and extinct species of the Cupressaceae s.l. (DOCX 34 kb)Additional file 2:Fig. S1. The strict consensus tree of Mesocyparis inferred from the 22 morphological characters. (TIF 337 kb)Additional file 3:Table S2. Morphological characters matrix of Mesocyparis and its outgroups. (DOCX 17 kb)Additional file 4:Table S3. The seed cones sizes of Cupressus, their geographic information and mean annual temperature (MAT) of their habitats. (DOCX 19 kb)Additional file 5:Table S4. Fossil localities of Mesocyparis and the estimates of their paleo-latitudes and palaeo-longitudes. (DOCX 15 kb)Additional file 6:Table S5. Fossil localities of Metasequoia and the estimates of their paleo-latitudes and palaeo-longitudes. (DOCX 20 kb)Additional file 7:Table S6. Fossil localities of Nordenskioldia and the estimates of their paleo-latitudes and paleo-longitudes. (DOCX 16 kb)


Depression is common among patients with axial spondyloarthritis (axSpA), but reports of its prevalence are highly variable. We performed a systematic review to (i) describe the prevalence of depression in axSpA, (ii) compare its prevalence between axSpA, ankylosing spondylitis (AS) and non-radiographic axSpA (nr-axSpA) cohorts, and (iii) compare disease activity and functional impairment between those with and without depression.We searched Medline, PubMed, Web of Science, PsycINFO, CINAHL Plus, the Cochrane library and conference abstracts of the European League Against Rheumatism, British Society for Rheumatology and American College of Rheumatology using a predefined protocol in accordance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. Meta-analysis was performed using quality-effects model.Fifteen original articles and one abstract were included for analysis; 14 studies described AS cohorts and two nr-axSpA. Three screening criteria and one diagnostic criterion were used to define depression. Prevalence ranged from 11 to 64% depending on criteria and thresholds used. Pooled prevalence of at least moderate depression was 15% using the Hospital Anxiety and Depression Scale (HADS) threshold of ≥ 11. The prevalence of depression was similar between axSpA, AS and nr-axSpA cohorts. Patients with depression had significantly worse disease activity, including higher BASDAI by 1.4 units (95% CI 1.0 to 1.9), ASDAS by 0.5 units (95% CI 0.3 to 0.7) and ESR by 3.5 mm/h (95% CI 0.6 to 6.4). They also had greater functional impairment with higher BASFI and BASMI by 1.2 units (95% CI 0.6 to 1.8) and 0.6 units (95% CI 0.3 to 0.8), respectively. Mean age of each study cohort inversely correlated with depression prevalence.Depression is common among axSpA patients and is associated with more severe disease activity and functional impairment. Identifying and managing depression should form part of their holistic care. Further longitudinal studies are needed to explore the impact of depression on treatment outcomes and axSpA treatment on symptoms of depression.The online version of this article (10.1186/s13075-018-1644-6) contains supplementary material, which is available to authorized users. Axial spondyloarthritis (axSpA) is a chronic inflammatory disease which predominantly affects the axial skeleton. It can be divided into ‘radiographic’  and ‘non-radiographic’ (nr-axSpA), depending on whether definitive structural changes are evident on plain radiographs of sacroiliac joints , 2. TheyLike other conditions for which chronic pain is a feature, axSpA is known to be associated with depression , 5. HoweEstimating the prevalence of depression in axSpA is further complicated by the distinction of AS and nr-axSpA. Although they can be considered entities along a spectrum, there are key differences: the proportion of male patients is higher in AS than nr-axSpA cohorts, functional impairment is greater in AS cohorts, and AS is more commonly associated with elevated inflammatory markers . Gender,In spite of these challenges, quantifying the prevalence of depression is an important first-step to improve awareness and management of this comorbidity. Depression has direct importance to rheumatologists since current methods of disease severity assessments, on which important treatment decisions are made, rely on subjective patient-reported measures, such as the Bath AS Disease Activity Index (BASDAI) and spinal pain visual analogue scale (spVAS). The presence of depression is known to influence the reporting of pain and impairment . EquallyTo date, depression in axSpA has not been systematically reviewed. Our aims were therefore to (i) report pooled prevalence of depression in axSpA, (ii) compare the prevalence of depression between axSpA, AS and nr-axSpA groups, and (iii) compare disease activity and functional impairment between those with and without comorbid depression.A systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The proStudies were included if they: (i) were cross-sectional in design or were longitudinal studies reporting baseline data, (ii) used a validated diagnostic or screening criteria for depression with a defined cut-off threshold, and (iii) recruited adult patients fulfilling the modified New York or ASAS criteria for AS or axSpA, respectively. Studies were excluded if they used non-representative sampling  or had a samples size of less than 30 . Publications in abstract form only were also considered, as some prevalence studies may not be published as full articles and may have sufficiently described methodology within the restrictions of an abstract. Reviews, comments and editorials were excluded.Two reviewers  independently assessed study eligibility and extracted data from qualifying studies. Any discrepancies were resolved through discussion moderated by a third reviewer. Information from included studies was extracted into predefined tabulated summaries containing data on: depression criteria and threshold used, study design, sample