Dlk1. There was a significant effect of maternal salt with increased hepatic expression of Dgat1 and Igfb2. Gut expression of inflammatory  and renin–angiotensin system  markers was significantly reduced in HFSD offspring compared to HF offspring. Therefore, salt mitigates some adverse offspring outcomes associated with a maternal HF diet, which may be mediated by altered adipose tissue morphology and gut inflammatory and renin–angiotensin regulation.Maternal high-fat or high-salt diets can independently program adverse cardiometabolic outcomes in offspring. However, there is a paucity of evidence examining their effects in combination on metabolic function in adult offspring. Female Sprague Dawley rats were randomly assigned to either: control , high-salt , high-fat  or high-fat and salt  diets 21 days prior to mating and throughout pregnancy and lactation. Male offspring were weaned onto a standard chow diet and were culled on postnatal day 130 for plasma and tissue collection. Adipocyte histology and adipose tissue, liver, and gut gene expression were examined in adult male offspring. HF offspring had significantly greater body weight, impaired insulin sensitivity and hyperleptinemia compared to CD offspring, but these increases were blunted in HFSD offspring. HF offspring had moderate adipocyte hypertrophy and increased expression of the pre-adipocyte marker  The incidence of obesity and related non-communicable diseases, including type 2 diabetes, cardiovascular diseases, and some cancers continues to rise. Although these conditions have a genetic component, the rapid rate at which these conditions have increased underscores the influence of a rapidly changing lifestyle. In parallel with the increase in obesity and related non-communicable diseases is a shift toward consumption of a Western-style diet, often composed of processed foods which are high in saturated fat and sodium –4. DespiThe Developmental Origins of Health and Disease hypothesis proposes that an adverse early life environment, such as suboptimal maternal nutrition, can program obesity and chronic disease in offspring . Both maTnfα, and there was a significant increase in expression of glucose and amino acid transporters in the HF-exposed groups . Rats were then randomly assigned to one of four experimental diets and fed ad libitum for 21 days prior to mating and throughout gestation and lactation. Experimental groups (n = 7/group) were fed either a: purified control , purified high salt , purified high fat , or purified high fat and high salt  diet and are further detailed by Reynolds et al. . Tissues were dissected, weighed and snap frozen and stored at −80°C or fixed in formalin. Trunk blood was collected in heparinized vacutainers  and plasma stored at −20°C until analysis.The procedures described were approved by the Animal Ethics Committee at the University of Auckland and were performed in accordance with relevant guidelines and regulations. The exper-imental protocol was performed as previously described by Gray et al.  Figure . Animalss et al.  . The cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor α (TNFα) were analyzed by Quantikine ELISA . Plasma was also analyzed for glucose, free fatty acids, triglycerides, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), total cholesterol, and lactate dehydrogenase by Hitachi 902 autoanalyzer .3, 0.1 M KH2PO4, 0.12 M KCl, 0.05 M CaCl2, 0.1 M MgS04, and 1 M HEPES, pH adjusted 7.35–7.45; 4% BSA, 2 mg/mL collagenase). The mixture was incubated in a shaking water bath at 37°C for 45 min and then filtered. Flow through was spun, and the SVF pellet was collected and stored in TRI Reagent  for subsequent analysis.Gonadal adipose tissue was immediately dissected from culled animals and placed in sterile phosphate-buffered saline. 1 g of adipose tissue was finely minced and placed in digestion buffer  were fixed in 10% neutral buffered formalin, paraffin embedded, and then sectioned (8 µm) using a Leica RM 2135 rotary microtome . Standard hematoxylin and eosin staining was performed. Slides were viewed under light microscopy at 10 × objective, and digital images were acquired with NIS Elements-D software . Images were blindly and manually analyzed by ImageJ 1.46v software . Four representative fields of view were analyzed from each section to determine average adipocyte area.Adipocyte histology was performed as previously described . RetropePpia), hypoxanthine–guanine phosphoribosyltransferase 1 (Hprt1), and/or glyceraldehyde 3-phosphate dehydrogenase (Gapdh) expression. The comparative CT method (2–ΔΔCT) was used to analyze data . RNA was isolated from the liver and the upper gut (duodenum) using the RNeasy Mini Kit . RNA concentration was determined using the NanoDrop 1000 Spectrophotometer . Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit . Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed on the ABI 7900HT Fast RT-qPCR System using Sequence Detection System 2.4 software to quantify mRNA expression using TaqMan Fast Advanced Master Mix and pre-designed TaqMan Gene Expression Assays . To control for variability between samples, the relative amounts of the genes were normalized to peptidylprolyl isomerase A , and statistical analysis was performed using SigmaPlot 12.5 . Data were analyzed by two-way analysis of variance, with maternal HF and maternal salt as factors. When data failed normality (Shapiro–Wilk test), it was log transformed. Holm–Sidak There were no significant differences in male offspring birth weights (data not shown). The physiological and metabolic profiles of adult male offspring are presented in Table 2 than the HF group. This was due to the HF group having a greater number of adipocytes with an area >15,000 μm2 compared to the SD group. These data suggest moderate adipocyte hypertrophy in HF offspring, with a greater proportion of smaller adipocytes in the SD group. Given the changes in adipocyte morphology, we examined gene expression in the retroperitoneal adipose tissue. There was a significant interaction in Dlk1, a pre-adipocyte marker and inhibitor of adipogenesis  in the SVF isolated from gonadal adipose tissue , but Igfbp2 was significantly increased by maternal salt intake, with a significant increase in the HFSD group compared to the HF group  or glucose (Glut2 and Pck1) transport and metabolism . There was a significant interaction in Cd36 expression, with significantly reduced expression in the HFSD group compared to SD  demonstrated improved insulin sensitivity concomitant with reduced adipose tissue inflammation  can impact digestive efficiency and weight gain.The RAS is typically known for its roles in cardiovascular homeostasis and fluid regulation. However, in recent years, components of the RAS have been shown to be expressed in multiple peripheral tissues, and it is clear that the RAS contributes to a range of physiological effects outside its classically described roles. Increased activation of the RAS is associated with obesity and T2DM, while blocking the RAS can be protective against diet-induced obesity and development of T2DM . AlthougCd36 and inflammatory markers in the gut. However, what is unclear from the present study is how this occurs. We have previously demonstrated in this model that dams consuming the HFSD diet during pregnancy gained significantly less weight than dams on the HF diet, although there were no significant differences in cumulative caloric consumption between the two groups (In conclusion, we have demonstrated that the addition of high salt to a maternal HF diet did not exacerbate the adverse effects of a maternal HF diet on regulation of metabolism in adult offspring. Rather, a maternal HFSD diet prevented some of the programmed effects in offspring as a consequence of a maternal HF diet, including impaired insulin sensitivity and hyperleptinemia. In line with this finding, a maternal HFSD diet prevented a maternal HF diet-induced increase in the key lipid transporter o groups . Indeed,o groups . Since do groups . In lineThis study and procedures described were carried out in accordance with the recommendations and regulations of the Animal Ethics Committee at the University of Auckland.Conceived and designed experiments: SAS, CMR, MHV and CG. Performed experiments: SAS, CMR, RP, CG and CJH. Wrote manuscript: SAS. Edited manuscript: CMR and MHV.