size, country setting, mean age, percentage of males, and depression prevalence.Studies were assessed for risk of bias  using the Health States Quality Index . When performing meta-analysis of proportions (variable bound between 0 and 1), traditional weighting methods based on inverse variance are problematic when the proportions are close to the bound limits, giving more weight to such studies [2 statistic. Funnel plot and the Doi plot/LFK index [Prevalence estimates were reported as percentages , I studies . The douFK index  were uses).Commonly used thresholds for each screening tool were used to categorise severity of depression. For the Hospital Anxiety and Depression Scale depression subscale (HADS) ≤ 7 was interpreted as no depression, 8 to 10 mild, 11 to 14 moderate and ≥ 15 severe . Studieshttp://www.epigear.com/).Differences in markers of disease activity and functional impairment were compared between groups with and without depression. Weighted mean difference (WMD) was calculated for each marker of disease activity and functional impairment, with pooled estimates shown in forest plots. Analyses were performed using MetaXL version 5.3  and two reported on nr-axSpA cohorts (including one axSpA study which reported on nr-axSpA participants separately). Sample size ranged from 60 to 1504. A total of 4753 axSpA patients were included across all studies, including separately described groups of 2857 AS and 334 nr-axSpA patients. Twelve studies were cross-sectional in design, three were longitudinal, and one was a randomised controlled trial (RCT). Four studies were from Turkey, five from China (including one from Hong Kong), three from the UK, and one each from Greece, Morocco and Spain (Canary Islands). The RCT recruited from Europe, Asia and South America.Three screening criteria and one diagnostic criterion were used for identifying depression. Nine studies used HADS with three different thresholds, five used SDS with three different thresholds, and one used PHQ-9. For the purposes of meta-analyses, HADS ≥ 7/8 were grouped together, and SDS ≥ 50/51/53 were grouped together. Only one study used diagnostic criteria for depression, the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders (SCID).Given the strict inclusion criteria employed for this meta-analysis, most studies had high quality scores with one scoring 6, three scoring 8, nine scoring 9 and three scoring ≥ 10 on the Health States Quality Index . Pooled prevalence of at least moderate depression, using HADS ≥ 11, was 15%  and using SDS was 52% . The study by Hyphantis et al. [Figure s et al.  reporteds et al.  reportedTwo studies reported disproportionately high prevalence of depression. Hakkou et al.  attributs = − 0.71, P = 0.003) but not with study size, BASDAI, year of publication or proportion of males (data not shown).Prevalence of depression was inversely associated with age .The studies by Chan et al. , Zou et Two studies reported depression prevalence for nr-axSpA cohorts. Pooled prevalence of mild depression (HADS ≥ 7/8) for nr-axSpA was similar to that of AS cohorts Table .Eight studies compared markers of disease severity between groups with and without depression  were similar. Pooling WMDs, BASDAI was 1.4 units (95% CI 1.0 to 1.9) higher in the depressed group. Of the six studies that reported spVAS, the groups with depression scored 1.2 units (95% CI 0.7 to 1.7) higher. Only two studies reported ASDAS, with a pooled WMD of 0.5 units (95% CI 0.3 to 0.7) between the two groups. ESR  was significantly higher in groups with depression, but not CRP .All studies reported significantly worse BASFI in the group with depression. The pooled difference in BASFI was 1.2 units (95% CI 0.6 to 1.8) but with more variation among the studies. The group with depression in the study by Hakkou et al. had much poorer function  compared to depressed groups of other studies, despite using a threshold for ‘mild’ depression. In contrast to other Bath indices, not all studies reported a difference in BASMI when comparing groups with and without depression. Nevertheless, the pooled estimate showed that axSpA patients with comorbid depression had significantly higher BASMI than those without .This systematic review and meta-analysis of nearly five thousand axSpA patients has shown that depression is highly prevalent and associated with greater disease activity and functional impairment. At least moderate depression was found in 15% of patients, although estimates varied depending on the criteria and thresholds selected; pooled estimates of prevalence of depression in axSpA were 38 to 52% using lower HADS thresholds and the SDS.The strength of this meta-analysis lies in its strict inclusion criteria, as prevalence estimates can vary significantly depending on variable sampling methods and definition for depression. The included studies had high quality overall and low risk of publication bias. However, overall heterogeneity of prevalence estimates remained high, largely due to the wide range of screening or diagnostic tools used to define depression. Heterogeneity was improved when estimates were pooled by depression definition, and further reduced in sensitivity analyses excluding the studies by Hakkou et al. and Jiang et al. These two studies highlight the importance of age, which was inversely associated with depression prevalence, and socioeconomic status, which future studies could approximate by grouping by continent or gross domestic product.The main limitation of the meta-analysis of prevalence was that almost all studies used screening criteria to detect depression. This may lead to over-estimation of the prevalence of true depression. Using diagnostic criteria, Chan et al. found one of the lowest prevalences of depression. However, they reported a sensitivity and specificity of 82 and 79%, respectively, for HADS ≥ 8 in their