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NBS1, was shown to be associated with increased susceptibility to lung and colorectal cancer. However, the relation between rs2735383 and susceptibility to breast cancer is not yet clear. Therefore, we genotyped rs2735383 in 1,170 familial non-BRCA1/2 breast cancer cases and 1,077 controls using PCR-based restriction fragment length polymorphism (RFLP-PCR) analysis, but found no association between rs2735383CC and breast cancer risk . Because we could not exclude a small effect size due to a limited sample size, we further analyzed imputed rs2735383 genotypes (r2 > 0.999) of 47,640 breast cancer cases and 46,656 controls from the Breast Cancer Association Consortium (BCAC). However, rs2735383CC was not associated with overall breast cancer risk in European  nor in Asian women . Subgroup analyses by age, age at menarche, age at menopause, menopausal status, number of pregnancies, breast feeding, family history and receptor status also did not reveal a significant association. This study therefore does not support the involvement of the genotype at NBS1 rs2735383 in breast cancer susceptibility.NBS1, also known as NBN, plays an important role in maintaining genomic stability. Interestingly, rs2735383 G > C, located in a microRNA binding site in the 3′-untranslated region (UTR) of  When left unrepaired, DSBs may cause genomic instability, cell death and cancer13The DNA damage response (DDR) pathway maintains the stability of the human genome via a complex network of pathways integrating signal transduction, regulation of the cell cycle and repair of DNA. Double-strand breaks (DSBs), a particularly severe form of DNA damage, arise as a consequence of cell replication, programmed DNA rearrangements  are involved in DNA repair7MCPH1as a novel breast cancer susceptibility gene illustrates that this hypothesis still holdsThe DDR plays an important role in susceptibility to breast cancer. In fact, all of the currently identified high- and moderate-risk breast cancer genes  and has its origin in the Slavic populationNBS1 mutations implicated in Nijmegen breakage syndrome are p.I171V and p.R215W. Although both mutations associate with an overall cancer risk of 4-fold and 2-fold, respectively, there does not seem to be an increased risk to develop breast cancer specificallyIn this respect, the NBS1  have also been reported to be associated with risks for several cancer types. Recent meta-analyses for rs1805794 have, however, shown that this variant does not associate with breast cancer risk131415131617NBS1, has been shown to modulate the binding ability of microRNA-629 in lung cancer cells and microRNA-509-5p in colorectal cancer cells, affect NBS1 transcriptional activity and decrease NBS1 mRNA and NBS1 protein levels1913NBS1 rs2735383 is associated with breast cancer risk in the Rotterdam Breast Cancer Study (RBCS) by RFLP-PCR and in 45 studies of BCAC through imputation of the iCOGS arrayBesides the rare Nijmegen breakage syndrome-associated mutations, two common variants in NBS1 rs2735383 and breast cancer risk, we analyzed NBS1 rs2735383 by RFLP-PCR in 1,269 non-BRCA1/2 familial breast cancer patients and 1,159 controls from RBCS. Since genetic risk factors are usually enriched in familial/early-onset breast cancer cases, specifically selecting these breast cancer patients improves statistical power. Among the cases, 516 had the GG genotype, 507 had the GC genotype and 147 had the CC genotype at rs2735383  = 0.342). Among the controls, 462 had the GG genotype, 501 had the GC genotype and 114 had the CC genotype (MAF = 0.338). For both cases and controls, the genotypes of rs2735383 were in Hardy-Weinberg equilibrium (HWE). Because rs2735383 CC was associated with an increased risk of lung cancer and colorectal cancer under a recessive genetic model13P = 0.144; P < 0.001), whereas the colorectal cancer risk had been associated with an OR of 1.55 13(i.e. OR < 1.28). Therefore, we cannot exclude rs2735383 CC is associated with breast cancer, but confers a smaller risk.To evaluate the association between NBS1 rs2735383 in BCAC studies through imputation. Since we had data available for RBCS on rs2735383 from both the PCR-based RFLP and from imputation, we first evaluated the concordance between the two methods. In total, from 1,313 samples (i.e. 646 cases and 667 controls) we had genotypes for rs2738353 available from both RFLP-PCR and imputation. Importantly, the agreement between the two methods was 97.1%  and was similar among cases and controls (i.e. 98.1% versus 96.1%). Moreover, case-control ORs for imputed data were comparable to ORs obtained by RFLP-PCR . Therefore, we used the imputed data on rs2735383 to evaluate further its association with breast cancer risk.For this reason we analyzed P = 0.556; P = 0.684; P = 0.984; 13P = 0.961; P = 0.900; P = 0.911; NBS1 rs2735383 is not associated with an increased risk to develop invasive breast cancer.For the overall analysis in Europeans we had 41,915 cases and 40,042 controls available from 36 case-control studies. However, rs2735383 was not associated with breast cancer risk in Europeans, neither under a recessive genetic model 13NBS1 rs2735383 with breast cancer risk. We found that the CC genotype of rs2735383 did not confer an increased breast cancer risk, neither in the overall analyses nor in the subgroup analyses.The CC genotype of the common variant rs2735383 in the 3′UTR of et al. consisting of 239 premenopausal breast cancer patients and 477 matched controls from the Nurses’ Health Study II showed that rs2735383 did not associate with breast cancer risk under an additive genetic model et al. consisting of 450 breast cancer patients and 450 cancer-free controls from the Henan Province in China also found no association with overall breast cancer riski.e. 450 cases and 450 controls in the study of Wu et al.), could have easily given rise to false positive findings. Therefore, one should be careful when reporting positive findings from multiple small subgroup comparisons and always use appropriate levels of statistical significanceet al. there is no mention of multiple testing correction.In agreement with these results, a small study by Han NBS1 gene through either binding of microRNA-629 to the 3′-UTR of NBS1 gene in lung cancer cells or the binding of microRNA-509-5p to the 3′-UTR of NBS1 gene in colorectal cancer cells19NBS1 may reduce the efficiency of DSB repair, this way the rs2735383CC genotype likely confers an increased lung and colorectal cancer risk. According to our study, however, the rs2735383CC genotype does not confer an increased breast cancer risk. Considering that in lung and colorectal cancer cells different microRNAs appear to be downregulating NBS1 expression, tissue specific expression of these microRNAs may likely play a role. Besides microRNA-509 and microRNA-629, the C allele at rs2735383 has also been predicted to enhance the binding of microRNA-499 and microRNA-508 to the 3′UTR of NBS1NBS1 cannot be downregulated by any of these microRNAs. At least in breast cancer cells, none of these microRNAs, except microRNA-629, are expressed at substantial levels . Since microRNAs are often deregulated between normal tissue and cancer tissue, this does not necessarily represent the situation in normal breast cells. Thus, further evaluation of the miRNA expression levels in normal (breast) tissue, but also their correlation with the genotype at rs2735383 should provide more insight for the tissue specificity of rs2735383 and cancer risk.It was found that the rs2735383CC genotype significantly decreased the expression of the i.e. OR > 1.04 for Europeans and OR > 1.11 for Asians) between the genotype at rs2735383 in the 3′UTR of NBS1 and breast cancer susceptibility.Importantly, in contrast to lung and colorectal cancer susceptibility, the results of this study do not support the presence of an association . RBCS controls  came from the same database and geographic location as the RBCS cases and included women from cystic fibrosis families who were either spouses of individuals at risk of being carrier of a CFTR mutation or individuals who were tested negative for a CFTR mutation. The median age of the RBCS controls was 41 years (range 10–97 years).RBCS cases  came from the database of the Clinical Genetics Department at Erasmus University Medical Centre in Rotterdam, representing the Southwestern part of the Netherlands. First, we selected families that presented with at least two cases of female breast cancer or at least one case of female breast cancer and one case of ovarian cancer in first- or second-degree relatives. In addition, at least one of these two cases needed to be diagnosed before the age of 60. For each selected family, the youngest breast cancer patient who had been tested for i.e. two studies) were not included because power in the analyses would be low due to a relatively low MAF (i.e. 0.123) and small amount of cases . Each study was approved by its relevant governing research ethics committee and all study participants provided written informed consent. The experimental protocol was approved by the Medical Ethical Committee of the Erasmus Medical Center Rotterdam and the study was carried out in accordance with the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands (http://www.fmwv.nl). Following genotyping on the iCOGS arrayBCAC consists of case-control studies of unrelated womenNBS1 including rs2735383 was amplified in a duplex PCR reaction together with a 713 bp fragment of the LRRC4 gene. Primers for NBS1 and LRRC4 were present in the PCR reaction at a final concentration of 0.25 and 1 μM, respectively, and sequences are available in LRRC4 fragment served as an internal digestion control and generated two fragments of 549 and 164 bp upon complete digestion with ScfI . The 324 bp amplified NBS1 fragment was only digested when the major G allele was present at rs2735383, thereby generating two fragments of 233 and 91 bp. Thus upon successful digestion with ScfI, samples with rs2735383 GG generated four fragments, samples with rs2735383 GC generated five fragments and samples with rs2735383 CC generated three fragments (A 324 bp fragment of the 3′UTR of ragments .i.e. when MAF >0.05) or <99% (i.e. when MAF <0.05), deviation from HWE (i.e. P < 10–7), or >2% duplicate discordance.Genotyping of BCAC studies was performed previously using the custom iCOGS Illumina Infinium iSelect BeadChipNBS1 rs2735383 was imputed with an imputation r2 > 0.999 in both Europeans and Asians.Imputation of genotypes was performed using 1000 Genomes Project data (v3 April 2012 release) as the reference panelNBS1 rs2735383 and invasive breast cancer risk was evaluated by logistic regression analysis providing ORs and 95% CIs. In the analyses of BCAC studies, ORs were adjusted for study, age, and PCs. In the analyses of RBCS, ethnicity was not a confounding factor thus reported ORs were unadjusted for PCs. For the European and Asian BCAC studies, we additionally performed study-specific logistic regression analysis adjusting for age and PCs, and pooled the log ORs in a fixed-effects meta-analysis. Subgroup analyses within the European BCAC studies were based on age (i.e. ≤50 years and >50 years), age at menarche (i.e. ≤13 years and >13 years), age at menopause (i.e. ≤50 years and >50 years), menopausal status , number of full-term pregnancies (i.e. ≤2 and >2), breast feeding (i.e. no and yes), first-degree family history of breast cancer and receptor status . Clinical and demographic characteristics of the BCAC cases are presented in NBS1 rs2735383 and the clinical and demographic characteristics were evaluated using a χ2 test. All P-values were two-sided and P < 0.05 was considered to be statistically significant after correction for multiple testing by the Bonferroni procedure. Logistic regression analyses were performed using SPSS statistics version 23  and fixed-effects meta-analyses using Stata version 13 .The association between How to cite this article: Liu, J. et al. rs2735383, located at a microRNA binding site in the 3′UTR of NBS1, is not associated with breast cancer risk. Sci. Rep.6, 36874; doi: 10.1038/srep36874 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Methods. The study consisted of 317 euthyroid subjects aged 48-49 from the Kaunas Cardiovascular Risk Cohort study. Thyroid-stimulating hormone (TSH), free thyroxine (FT4), and antithyroid peroxidase antibody (ATPO) levels, as well as anthropometric and metabolic parameters and smoking information, were evaluated. Results. In subjects with and without thyroid nodules, thyroid volume correlated with components of metabolic syndrome, body mass index (BMI), smoking, and TSH levels. In the nonnodular thyroid group, thyroid volume was also positively related to serum insulin and HOMA-IR, whereas a negative correlation between thyroid volume and leptin was identified in the nodular thyroid group. The goiter was identified in 12.3% of subjects. Female gender, thyroid nodules, smoking, BMI, and levels of TSH were independent predictors for goiter. Thyroid nodules were found in 31.2% of participants. Female gender, higher TSH levels, and thyroid volume were independent risk factors for thyroid nodules. Conclusions. Female gender, thyroid nodules, smoking, BMI, and TSH levels were identified as potential predictors of goiter. Female gender, TSH levels, and thyroid volume predicted the presence of thyroid nodules.The aim of the present study was to determine associations of thyroid hormone levels and different metabolic parameters and anthropometric measurements with volume of nodular and nonnodular thyroid as well as with prevalence of goiter and thyroid nodules in middle-aged euthyroid subjects.  The most common thyroid disorders are thyroid nodules and goiter that develop as a result of the interplay between genetic, environmental, or endogenous factors. Goiter affects approximately 15.8% of the general population and iodine is the main environmental factor that determines goiter prevalence . AlthougWomen are more likely to have thyroid disorders, including goiter and thyroid nodules, although some authors could not confirm any gender-related associations , 10. AssThe aim of the present study was to determine associations of thyroid hormone levels and different metabolic parameters and anthropometric measurements with volume of nodular and nonnodular thyroid as well as with prevalence of goiter and thyroid nodules in middle-aged euthyroid subjects.n = 19; 3.7%), those having elevated levels of ATPO (presence of ATPO is an early sign of autoimmune thyroid diseases) , TSH or FT4 levels outside the reference range , or who did not undergo thyroid ultrasound examination  were excluded from the analysis. Finally, data of 317 subjects (142 men and 175 women) were analyzed in this study.The study subjects were participants of the Kaunas Cardiovascular Risk Cohort study started in 1977, when a random sample of 1082 Kaunas schoolchildren aged 12-13 years was screened [The study protocol was approved by the Lithuanian Bioethics Committee (permission number BE-2-30) and written informed consent was obtained from all participants.Thyroid ultrasonography was performed by a single physician who was unaware of the patient clinical conditions. The real-time 3D/4D imaging diagnostic ultrasound system Mindray CD-7 with a multifrequency transducer (L12-4 MHz HiRez Linear) was used. Subjects were examined in the supine position with the neck hyperextended. Thyroid volume was calculated according to the formula: width × length × thickness × 0.479 for each lobe. Goiter was determined as a thyroid volume exceeding 18 mL for women and 25 mL for men. Thyroid nodules greater than 3 mm in diameter were registered. In patients with multiple thyroid nodules, the largest diameter of the largest nodule was considered. Thyroid nodules 10 mm or larger in diameter and smaller nodules (6 mm or greater) with suspicious features for malignancy underwent a fine-needle aspiration biopsy. Based on the thyroid nodularity, participants were split into two groups: nodule-free or those with at least one nodule.2). Obesity was defined as BMI of 30 kg/m2 or higher. Waist circumference (WC) was measured at the midpoint between the lower margin of the last palpable rib and the top of the iliac crest using a stretch-resistant tape. Blood pressure (BP) was measured from the right brachial artery with a standard mercury sphygmomanometer in the sitting position after 5 minutes of rest. BP measurements were performed to the nearest 2 mmHg. Three consecutive BP measurements were taken. The average of these three measurements was used in the analysis. Information on cigarette smoking was collected using a standard questionnaire. Participants were grouped in daily smokers and others.The height of participants, without shoes, was measured to the nearest centimeter with a stadiometer. The body weight of participants was measured to the nearest 0.1 kg with standardized medical scales. Body mass index (BMI) was calculated as weight (kg) divided by squared height (mγ-ray counter Berthold (Germany). Analytical sensitivity of reagents (lowest detection limit) was 0.11 mU/L for TSH, 0.7 pmol/L for FT4, 2 kU/L for ATPO, 0.1 pg/L for leptin, and 1 mU/L for insulin. The coefficients of variation within a set of reagents/between kits (intra/interassays CV) were 4.7/8.6% for TSH, 2.6/5.7% for FT4, 6.7/7.5% for ATPO, 1.8/2.5% for leptin, and 2.1/6.5% for insulin. The laboratory reference values were 10.1–22.0 pmol/L for FT4, 0.27–3.75 mIU/L for TSH, and 4–16 mU/L for insulin. The reference value for ATPO was <78 kU/L. All measurements for lipids and glucose were performed on an automatic analyzer Cobas Integra 400 plus. Serum lipid levels were determined using conventional enzymatic methods. Hexokinase was used for measurement of glucose level.Venous blood