axSpA cohort, using SCID as the gold standard . Almost The prevalence of depression in axSpA was higher than that of the general population , but verPatients with depression had significantly worse disease activity and functional impairment across most indices. As an example, the pooled difference in BASDAI was 1.4 units, which is clinically relevant as ≥ 2-unit improvement is considered as response to TNF inhibitors (TNFi) . The dirIn healthcare systems where TNFi can only be continued on the basis of demonstrable response, such as in the UK, efforts should be made to identify and manage comorbid depression to avoid withdrawing efficacious therapy inappropriately. More objective measures of disease activity, such as ASDAS, may be more robust to the influence of depression. Longitudinal axSpA studies are needed to evaluate the impact of depression on treatment outcomes; we have previously demonstrated, in a longitudinal RA cohort, that depression at baseline adversely affects treatment outcomes . EquallyDepression is common among axSpA patients and is associated with worse disease activity and functional impairment. Clinicians should be mindful of comorbid depression when managing axSpA patients, especially for younger patients and those with severe disease activity and functional impairment. Patients with depression should be appropriately referred and managed. This is especially pertinent if depressive symptoms are thought to adversely influence assessments of treatment response.Additional file 1:Tables S1-S3, Figures S1-S3. (DOCX 71 kb)


Palaemonetes sinensis, is an economically important freshwater shrimp in China, and the study of genetic diversity and structure can positively contribute to the exploration of germplasm resources and assist in the understanding of P. sinensis aquaculture. Microsatellite markers are widely used in research of genetic backgrounds since it is considered an important molecular marker for the analyses of genetic diversity and structure. Hence, the aim of this study was to evaluate the genetic diversity and structure of wild P. sinensis populations in China using the polymorphic microsatellite makers from the transcriptome.The Chinese grass shrimp, P. sinensis from transcriptome, and analyzed for differences in genetic diversity and structure in multiple wild P. sinensis populations in China. Totally of 319 individual shrimps from seven different populations were genotyped to find that allelic polymorphisms varied in two to thirteen alleles seen in the entire loci. Compared to other populations analyzed, the two populations including LD and SJ showed lower genetic diversity. Both the genetic distance (D) and Wrights fixation index (FST) comparing any two populations also indicated that LD and SJ populations differed from the other five populations. An UPGMA tree analysis showed three main clusters containing SJ, LD and other populations which were also confirmed using STRUCTURE analysis.Sixteen polymorphic microsatellite markers were developed for P. sinensis populations. All the polymorphic microsatellite makers are believed useful for evaluating the extent of the genetic diversity and population structure of P. sinensis. Compared to the other five populations, the LD and SJ populations exhibited lower genetic diversity, and the genetic structure was differed from the other five populations. Therefore, they needed to be protected against further declines in genetic diversity. The other five populations, LP, LA, LSL, LSY and LSH, are all belonging to Liaohe River Drainage with a relatively high genetic diversity, and hence can be considered as hot spots for in-situ conservation of P. sinensis as well as sources of desirable alleles for breeding values.This is the first study where polymorphic microsatellite markers from the transcriptome were used to analyze genetic diversity and structures of different wild  Palaemonetes sinensis , also known as Chinese grass shrimp, a small freshwater shrimp belonging to the Palaemonidae family distributed in China, Myanmar, Vietnam, Japan, southeastern Siberia and Sakhalin and has both ecological and ornamental value [P. sinensis is commonly distributed in the Liaoning, Jilin, Heilongjiang, Hebei, Jiangsu, Fujian and Yunnan provinces [P. sinensis is an attractive shrimp due to its striking appearance, appealing flavor [P. sinensis is very popular in both domestic and foreign markets [P. sinensis population has gradually diminished. As such, there has been interest in P. sinensis aquaculture to potentially alleviate fishing pressure on the wild population by meeting consumer demand with farmed shrimp and through stock enhancement. Nevertheless, there are only a handful of studies investigating P. sinensis morphology and more work is needed to understand the biology of this species [P. sinensis using molecular biology are rarely reported, only some studies have used microsatellite markers in other species of the Palaemonidae family, including Macrobrachium rosenbergii [Macrobrachium nipponense [Scylla paramamosain). Research in related species of snapping shrimps with highly duplicated genomes proved that microsatellites acquired from the transcriptome were more likely to work effectively than those developed through traditional methods from the genome [P. sinensis [P. sinensis, which has the potential to aid in the conservation and improvement of this shrimp species. In view of the broad distribution of its wild populations, the questions over whether they are genetically similar or do exhibit differences attributable to their locations or drainage basins are still unsolved. The objectives of the current study were to validate variations of transcriptome-derived microsatellite markers and to analyze their underlying genetic background of different wild P. sinensis populations. It is hoped that this work could make a positive contribution