samples for laboratory analysis were obtained by venipuncture from all patients between 08.00 and 10.00 h after a minimum of 12 h overnight fast. Hormone levels were analyzed by immunoradiometric assay for TSH and radioimmunoassay for FT4 and ATPO using commercial reagent kits of the IZOTOP (Hungary). Insulin and leptin were investigated by immunoradiometric assay using commercial reagent kits of the DiaSource (Belgium) and evaluated using means of IR was determined according to HOMA-IR (homeostasis model assessment of IR). HOMA-IR was calculated using the following formula: fasting insulin (mU/L) × fasting glucose (mmol/L)/22.5 . HOMA-IRMetS was determined according to the criteria of the International Diabetes Federation: central obesity defined as waist circumference ≥94 cm for men and ≥80 cm for women plus any two of the following four factors: raised triglycerides level (≥1.7 mmol/L), reduced high-density lipoprotein (HDL) cholesterol level (for men <1.03 mmol/L and for women <1.29 mmol/L), raised blood pressure , and raised fasting plasma glucose (≥5.6 mmol/L or previously diagnosed type 2 diabetes) [χ2) test was used for analysis of data when variables were categorical. Continuous variables were tested for normality according to the Kolmogorov-Smirnov test. The results were presented as means ± standard deviation (SD) if the distributions were normal or median and interquartile range (IQR) if the distributions did not meet the criteria of normality. Student's t-tests  or the Mann-Whitney tests  were used to compare two continuous variables. For bivariate correlations, Pearson correlation tests (for parametric data) or Spearman correlation tests (for nonparametric data) (ρ) were used. Odds ratios (OR) with 95% confidence interval (CI) were calculated using univariate and multivariate binary logistic regression analysis to estimate associations between different variables and goiter or thyroid nodules. Data were analyzed using IBM SPSS Statistics for Mac (V20.0). p value less than 0.05 was considered as statistically significant.Chi-square (p < 0.001). Women had a higher prevalence of thyroid nodules than men (p < 0.001). Serum concentrations of TSH and ATPO were similar in both genders, whereas mean levels of FT4 were higher in men than in women. BMI; waist circumference; blood pressure (both systolic and diastolic); and levels of fasting plasma glucose, fasting insulin, and HOMA-IR values were significantly higher in men than in women. Although levels of total cholesterol were similar in both genders, men had significantly higher levels of LDL cholesterol and triglycerides, whereas HDL cholesterol levels were lower. Proportion of current smokers and prevalence of metabolic syndrome were higher in men than in women.Characteristics of the study population are presented in Table Participants were split into two groups according to the presence or absence of thyroid nodules. In both groups, thyroid volume significantly correlated with components of metabolic syndrome , BMI, smoking status, and levels of TSH Table . In the We also analyzed the relationship between thyroid size, prevalence of thyroid nodules, and a number of previous pregnancies; however, no significant associations among these factors were determined (data not shown).p < 0.001), lower levels of TSH (median (IQR): 0.9 (0.6) versus 1.3 (0.8) mU/L, p < 0.001), higher HOMA-IR values (median (IQR): 2.3 (1.2) versus 2.0 (1.3), p = 0.046), and higher BMI (median (IQR): 28.5 (7.8) versus 25.6 (5.9) kg/m2, p = 0.018) compared with goiter-free subjects. Also the percentage of daily smokers was higher among subjects with goiter than goiter-free subjects . The multivariate logistic regression analysis showed that female gender, thyroid nodules, smoking, BMI, and levels of TSH were independent predictors for goiter. There was a negative association between the presence of goiter and the levels of TSH (The goiter was identified in 12.3% of subjects with similar proportions in men and women . Subjects of TSH .p = 0.432). Thyroid nodule size was positively correlated with thyroid volume . No significant differences were observed in thyroid hormone levels, blood pressure, different metabolic parameters, and BMI between the subjects with larger thyroid nodules (≥10 mm in size) and the subjects with smaller nodules. In the multivariate analysis, female gender, TSH levels, and thyroid volume were independent risk factors for thyroid nodules . In men, thyroid volume was also positively correlated with levels of ATPO .In an analysis limited only to a subgroup of subjects with goiter and/or larger thyroid nodules (≥10 mm in size), a negative correlation between thyroid volume and levels of TSH remained significant for both genders  but suspicious ultrasound features of malignancy . None of biopsied subjects with thyroid nodules were found to have malignant lesions.Although nontoxic goiter and thyroid nodules are the most common thyroid disorders in adult population, the etiology of both conditions is not completely understood. In this study, we identified some risk factors for goiter and thyroid nodules in middle-aged euthyroid subjects. Female gender, thyroid nodules, smoking, BMI, and lower TSH levels were found to be independently associated with higher risk of goiter. The risk of increased prevalence for thyroid nodules was significantly associated with thyroid volume, female gender, and TSH levels.A number of earlier studies have analyzed factors that can affect thyroid morphological abnormalities. Iodine undoubtedly plays the most important role in function and structure of the thyroid gland. Epidemiological studies have shown that the abnormality of iodine intake in population may result in thyroid dysfunction and/or abnormal thyroid structure , 16. TheAn association between gender and thyroid volume or goiter prevalence has been reported. Knudsen et al. noted that thyroid volume was larger in men than in women, yet women were 2- to 10-fold more likely to have goiter than men . AssociaSome researchers reported that men had a larger thyroid gland than women due to the difference in body mass . The relThyroid nodules are 4 times more common in women than men and their frequency increases with advancing age and iodine deficiency . Causes TSH plays a major role in regulation of thyroid cell growth and differentiation and may play a direct role in nodule formation . ConflicSome authors highlighted importance of MetS and IR in thyroid volume increase and higher risk for formation of thyroid nodules , 44. RezConflicting data exist regarding the relation between tobacco smoking and goiter. Some researchers have found a positive association between smoking and higher prevalence of goiter , 47 and/Our study has some limitations that offer opportunities for future investigations. We acknowledge that a major limitation of our study is a relatively small number of subjects. Moreover, we cannot currently provide data on iodine intake. Studies directly assessing iodine intake in Lithuanian population after introduction of iodized salt are lacking. Therefore, we can only speculate that our subjects are not currently iodine deficient, although they had been exposed to an iodine-deficient environment previously. We also did not analyze effects of selenium, zinc, or other factors that are also important in thyroid metabolism and may affect thyroid function, size, and nodules. Lack of repetitive TSH testing in subjects should be also mentioned as a possible limitation of our study. Repetitive TSH testing may help to obtain a more accurate evaluation of thyroid status and to avoid discrepancies between the results. Another limitation of our study is that we used HOMA-IR for assessment of IR, which is a surrogate marker of IR. The “gold standard” for IR assessment today is the hyperinsulinemic-euglycemic clamp, which is more accurate in evaluating insulin resistance .The strength of the present study includes the use of the data from a randomly selected cohort that has been prospectively followed up for 35 years. A further strength is extensive phenotypic characterization of participants, which includes an elaborate set of anthropometric measurements and metabolic factors, as well as a complete thyroid status assessment with measurements of TSH, FT4, and antithyroid peroxidase antibodies. The narrow age range of subjects eliminates the potential effect of age that may influence the observed associations among variables.This study showed that female gender, thyroid nodules, smoking, BMI, and lower levels of TSH are independently associated with an increased risk of goiter. Prevalence of thyroid nodules was found to be significantly associated with female gender, higher levels of TSH, and increased thyroid volume.


This study investigated the effect of vitamin D supplementation in patients with dry eye syndrome (DES) refractory to conventional treatment with vitamin D deficiency. A total of 105 patients with DES refractory to conventional treatment and vitamin D deficiency that was treated with an intramuscular injection of cholecalciferol . Serum 25-hydroxyvitamin D (25(OH)D) levels were measured. Eye discomfort was assessed using ocular surface disease index (OSDI) and visual analogue pain score (VAS). Tear break-up time (TBUT), fluorescein staining score (FSS), eyelid margin hyperemia, and tear secretion test were measured before treatment, and 2, 6, and 10 weeks after vitamin D supplementation. Mean serum 25(OH)D level was 10.52 ± 4.61 ng/mL. TBUT, and tear secretion test showed an improvement at 2 and 6 weeks after vitamin D supplementation compared to pretreatment values . Eyelid margin hyperemia and the severity of symptoms showed improvement at 2, 6, and 10 weeks after vitamin D supplementation . Compared to pre-treatment values, FSS, OSDI and VAS were decreased at 2 weeks . In conclusion, vitamin D supplementation is effective and useful in the treatment of patients with DES refractory to conventional treatment and with vitamin D deficiency. The ethics committee/IRB waived the need for informed consent because this study is retrospective. We reviewed the medical charts from patients who had visited the Hallym University Kangnam Sacred Heart Hospital from June 2015 to March 2016. Patients with DES that was refractory to artificial tear treatment  and liposic EDO  and with demonstrated vitamin D deficiency were included. Serum 25(OH)D concentration was measured. Patients with vitamin D deficiency or insufficiency were treated by an intramuscular injection of 200,000 IU cholecalciferol. Exclusion criteria were autoimmune diseases such as Sjogren’s syndrome or lupus syndrome; corneal surgery such as penetrating keratoplasty, corneal limbal allo-transplantation or corneal laceration repair; corneal diseases such as recurrent corneal erosion syndrome or keratoconus; and corneal opacity. Data was obtained pre-treatment, 2 weeks, 6 weeks, and 10 weeks after vitamin D supplementation.Tear break-up time (TBUT), fluorescein staining score (FSS), and the Schirmer tear secretion test were used to evaluate the tear film. TBUT evaluation was performed in a dimly lit room. Fluorescein was placed in the lower conjunctival sac using a fluorescein strip . The subjects were then asked to blink, and the time before the first defect appeared in the stained tear film was measured as TBUT. Fluorescein staining was performed as previously describedHyperemia and telangiectasia of the eyelid margin was graded as follows: 0 = none, 1 = mild, 2 = moderate, and 3 = severe. Conjunctivochalasis (CCH) was graded on the basis of the extent of inferior eyelid margin involvement as follows: 1 = single  location, 2 = two locations , and 3 = whole eyelid.Eye discomfort was assessed by the ocular surface disease index (OSDI), visual analogue pain score (VAS), severity, and duration of symptoms. An OSDI questionnaire was used to quantify the dry eye symptoms. Subjects were asked questions regarding the symptoms of dry eye that they had experienced during a 1-week recall period; the OSDI questions comprised three different subscales: ocular symptoms, vision-related functions, and environmental triggers. Each answer was scored on a 4-point scale that ranged from zero (indicating no problems) to four (indicating a significant problem). Responses to all of the questions were combined to generate a composite OSDI score that ranged from 0 to 100, with higher OSDI scores indicating more severe symptoms23All data are presented as mean and standard deviation. Paired-sample t-tests were used to compare the TBUT, FSS, eyelid margin hyperemia, Schirmer test, OSDI, VAS, and severity and duration of symptoms between pre-treatment, and 2, 6 and 10 weeks after vitamin D supplementation. SPSS version 23 for Windows  was used for all analyses. P < 0.05 was considered to be statistically significant.A total of 105 patients were included in the study . The meaThe effects of vitamin D supplementation on DES were analyzed according to gender , Table 3The effects of vitamin D supplementation on DES were analyzed according to age group , Table 4DES has been reported to be associated with a variety of factors16In this study, vitamin D supplementation improved TBUT, FSS, eyelid margin hyperemia, and tear secretion. TBUT is the most frequently employed method for assessing tear instability3032et al. reported a case of putative corneal neuralgia accompanying hypovitaminosis D and responding to vitamin D supplementationIn this study, vitamin D supplementation improved subjective symptoms including OSDI score, VAS score, severity, and duration of symptoms. A significant proportion of patients with DES complained of moderate or greater ocular pain intensity1245TBUT increased in both male and female patients, whereas FSS and tear secretion was only improved in women. The effect of vitamin D has been reported to differ according to gender4951In summary, vitamin D supplementation promoted tear secretion, reduced tear instability, and reduced inflammation at the ocular surface and eyelid margin. Furthermore, vitamin D supplementation improved the symptoms of DES. In conclusion, vitamin D supplementation is an effective and useful treatment for patients with DES that is refractory to conventional treatment.How to cite this article: Bae, S. H. et al. Vitamin D Supplementation for Patients with Dry Eye Syndrome Refractory to Conventional Treatment. Sci. Rep.6, 33083; doi: 10.1038/srep33083 (2016).


Fingolimod (Fg), a sphingosine 1-phosphate receptor modulator, decreases the annual relapse rate (ARR) in relapsing-remitting multiple sclerosis (RRMS). The aim of this study was to assess the efficacy and safety of Fg in daily practice in patients with RRMS, previously treated with natalizumab (Nz) or not, and systematically followed during at least 1 year.Data were collected from the patient files. Primary endpoint was the comparison between the ARR the year before Fg onset and after 1 and 2 years of Fg treatment. The secondary endpoints were the difference between Expanded Disability Status Scale (EDSS) at Fg onset and after 1 and 2 years of treatment, and safety.p < 0.0001). Between our two groups (with or without Nz before Fg), the ARR was higher in the Nz group during the first year but similar during the second year. The EDSS was stable during the first year of Fg but significantly higher after 2 years . Concerning safety, only three patients had to discontinue Fg because of tolerance issues.In the whole sample, we confirmed Fg efficacy on the ARR (0.895 before vs. 0.364 1 year after, Our study showed that Fg is safe in RRMS and can be used either after first-line treatments or after Nz. However we observed a mild disability progression after 2 years. Fingolimod (Fg), a sphingosine 1-phosphate receptor modulator, reduces the release of lymphocytes from secondary lymphoid organs, thereby their infiltration in the central nervous system , 3. Its In this retrospective and monocentric study, every patient fulfilling RRMS 2005 McDonald criteria  and treaData were collected retrospectively from MS patient files. Personal characteristics of the patients, previous history of MS, previous and ongoing disease modifying drugs (DMDs), pretreatment blood panel including JCV serology, and all data from the follow-up by the treating neurologist were systematically reported. Patients’ follow-up was scheduled according to the French authority recommendations in our MS clinic. At Fg onset, Expanded Disability Status Scale (EDSS)  was scorA relapse of MS was defined as occurrence, recurrence, or worsening of symptoms of neurological dysfunction lasting over 24 h and usually ending up in a partial or complete remission , 7. FatiThe MS course was categorized according to 1996 classification . Patient3), elevated liver enzymes (>3 N).Due to the amount of missing data (more than 50%), brain MRI results were not analyzed. Safety data were also reported: cardiac events at Fg onset, infections, high blood pressure, macular edema, lymphopenia (<500/mmThe primary objective was to assess the efficacy and the safety of Fg in patients with MS in daily practice. We focused on (i) the comparison between ARR the year before Fg onset and after 1 and 2 years of Fg treatment, (ii) the difference between EDSS at Fg onset and after 1 and 2 years of treatment, (iii) the reasons to Fg discontinuation, and (iv) the number and the types of adverse events.n (%)]. The sample was divided in two subgroups according to the use of Nz during the 6 months preceding Fg onset (Nz and no-Nz groups). General characteristics were calculated in the whole sample and according to Nz groups, and comparison was made using χ2 test or T-test for categorical and continuous variables, respectively.Data are expressed as the mean (SD) [M (SD)] or percentages and numbers  and started Fg older  . The mean follow-up was 22.0 (7.0) months.The mean age at Fg onset was 35.1 (8.5) years, the mean disease duration was 9.43 (4.92) years, and JCV serology was found positive in 71 (92.2%) patients tested (n = 42), (ii) after immunosuppressants (n = 54) , or (iii) in naïve patients (n = 11). The demographic and clinical characteristics according to Nz treatment before Fg are summarized in Table p ≤ 0.0001] and a higher EDSS  at Fg onset. In the Nz group, the mean washout duration was 101 (30) days. The switching protocol was carefully applied in our unit and almost every patient