to the molecular genetic analyses of P. sinensis populations and eventually serve as a basis for the improvement and sustainable conservation of P. sinensis aquaculture in China.Sollaud, 911, alsoal value . In Ching flavor  and use g flavor . Due to  markets –6. Howev species , 6. At penbergii  and Macrpponense . In the pponense  identifisinensis . MoreoveP. sinensis, totaling 319 individual shrimps , NE (3.628), He (0.724) and Ar (7.478), and c2591_g1_i1 was the least polymorphic locus, with the lowest NA (2), NE (1.006), He (0.006) and Ar (1.213) values.Seven wild populations of P < 0.01), whereas the other seven loci showed no significant differences (Table P < 0.01)  (es Table . All pop1) Table . HoweverSY Table . In geneP. sinensis populations were shown in Table NA, He and Ar values, while the LSH population exhibited highest NE and Ho values. In the LD, LP, LA, LSL and SJ populations, FIS coefficients varied from 0.093 to 0.322, suggesting significant deficiencies of heterozygotes with 95% confidence interval. In the other two populations, FIS values did not show significantly different from zero.Data for all parameters of genetic diversity for the seven Ne) and the mean ratio of the number of alleles to the range in allele size (M ratios) of each population were also listed in Table Ne values for LP and LSH showed to be the highest (infinite), whereas the lowest Ne value was 106.1 as shown in LA. M ratios among all populations ranged from 0.826 to 0.908, which revealed that no population experienced reduction in effective size. Reduction in effective size only occurs when M < 0.68 [P > 0.05).Estimated of effective population size (M < 0.68 . PotentiFST) and Cavalli-Sforza and Edwards’ genetic distance (D) [P < 0.01) (Table The pairwise Wrights fixation index (ance (D)  values w1) Table .Table 4FST values among the seven populations reflected their geographic relationships. Firstly, there was a very great genetic differentiation between SJ (the only population from Huaihe Drainage Basin in Shandong Province) and the other six populations  (FST > 0.25). Secondly, LD (Related River Drainage Basin) and all populations from Liaohe River Drainage Basin  exhibited a great genetic differentiation between (0.15 < FST < 0.25). Finally, in Liaohe River Drainage Basin, there was a moderate genetic differentiation among LP, LA, LSL, LSY and LSH (0.05 < FST < 0.15), meanwhile, LSL and LP, LSL and LSY populations showed little genetic differentiation (FST < 0.05) [FST values revealed significant differences among the populations (P < 0.01).Basically, the D values) was significantly correlated . The D value between the populations displayed similar pattern as FST values. For example, most D values between SJ and the other six populations including SJ vs LA, SJ vs LSL, SJ vs LYS, and SJ vs LSH, were larger than 0.20, suggesting that they were genetically disparate populations [D values between LD and populations of Liaohe Drainage Basins were also higher than those between latter populations.As analyzed by the Mantel test, the correlation between geographical distances and a pairwise comparison with genetic distances  to 3.545 (SJ into LP). Most long-term gene flow between populations were symmetric , except those between SJ and LP, LSY and LSL, and SJ and LSY  related with different numbers of genetic clusters K, calculated from structure analysis of 319 individuals of NA value ranging from 2 to 13 (mean = 5.8), which is similar with studies investigating transcriptome-derived microsatellite markers in other decapod [M. rosenbergii [M. nipponense [P. sinensis so far, these polymorphic microsatellite makers and further mining of transcriptomic data may helpful for future research of P. sinensis and its related species.One of the main objectives of this study was to detect polymorphisms in transcriptome-derived microsatellite makers and to further understand the genetic diversity and structure of the wild populations from different locations. Compared with genomic derived microsatellites, transcriptome-derived microsatellites have several advantages including high efficiency, strong transferability, and correlation with potential genes . Althoug decapod . Additioenbergii  and M. nmean = 5., which iNE, Ho and He values in seven P. sinensis populations ranged from 1.441–1.867, 0.158–0.369 and 0.226–0.379, respectively, showing that the genetic diversity of the populations was lower than that of other species of the Palaemonidae family [FIS values. The LD and SJ populations showed lower genetic diversity as indicated by NE, Ho and He values, which may be a result of a small Ne value, inbreeding and a limited number of shrimp samples. Results in the current study indicated that the Ne values of the four populations LD, LA, LSY and SJ, were lower than the values suggested by Franklin et al. [P. sinensis. In addition, due to time and space constraints in sampling, a certain number of individuals may have been from a few number of spawning shrimps, which would affect genetic diversity through an increase in genetic identity [The mean n et al. . This maidentity .P. sinensis populations did not experience a bottleneck effect or a recent decline in quantity. Therefore, in future breeding projects, the level of inbreeding, brood shrimp population size and genetic diversity must be considered since domestication probably leads to a reduction in genetic variation due to genetic drift, selection and inbreeding [Most of the populations analyzed in this study showed a departure from HWE. This result was similar to several other studies which microsatellites derived from transcribed sequence data significantly depart from HWE –23. Thisbreeding .In this present study, subjects are collected from three geographically isolated drainage basins. LD population belongs to Related River Drainage in East Liaoning Peninsula; the