started Fg 3 months after the last Nz infusion. Patients discontinued Nz because of positive JCV serology (n = 46), drug inefficacy (n = 1), based on a personal preference (n = 3), adverse effects (n = 1), unknown reason (n = 1).Fingolimod was introduced (i) after injectable DMD n = 4, (ii) afConcerning the whole population, 42 patients (39.3%) had one or more relapse under Fg including 24 patients (22.4%) with at least one relapse within the 3 months after Fg onset. In the Nz group, nine patients (14.8%) relapsed during the washout between Nz and Fg, but their ARR remains similar to other patients at 1 and 2 years (data not shown). Eleven patients (21.2%) experienced at least one relapse within the 6 months after Fg start, and four patients (7.7%) experienced their first relapse between 6 and 12 months after Fg start.p < 0.0001) and 0.423 (0.696) during the second year after Fg onset  during the year before Fg onset, 0.364 (0.620) during the first year after Fg onset , p = 0.41].In the Nz group, the ARR was 0.340 (0.626) the year before Fg onset. Compared to the year before Fg onset, there were no significant changes during the first year of Fg . Then, the ARR remained stable during the second year of Fg .In the no-Nz group, the ARR was 1.400 (0.807) during the year before Fg onset. Compared to the year before Fg onset, the ARR was significantly lower during the first year of Fg  and a higher ARR during the first year of Fg . However, there was no significant difference between the two groups for the ARR during the second year of Fg .Compared to the patients who did not receive Nz before Fg, those who received Nz before Fg had, on average, a lower ARR during the year before Fg onset  but significantly higher after 2 years of Fg .In the whole sample, the EDSS was 3.33 (1.94) the year before Fg onset. Compared to the year before Fg onset, the EDSS was not different after 1 year of Fg . It stresses the fact that brain MRI is not performed as scheduled despite its high priority. Yet, brain MRI is a key point in the evaluation of a treatment efficacy, like it is used in the modified Rio score with interferon treatment . Indeed,The JCV serology was found positive in 92.2% of the patients who were tested, which is more than expected in the general and MS populations . This reIn the Nz group, the mean washout duration was 101 (30) days, which is similar to others’ studies in literature , 23, 27,Besides, the first relapse of the patients previously treated with Nz mostly happened within the 6 months after Fg start. This is consistent with the results of a large French post-marketing cohort including 715 patients who stopped Nz . In thisAs expected, the safety is correct, with only three Fg discontinuation after adverse events, as described in other studies , 27, 29.We are aware that our study has many biases, including a retrospective analysis and a small size of the cohort. However, it provides real-life information about Fg treatment, especially with the patients who were not included in the phase III studies . It highlights that, in daily practice, Fg is efficient and safe in RRMS. Our study also revealed that Fg can be used either after first-line DMDs or after Nz. However, we observed a mild disability progression after 2 years. Controlled randomized trials, with longer follow-up and larger samples, are needed.Clinical data were obtained in accordance with ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients were informed and approved to participate to this study prior inclusion. In this retrospective clinical study, no authorization from an ethical committee was required.TR, EM, and CP acquired and analyzed the data. J-SV, STM, and CL analyzed the data. All the authors revised and approved the manuscript.EM has received conference fees for Biogen, Genzyme, Novartis, and Teva. CP served on the scientific advisory board for Bayer Schering, Novartis, and Teva and was employed for educational activities from Bayer Schering, Sanofi-Genzyme, Biogen Idec, Novartis, Merck Serono, and Roche. CL served on the scientific advisory board for Vertex, Biogen, Novartis, and Genzyme; served on the editorial board for MSJ and MSJ and Related Disorders; was associate editor for Brain; consulted for Biogen, Roche, and Genzyme; and had a scientific collaboration with EMD Serono. The other authors declare no conflict of interest.


Dicentrarchux labrax) fed a low fish meal diet (5%), for 65 days.Aquaculture production is expected to double by 2030, and demands for aquafeeds and raw materials are expected to increase accordingly. Sustainable growth of aquaculture will require the development of highly nutritive and functional raw materials to efficiently replace fish meal. Enzymatic hydrolysis of marine and aquaculture raw materials could bring new functionalities to finished products. The aim of this study was to determine the zootechnical and transcriptomic performances of protein hydrolysates of different origins  in European seabass  contains supplementary material, which is available to authorized users. Fish captures have been stabilizing since the 1980’s, and in most parts of the world seafood production is now supplied by the aquaculture industry . OriginaThe large availability of marine and aquaculture by-products could allow for partial replacement of wild fish-derived FM , 20. By-Dietary shrimp hydrolysates stimulate growth performances in fish, and also possess antimicrobial properties against aquaculture pathogens –39. SimiDicentrarchus labrax, Linnaeus, 1758) is a marine fish widely reared in the Mediterranean sea, with more than 156,449 tons produced in 2014  × 100.At the end of the trial, we measured the BW and SL of all sea bass from each tank (the fish were fasted for 24 h prior to sampling). Specific Growth Rates (SGR), Fulton’s condition factors (K), feed conversion ratio (FCR) and Survival Rates (SR) were calculated as follows: SGR (% BW/day) = 100 x [ln final BW - ln initial BW]/duration of the trial (days); K = [BW/SLAll experimental procedures involving sea bass were conducted in compliance with the experimental research protocol approved by the Committee of Ethics and Animal Experimentation of the IRTA, the Departament Agricultura, Ramaderia, Pesca, Alimentació i Medi Natural (permit number 7962) and in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals.per diet were dissected and preserved in a 10% phosphate-formaldehyde buffer (pH = 7.2) for histological purposes. In addition, the anterior intestine from nine fish per dietary treatment was sampled and immediately flash-frozen in liquid nitrogen for transcriptomic analysis. These samples were kept at − 80 °C until RNA extraction.During handling and weighing, sea bass were anesthetized with 50 mg/L of MS-222 . At the end of the trial, they were sacrificed with an overdose of MS-222 (100 mg/mL) to collect the different tissues for analytical purposes. Sea bass were sampled on a cold plate (0–4 °C), and the intestines from 15 individuals per diet. Goblet cell counts in intestinal villi were expressed over a contour length of 100 μm, whereas villi height and width were calculated according to the method of Escaffre et al. [After fixation, samples were dehydrated in a graded series of ethanol, cleared with xylene, embedded in paraffin  and cut into serial sections (2–3 μm thick) . Sections were stained with Hematoxin-Eosin for general histological descriptions, whereas slides were stained with Periodic Acid Schiff (PAS) and Alcian blue (AB) at two different pH values (1.0 and 2.5) to stain different types of mucins produced by goblet cells . PAS stae et al. .260/A280 ratios, and RNA integrity was assessed by denaturing gel electrophoresis. The three RNA samples from same replicate tank were then pooled before generating cDNA libraries. The total RNA concentration of each sample was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA libraries were built, and sequencing was performed as described in [per second, duration: 50 s). Eight pM of cDNA libraries per lane were loaded onto flow cells . The sequencing of 100-pb paired-end reads was performed on an Illumina Miseq Sequencer at the SéSAME Platform .Total RNAs from a pool of the proximal intestines of three sea bass from the same replicate tank were extracted separately. Tissues were homogenized  in 1 mL of TRIzol , and solvent extraction was performed following the manufacturer’s instructions. Final RNA concentrations were determined by spectrophotometry . RNA quality was assessed from Aribed in , with slhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc). Global assembly was conducted using Trinity 2.1.1. [http://trinotate.github.io), as described in [− 3, and against the human proteome Ensembl database (release 82) (BLASTX). Only hits with E-values < 0.001 were kept. Peptide prediction was performed using Transdecoder [− 6, annotation cut-off: 55, GO weight: 5). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was based on best BLASTX and PFAM search results.Bioinformatic analysis was performed on RNA sequencing data from European seabass anterior intestine to study their physiological and molecular responses to dietary hydrolysate inclusion in a low fish meal diet. We tested 5 dietary conditions on the transcriptomic response of sea bass intestine: FM5 and FM20 diets as negative and positive control groups, respectively, and the SH, TH and MH hydrolysate-based diets as experimental groups. The whole raw dataset was filtered and trimmed using Trimmomatic v(0.30) , using ty 2.1.1. , a dedicy 2.1.1. , and esty 2.1.1.  to get ty 2.1.1.  