SJ population belongs to Huaihe River Drainage in north China and the other five populations  come from Liaohe River Basin in central Liaoning Province. Specifically, LSL and LSY come from a closed lake and a semi-closed reservoir respectively; LA and LSH belong to inland tributaries; LP locates close to the mouth of Liaohe River.P. sinensis, which accounted for 17.24% of the total genetic variation, was much lower than that within populations. Pairwise D and FST values were consistent with these results. According to Thorp [D values between most of the populations of P. sinensis indicated that they were closely related populations. Likewise, excluding SJ and LD populations, the pairwise FST values among the other five populations were low to moderate [FST values among five populations indicated they might share the same ancestors. Even so, the significant correlation between geographical and genetic distances as well as the gene flow values indicated that five populations from Liaohe River Basin were separate due to the habitat fragmentation. The divergence between SJ and the other six populations as well as the divergence between LD and the other five populations were due to long-term geographic separation. These outcomes were consistent with the Bayesian analysis in genetic structure simulations, which also revealed that the SJ and LD populations were much different from the other populations.AMOVA analysis revealed that the genetic differentiation among populations of to Thorp , the paimoderate . The sliP. sinensis populations in China. All the polymorphic microsatellite makers are believed useful for evaluating the extent of genetic diversity and population structure of P. sinensis. Compared to the other five populations, the LD and SJ populations exhibited lower genetic diversity due to the lower NE, Ho and He. All populations showed normal L-shaped distributions and may not have experienced a bottleneck or recent reductions in effective size. The AMOVA analysis revealed that genetic variation among populations was 17.24% and much lower than that within populations. D and FST values between any two populations indicated that the LD and SJ populations differed from the other five populations. The UPGMA tree and the STRUCTURE analysis also supported the result. Therefore, they needed to be protected against further declines in genetic diversity. Among the seven populations, LP, LA, LSL, LSY and LSH populations were all from Liaohe River Drainage with a relatively high genetic diversity, and hence can be considered as hot spots for in-situ conservation of P. sinensis as well as sources of desirable alleles for breeding values. In future, further development of transcriptome-derived microsatellite markers is necessary for more detailed investigation on the genetic variation, genetic structure, and molecular markers-assisted selection (MAS) of P. sinensis. Overall, this study provided a theoretical basis for the protection, rational use and genetic breeding of germplasm resources.In this study, 16 polymorphic transcriptome-derived microsatellites were screened and used to assess the genetic diversity and structure among wild P. sinensis were collected in 2016 from Sha River in Liaoning Dalian , Shuangtaizi River in Liaoning Panjin , Yangliu River in Liaoning Anshan , Longwei Lake in Liaoning Shenyang , Yangshi reservoir in Liaoning Shenyang , Liao River in Liaoning Shenyang Huangjia  and Dushan Lake in Shandong Jining   [http://bioinfo.ut.ee/primer3-0.4.0/). Primers were examined using varying PCR conditions and PCR amplified products were evaluated by agarose gels. PCR reactions were performed by ABI 2720 thermocycler . DNA samples were subsequently amplified for the polymorphic loci in single PCR reactions. PCR reactions were 15 μL, containing 50-100 ng template, 10 × buffer 1.5 μL, Mg2+ (25 mmol·L− 1) 1.5 μL, dNTPs (each of 10 mmol·L− 1) 0.25 μL, each of forward primer and reverse primer (10 μmol·L− 1) 0.15 μL, Taq polymerase 1 U, and ddH2O. Denaturation for 3 min at 94 °C was followed by 35 cycles made up of 25 s at 94 °C, 25 s at the 54 °C and 60 s at 72 °C. The final step was a prolonged extension of 10 min at 72 °C, followed by 4 °C hold. All individual genotypes were scored after the PCR products were resolved on Applied Biosystems 3730XL Genetic Analyzer  and the product size was analyzed by GeneMarker version 2.2.0. .More than 50 microsatellite loci were selected from the data of P. sinensis populations (Table NA), the number of effective alleles (NE), observed heterozygosity (Ho) and expected heterozygosity (He) of each locus of each population was calculated using POPGENE 1.32 [r) in each locus was estimated using software FreeNA [Ar) and Wright’s FIS values with 95% confidence intervals were calculated according to Weir and Cockerham using FSTAT version 2.9.3.2 software [Sixteen polymorphic loci were used to detect genetic variation among ns Table . The numENE 1.32 . Null ale FreeNA  in whichsoftware . Tests fsoftware  and a MaNe) for each population was calculated using the gametic disequilibrium method implemented in LDNe 1.31 [Ne value using Arlequin 3.5.2.2 [The estimates of effective population size  and analysis of molecular variances (AMOVAs) were calculated using Arlequin 3.5.2.2 [Pairwise Wrights fixation index .Pairwise gene flow among all seven populations was calculated by using software Migrate-n 4.4.3 , 33 throGenetic structures among populations analysis was performed via STRUCTURE v2.3.3  using BaAdditional file 1: Table S1.P-value for deviation from Hardy-Weinberg equilibrium for each locus in each population.Additional file 2: Table S2. Comparative migration estimation among all pairs of P. sinensis populations for coalescent (MIGRATE) estimator along with 95% confidence intervals.Additional file 3: Table S3. Cumulative gene flow for each population of P. sinensis.