and the ribed in . Sequencsdecoder . Similarsdecoder . Transmesdecoder . Proteinsdecoder . FinallyP-values < 0.2 and absolute fold changes ≥1.4 were considered as differentially expressed. The relatively low stringency of the cut-off criteria is consistent with other nutrigenomic studies [Differential gene expression between dietary treatments was identified using R software  and the  studies . The impP < 0.05. For GO term comparison with Wego tool, Pearson Chi-Square tests were performed to show significant differences between diets (P < 0.05) [Final BW, SL and K results were expressed as mean ± standard error of the mean (SEM) from all fish from each treatment. SGR, intestinal villi height, number of goblet cells, proximal composition and survival rates were expressed as mean ± standard error of the mean (SEM) from the value of each replicate tank. All data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test . Differences were considered significant at  < 0.05) .P < 0.05). However, diets including protein hydrolysates  significantly improved growth performances as compared to FM5 (P < 0.05) and allowed for similar growth performances as in sea bass fed the FM20 diet. The condition factor (K) and the feed intake were not affected by dietary FM reduction or dietary hydrolysate inclusion (P > 0.05). Even if FCR values were slightly improved in fish fed dietary hydrolysates compared to negative control (FM5), the only statistical difference was between the negative and the positive controls (P < 0.05). Dietary treatments did not impact fish survival (P > 0.05).Sea bass and feed performances are summarized in Table P < 0.05). Inclusion of protein hydrolysates in the FM5 diet significantly increased intestinal villi height to reach values close to or even higher than in sea bass fed the FM20 diet (P < 0.05). The response of intestinal globlet cell density to dietary FM reduction and protein hydrolysate supplementation showed the same trend as intestinal villi height.Figure http://seabass.mpipz.de/DOWNLOADS/) and on the de novo assembled transcriptome from all samples, corresponding to 36 experimental conditions (6 diets * 3 tissues * 2 fish conditions) realized in triplicate.The whole European seabass transcriptome project includes 108 Illumina libraries corresponding to the anterior intestine, but also the liver and kidney for each diet, for a total of 832,132,824 paired-end reads of 100 bp. Table Mapping scores were as follows: 83.55% for the mRNA extracted from the reference seabass genome annotation, and 92.77% for the de novo assembled transcriptome of the anterior intestine. In addition, the numbers of DE genes and annotations were higher with the de novo assembled transcriptome of the anterior intestine. Further differential expression analyses were then conducted on the global de novo transcriptome, composed of 56,246 transcripts after filtering with FPKM < 1 and isoform < 1%. The transcriptome corresponding to the total expression in the anterior intestinal mucosa was composed of 34,174 transcripts. More precisely, 99% of total expression was present in 49,797 for the whole project and in 10,003 transcripts for the anterior intestinal mucosa respectively.Differential gene expression analysis of RNA-seq data was performed to compare diets containing hydrolysates included to low (FM5) and high FM20) fish meal diets. A total of 383 unique genes were differentially expressed . In particular, under the TH diet, the level of GO term annotation was higher for the following categories: macromolecular complex, non-membrane-bounded organelle, intracellular non-membrane-bounded organelle, hydrolase activity, and ribonucleotide binding. In contrast, under the SH diet, the differentially regulated genes belonged mostly to the immune response and response to stimulus. Finally, under the MH diet, lipid metabolism-related GO terms were over-represented.GO term analysis of the genes regulated by hydrolysate-supplemented diets is presented in Fig. Based on GO term annotations, 74 differentially expressed transcripts were linked to nutrition as compared to the FM5 diet: 15 transcripts related to digestion, 15 to carbohydrates, 7 to starvation, and 37 to carriers (Table Table Transcripts related to fish immunity and/or the stress response are presented in Table https://string-db.org/cgi/input.pl?) to reveal relationships between regulated genes and a defined enrichment . The g6pc, aldob, eif2ak4, ace, apob and hba1, scn2a, junb, fdp5, dhx58 genes were connected to the TH and SH diets, respectively. Under the MH diet, many more regulated genes were connected (87), with the ACLY gene at the center of the gene network. The enrichment scores calculated for biological processes, molecular functions, cellular component and KEGG pathways are shown in Table We conducted various analyses to study the metabolic pathways impacted by dietary hydrolysates. Firstly, we performed a string analysis  goblet cells regulate proteins or peptides as well as ion and fluid transport, and also provide an effective immune barrier against potentially pathogenic gut bacteria [Feed intake was not modified by diets as fish were fed in excess to guarantee a high consumption. Feed conversion ratio improvement of fish fed the FM20 diet compared to the FM5 diet could be the result of a better nutritional balance of the diet combined to a better feed consumption. Dietary inclusion of hydrolysate allowed to recover a good feed intake as well as to improve enough feed efficiency to reach fish and feed performance very closed to the positive control. The high degree of protein hydrolysis of our hydrolysates , 40, resbacteria , 76. Furbacteria . In the bacteria , 78–80 abacteria , 39 in fThe intestine is a complex organ because many metabolic interactions take place inside it . We analThe different transcriptomic data analyses evidenced a few discrepancies in sea bass responses depending on dietary hydrolysate origin Tables  and 9. TP-adj < 0.2). Hence, the low number of regulated genes did not allow us to highlight any group of genes to explain the phenotypic differences observed between dietary treatments at a transcriptomic level.String analysis did not evidence any GO term or metabolic enrichment in diets containing hydrolysates of a specific origin (shrimp or tilapia). This result is surprising given the marked difference in growth responses between sea bass fed the FM5 diet and sea bass fed diets containing hydrolysates, as well as the positive response of intestine histological organization in sea bass fed the SH diet. For the SH diet, this may be due to the fact that sea bass fed the FM5 diet did not show any histological signs of severe enteritis or inflammation of the intestinal mucosa; besides, there was no change at a molecular level. Enteritis induces the regulation of a large number of genes  as already shown in a study on the replacement of FM by soybean meal in Atlantic salmon . In our On the contrary, in sea bass fed the diet containing the mixed hydrolysate (MH diet), the much larger number of differentially regulated genes (270 as compared to the FM5 diet) made it possible to carry out an enrichment analysis with String both in GO terms and in metabolic pathways. The analysis showed that the lipid metabolism was strongly impacted, and acetyl-CoA appeared to play a central role in this process. Interestingly, the MH diet decreased cholesterol and long-chain fatty acid metabolism , regardless of the included hydrolysate. Compared to the FM20 diet, the number of differentially regulated genes was very low for all hydrolysate diets, with 6, 8, and 6 genes for the TH, SH, and MH diets, respectively  restored European seabass performances to a similar high level to those recorded with the high FM diet (FM20). In addition, this study demonstrates that the raw material used to manufacture protein hydrolysate is an important determinant in the transcriptomic response of fish.Although the hydrolysates promoted the same zootechnical performances, the transcriptomic response of the intestine showed that genes and metabolic pathways were modulated in a hydrolysate-dependent manner. Moreover, including a combination of tilapia and shrimp hydrolysates into European seabass feed regulated a higher number of genes than the independent hydrolysates. It would be interesting to assess hydrolysate performances in fish grown under more challenging conditions  to check how these differences in transcriptomic profiles are translated into phenotypic responses. These results offer an interesting scenario for the formulation of new high-performance feeds with a low level of FM inclusion. The analysis of the transcriptomic responses of liver and kidney sampled in the present study will allow us to complete this work and to better understand how metabolic pathways are modulated by the replacement of FM by protein hydrolysates in European seabass.Additional file 1:Diet formulation and proximal composition. FM5, 5% fish meal diet; FM20, 20% fish meal diet; TH, tilapia hydrolysate diet; SH, shrimp hydrolysate diet; MH, mixed hydrolysate diet. (XLSX 10 kb)Additional file 2:Global list and fold changes of differentially expressed genes versus FM5. Values correspond to fold changes between diets and the low fish meal control diet. Positive values indicate upregulation, and negative values down regulation. FM5, 5% fish meal diet; FM20, 20% fish meal diet; TH, tilapia hydrolysate diet; SH, shrimp hydrolysate diet; MH, mixed hydrolysate diet. Values with “*” correspond to fold change means of isoforms. If isoforms were at the same time up and down regulated each values appear. (XLSX 37 kb)Additional file 3:Global list and fold changes of differentially expressed genes versus FM20. Values correspond to fold changes between hydrolysate diets and the high fish meal control diet. Positive values indicate upregulation, and negative values down regulation. (XLSX 10 kb)Additional file 4:Homo sapiens background. Only connected genes are presented. A. relation between the regulated genes by shrimp hydrolysate diet. B. relation between the regulated genes by tilapia hydrolysate diet. C. relation between the regulated genes by mixed hydrolysate diet. (TIF 2612 kb)Relationship between regulated genes by hydrolysate diets. Differentially expressed gene compared to low fish meal diet have been tested against Additional file 5:KEGG analysis: list of genes differentially expressed by hydrolysate and high fish meal diets compared to low fish meal diet. (XLSX 63 kb)Additional file 6:IPA analysis: list of main pathways impacted by hydrolysate and high fish meal diets compared to low fish meal diet. (XLSX 57 kb)