Transforming growth factor (TGF)-β1 contributed to angiotensin II (Ang II)-mediated collagen accumulation after myocardial infarction (MI). The present study aimed to investigate the association of genetic variant of TGF-β1 gene with the risk of MI. The present study recruited a total of 530 MI patients and 651 healthy controls. The genomic DNA was extracted and subjected into polymerase chain reaction (PCR) and Sanger sequencing. The present study indicated that TGF-β1 -913G/C polymorphism was associated with increased risk for MI under the co-dominant, dominant and allelic models. The increased risk effect was also evident among the females, younger subjects (age < 60 years), smokers, non-drinkers and individuals with hypertension. Additionally, the present study observed significant differences among cases and controls in terms of total cholesterol (TC). In conclusion, TGF-β1 -913G/C polymorphism is associated with increased risk for MI. TGF-β1 -913G/C polymorphism may be a potential prognostic biomarker for MI. Myocardial infarction (MI), a clinical complication of coronary atherosclerosis, primarily develops from the rupture of an atherosclerotic plaque with thrombus formation and the occlusion of the coronary vessel , therebyTransforming growth factor (TGF)-β1 is a cytokine expressed in platelets, hematopoietic, connective and endothelial tissue cells . TGF-β1 The present study of a total of 530 patients diagnosed with MI and 651 gender- and age-matched controls were recruited from the Affiliated Hospital of Hangzhou Normal University, and the Second Affiliated Hospital of Zhejiang Chinese Medical University from April 2013 to April 2018. The typical ECG changes, elevated cardiac markers and clinical history were used for diagnosis. Coronary angiography was used to identify the responsible stenosis in any of the major coronary arteries or in the left main trunk. Patients with history of coronary artery disease, renal or hepatic disease, cardiomyopathy and malignant tumor were excluded from case groups.The control groups were recruited from individuals who had physical examinations in our hospital. Controls with sinus rhythm (ECG data) and normal heart function (UCG data) in the normal condition were enrolled in this case–control study. The controls excluded individuals with a history of atherosclerosis, rheumatic heart disease and liver, and kidney dysfunction by medical history. A standard questionnaire was used to obtain demographic of all participants, including smoking status, drinking status, history of MI, hypertension and diabetes mellitus. All MI patients and healthy controls were interviewed and recorded about demographic and risk factors. Individuals were defined as smoker if they smoked at least one cigarette per day for more than 1 year. Individuals who drank three alcoholic drinks in a week. If the systolic/diastolic blood pressure exceeded 140/90 mmHg ,19 or thWe obtained written informed consent from all subjects. All procedure has been reviewed in line with Ethical Standards of the Affiliated Hospital of Hangzhou Normal University, and the Second Affiliated Hospital of Zhejiang Chinese Medical University. The present study was in accordance with the Helsinki declaration.Peripheral blood samples (2 ml) were collected in tubes containing EDTA for genotype analysis. Genomic DNA was extracted using the TIANamp Blood DNA kit  in accordance with manufacturer’s instructions. TGF-β1 gene -913G/C polymorphism was genotyped by using polymerase chain reaction (PCR) and Sanger sequencing. The primers were 5′-TGGCATGAACCGGCCTT-3′ (forward) and 5′-GCCGGGAGCTTTGCAGAT-3′ (reverse). PCR was performed under the following conditions: an initial holding at 95°C for 10 min, 40 cycles of denaturation at 92°C for 15 s and annealing and extension at 60°C for 1 min, and a final holding at 60°C for 1 min. The PCR products were sequenced by using ABI 3500 Genetic Analyzer. The sequence data were analyzed by using Sequence Scanner Version 1.0. Further, 2% of samples were repeatedly genotyped for quality control.P-value less than 0.05 was considered as statistically significant. All statistical analyses were conducted using SPSS 20.0 software .The differences of the demographic characteristics and risk factors between MI patients and controls were analyzed using chi-square test  and Student’s test and one-way ANOVA (for continuous variables). Departure from the Hardy–Weinberg (HWE) among controls was evaluated using a goodness-of-fit chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated to evaluate the effect of TGF-β1 -913G/C polymorphism on the risk of MI by using logistic regression analysis adjusted for age and sex. P>0.05). There were more smokers, alcohol consumers and individuals with hypertension or diabetes mellitus among the MI patients than those among the healthy controls. Additionally, the MI patients had higher levels of body mass index (BMI), TC, and LDL, but lower HDL.The demographic characteristics of participants are shown in P=0.546). Our results indicated that TT genotype , but not CT genotype, had a significantly elevated risk for MI, compared with CC genotype. Similarly, CT+TT genotype had an increased susceptibility to MI . The association was also significant after adjusting sex and age. Additionally, TGF-β1 -913G/C polymorphism was also associated with increased risk for MI in the allelic model .The distribution of genotypes and alleles of the TGF-β1 -913G/C polymorphism in MI cases and controls are provided in P=0.001)  . Next, wP=0.001) .In the present study, our data showed that TGF-β1 -913G/C polymorphism was associated with MI risk in this Chinese Han population. This increased effect was more evident among the females, younger subjects (age < 60 years), smokers, non-drinkers, and individuals without hypertension. Furthermore, this SNP was demonstrated to be associated with the TC level.Koch et al.  first inIn the present study, we found that TGF-β1 -913G/C polymorphism was related to increased risk for MI in a Chinese Han population. It is obvious that abovementioned studies  yielded Several limitations should be considered in the present study. One, the sample size of this case–control study was limited, which may make the findings of the present study underpowered. A multi-center case–control study with larger sample size is warranted in the future. Two, we only explored one SNP of TGF-β1 gene. Three, selection bias in this retrospective study should not be ignored. Four, further study uncovering the underlying mechanism why TGF-β1 gene polymorphism conferred susceptibility to MI was warranted. Five, the follow-up data were missing, which restricted further analysis about the relationship between this SNP and other clinical symptoms. Last, the interaction between genetic factors and environmental factors should be performed.In conclusion, the present study finds that TGF-β1 -913G/C polymorphism is associated with increased risk for MI in a Chinese Han population. Other studies with larger sample sizes in Chinese Han population are urgently needed.


Metabolic syndrome (MetS) is characterized by a clustering of cardiovascular risk factors that include: abdominal obesity, dyslipidemia, hypertension and glucose intolerance. Angiopoietin-like protein 4 (ANGPTL4) is a circulating peptide that is an inhibitor of lipoprotein lipase, a key enzyme in lipid metabolism. The objective of this study was to investigate the association of ANGPTL4 gene variants (E40K) with fasting serum triglyceride levels and with cardiovascular risk factors, that included the presence of MetS in 817 subjects recruited from the Mashhad stroke and heart Atherosclerosis Disorders (MASHAD) cohort Study.ANGPTL4 genotypes were determined using a TaqMan genotyping based real time PCR method. The association of the genetic variant with the risk of metabolic syndrome and its relationship with lipid profile were determined.p < 0.0001). Subject with an A allele had a higher risk for MetS , p < 0.0001). There was a significant difference in fasted lipid profiles across the genotypes for ANGPTL4. Carriers of the AG genotype had higher levels of serum HDL-cholesterol (HDL-C) and lower TG, compared to the GG homozygotes genotype.The frequency of GG, GA and AA genotypes were 96.9, 2.7 and 0.4% in individuals with MetS, and 78.8, 20.8, 0.4%, in those without MetS. The GA genotype of the rs116843064 polymorphism was associated with a lower risk for MetS , The G allele at the rs116843064 polymorphic locus of the ANGPTL4 gene was associated with a lower prevalence of MetS. Metabolic syndrome is increasing globally, and is important because of its association with increased cardiovascular disease (CVD) risk . It affeThe development of MetS depends on environmental  and genetic factors –16. ChroLong periods of fasting, low calorie diet and a diet high in fat and energy increase plasma free fatty acid concentrations and levels of ANGPTL4, most probably by activation of PPARs in liver and adipose tissue , 25. ANGHowever, other reports have suggested that the serum ANGPTL4 is positively associated with serum triglycerides in individuals with MetS . These vThe study population comprised 260 with, and 557 individuals without MetS, who were recruited from the Mashhad Stroke and Heart Atherosclerosis Disorder (MASHAD) cohort study. They were recruited through a cluster-randomized-recruitment in 2007–2008, as described previously , 34. MetInformed written consent was obtained from all individuals, and the study protocol was approved by the Ethics Committee of the MUMS. All participants were able to read and understand and were willing to provide written, informed consent.General entry and exclusion criteria for this study and public specifications of the sample participants, including marital status, occupational status, drug use and biochemical measurements were obtained as previously described (32–33).2), systolic blood pressure (mmHg) and diastolic blood pressure (mm Hg) were determined for all participant as previously described [2) was used. Biochemical parameters including fasted serum triglyceride(mg/dl), cholesterol (mg/dl), HDL-C(mg/dl), fasting blood glucose (mg/dl), serum Hs-CRP (mg/dl) and uric acid were measured as explained previously [Anthropometric parameters containing weight (kg), height (m), waist circumference (cm), hip circumference (cm), waist/hip (cm), BMI  and the manufacturer’s protocol. Using the NanoDrop®-1000-Detector  the purity and concentration of DNAs were evaluated. The genotyping of the ANGPTL4 rs116843064 polymorphism was performed using Taqman®-probes-based assay. The PCR reaction was performed in a total volume of 12.5 μl through 10 ng DNA in TaqMan® Universal Master Mix with particular primes and probes . ABIPRISM tools with SDS-version 2.0 software has been used to examine the content of allelic samples.P < .05.The analysis of the data was performed using SPSS 22 software . A Kolmogorove Smirnov test was untaked to determine the normality of the data within groups. The genotype and allele frequencies of the ANGPTL4 rs116843064 polymorphism were determined for analysis for the Hardy–Weinberg equilibrium (HWE) through Pearson χ distribution. Kruskal-Wallis and Mann-Whitney U tests, or ANOVA and t tests were used to evaluate the differences between groups. The relationship between the risk of MetS with GG and GA genotypes, versus the AA genotype under recessive genetic model, were investigated through various genetic models. MetS risk was expressed as the odds ratio (OR) and its corresponding 95% confidence interval (CI). The analyses were two-sided and statistical significance was set at p < 0.05). Also fasting blood glucose was higher in MetS patients but there was no significant difference between the two groups.Anthropometrics and biochemical characteristic of MetS and healthy subjects are presented in Table However height, serum cholesterol, HDL and LDL were higher in the non-MetS group Table .P < 0.05).We have found that the genotype frequency of GG, GA and AA were 96.9, 2.7 and 0.4% in MetS group and 78.8, 20.8, 0.4%, respectively in those without MetS (Table p < 0.0001). Also subjects with an A allele had a higher risk for MetS , p < 0.0001).The distribution of the ANGPTL4 gene rs116843064 polymorphism genotypes were investigated in genetic various models Table . These dp < 0.0001).The relationship between the ANGPTL4 gene rs116843064 polymorphism and serum fasted TG and HDL level in total population and non-MetS group is presented in Table ANGPTL4 gene is linked with significant differences in serum triglycerides and HDL cholesterol. This relationship can be explained at least in part by its function in the inhibition the activity of lipoprotein lipase and stimulates adipose tissue lipolysis and relates to dyslipidemia [Multiple studies have revealed that metabolic syndrome is a risk factor for diabetes mellitus, hypertension, pro-inflammatory state, central adiposity, dyslipidemia and high concentrations of ANGPTL4 , 36. A lipidemia , 24. A mipidemia . Genotypipidemia .ANGPTL4 [Free fatty acids through the lipid-sensing peroxisome proliferator-activated receptors (PPARs) play a main role in the expression regulation of ANGPTL4 . On the ANGPTL4 .ANGPTL4 gene have higher levels of HDL but lower TG levels, compared to those with GG homozygote genotype. Moreover the GA genotype was associated with reduced risk of MetS. Some other studies showed that the E40K variant was linked with lower serum TG and higher HDL-cholesterol [Sadeghabadi et al., have reported an association of plasma levels of ANGPTL4 and expression of PPARγ gene with metabolic characteristics in obese children and adolescents. Although both of PPARγ gene expression and levels of ANGPTL4 were decreased in individuals with obesity, significant association were not found in obese children with insulin resistance as compared to those without insulin resistance and also in obese children with or without metabolic syndrome . Althouglesterol , 28. Thelesterol . Experimlesterol . Abid etlesterol . A prosplesterol . Stejskalesterol . Smart-Hlesterol . An invelesterol . Other slesterol .Miida et al. observed that in individuals with MetS, serum ANGPTL4 was directly associated with serum triglycerides . MuendleIn summary our results supports a significant relationship between the rs116843064 genetic polymorphism within the ANGPTL4 gene with the presence of MetS and lipid profile.