P < 0.05). Kaplan–Meier analysis showed that the patients with IMN+IgAN had significantly higher cumulative incidence rates of partial or complete remission . Multivariate Cox model analysis revealed that old age at biopsy and high baseline serum creatinine and uric acid levels were significantly associated with poor prognosis , and increased IgA expression correlated significantly with PR or CR (P < 0.05). The present study found that overlapping IMN and IgAN presents with unique renal histopathology and appears not to cause a poorer prognosis than IMN.Overlapping idiopathic membranous nephropathy (IMN) and immunoglobulin A nephropathy (IgAN) is rare. This study aims to investigate the unique prognostic, clinical, and renal histopathological characteristics of IMN+IgAN. This retrospective observational study included 73 consecutive cases of IMN+IgAN and 425 cases of IMN treated between September 2006 and November 2015. Prognostic and baseline clinical and histopathological data were compared between the two patient groups. Poor prognostic events included a permanent 50% reduction in eGFR, end-stage renal disease, and all-cause mortality. Renal histopathology demonstrated that the patients with IMN+IgAN presented with significantly increased mesangial cell proliferation and matrix expansion, increased inflammatory cell infiltration, and higher proportions of arteriole hyalinosis and lesions than the patients with IMN (all  Approximately 75% of patients with MN have idiopathic or primary membranous nephropathy (IMN), and the remaining 25% of cases are associated with various secondary causes6. The incidence of MN peaks in the fourth and fifth decades of life and appears higher in men than in women (men: women = 2-3:1)5. The exact etiology of MN remains unclear, and the disease course varies substantially. Approximately one-third of patients with MN experience spontaneous remission of proteinuria; another one-third develop persistent proteinuria; and the remaining one-third progress to end-stage renal disease (ESRD)9.Membranous nephropathy (MN), an autoimmune glomerular disease with a characteristic renal histopathology of immunoglobulin deposition along the extracapillary side of the glomerular basement membrane, is one of the most common causes for nephrotic syndrome in adults11. Although both IMN and IgAN are common primary glomerular diseases, coexisting MN and IgAN in adults is very rare14. Because of the low incidence, reports on overlapping MN and IgAN are limited to sparse case reports focusing on histopathological features15. In this study, we retrospectively reviewed 73 cases of overlapping IMN and IgAN and compared the renal histopathology of IMN only versus overlapping IMN and IgAN. In addition, we further examined the follow-up clinical data and compared the prognosis and outcomes of overlapping IMN and IgAN versus IMN only.Immunoglobulin A nephropathy (IgAN), also known as Berger’s disease, is the most common primary glomerulonephritis, which is characterized by dominant or codominant IgA mesangial depositionThe study was carried out in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of The First Affiliated Hospital of Wenzhou Medical University . All the study participants signed the informed consent form.16 and was determined by a careful examination of kidney biopsies using light, immunofluorescence, and electron microscopy and an exclusion of secondary causes for MN. The pathology of the patients’ renal biopsies was characterized by (1) thickened glomerular capillary walls revealed by hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), periodic acid silver methenamine (PASM), and Masson trichrome (Masson) staining; (2) positive IgG and C3 immunofluorescence staining along the capillary walls; and (3) subepithelial deposits and fusion in podocyte structure revealed by electron microscopy16. At the initial diagnosis, 523 patients with MN did not present with secondary causes for MN, such as malignancy, systemic lupus erythematosus, hepatitis B or C, diabetic glomerulosclerosis, adverse responses to medications, or renal damage from toxic agents. The authors had access to information that could identify individual participants during or after data collection. However, during follow-up examinations, 17 of the 523 patients were found to have a secondary cause for MN, including 9 cases of cancer and 8 cases of systemic lupus erythematosus. Thus, 506 patients with IMN were analyzed. The flow chart of patient selection is displayed in Fig. 2), course of disease, and comorbidities such as diabetes, hypertension, infections, embolism, edema, nephrotic syndrome, and microscopic hematuria were collected.A total of 523 patients with IMN were identified from 4694 consecutive patients who underwent renal biopsies and were treated in the Department of Nephrology of The First Affiliated Hospital of Wenzhou Medical University between September 11, 2006 and November 25, 2015. The diagnosis of IMN followed the 2012 KDIGO Clinical Practice Guideline for GlomerulonephritisFormalin-fixed paraffin-embedded biopsy tissue specimens were sectioned into 3-μm-thick sections. The tissue sections were stained with H&E, PAS, PASM, and Masson according to the standard protocols. Based on these stains, the extent of mesangial cell proliferation and matrix expansion was graded as 0, 1, 2, and 3, representing negative, mild, moderate, or strong, respectively. In addition, the extent of inflammatory cell infiltration, interstitial fibrosis, tubular atrophy, and renal interstitial lesions were evaluated semi-quantitatively according to a scale 0 to 3, representing very small , small , medium , and large affected area . Two pathologists scored the stained sections, and the average score was used. Other histopathological characteristics, including segmental sclerosis, crescent, arteriole hyperplasia, arteriole hyalinosis, and arteriole lesions, were also evaluated.The expression of IgG, IgA, IgM, C3, C4, C1q, and fibrinogen was determined by fluorescence immunohistochemical (IHC) staining. Frozen tissue sections (5 μm) were incubated with the following primary antibodies: anti-human IgG (1:50), IgA (1:50), IgM (1:50), C3 (1:50), C4 (1:50), C1q (1:90), and fibrinogen (1:90) at 4 °C overnight. All the antibodies were purchased from Gene Tech Company . The tissue sections were washed with PBS buffer and then incubated with FITC-labeled secondary antibodies  at room temperature for 45 minutes. The stained slides were observed under an Olympus BX41 fluorescence microscope . Images (200x magnification) were captured using the digital camera attached to the microscope. The intensity of the staining signals was scored as 0, 1, 2, 3, and 4, representing very weak, weak, moderate, strong, and very strong, respectively. IgA positive staining was defined as an intensity score ≥2. The staining intensity was scored by two pathologists, and the average score was used.17.Urine protein quantification was determined using a 24-hour collection. Nephrotic syndrome was defined as proteinuria ≥3.5 g/d and hypoalbuminemia <30 g/L. Subnephrotic status was defined as >0.5 g proteinuria and <3.5 g per day. Proteinuria was graded as stage 1, 2, 3, and 4 when proteinuria was <3.5 g/d, 3.5–8 g/d, 8–15 g/d, and 15 g/d, respectively. Proteinuria and microscopic hematuria were evaluated by urinary sediment examination. The severity of proteinuria and microscopic hematuria was graded 0–4 with 0 representing the mildest and 4 representing the most severe stage. The estimated glomerular filtration rate (eGFR) was calculated according to the abbreviated Modification of Diet in Renal Disease (MDRD) study formula and serum creatinine levelsEpon-embedded biopsy tissue specimens were cut into ultrathin sections. The sections were fixed in 2.5% glutaraldehyde and then in 1% osmium tetroxide. The tissue sections were stained with uranyl acetate and lead citrate and examined under a Hitachi H7500 electron microscope . The ultrastructural stage of MN was determined according to the Ehrenreich and Churg four-stage classification.18. ESRD was defined as a permanent drop in eGFR to <15 mL/min/1.73 m2 requiring dialysis or kidney transplantation.The following outcome and prognostic parameters were recorded and used for statistical analysis: development of end-stage renal disease (ESRD), all-cause mortality, a permanent 50% reduction in eGFR compared with the baseline value, complete remission (CR), and partial remission (PR). CR was defined as urinary protein excretion <0.3 g/d (uPCR <300 mg/g or <30 mg/mmol) accompanied by a normal serum albumin concentration and a normal serum creatinine. PR was defined as urinary protein excretion <3.5 g/d (uPCR <3500 mg/g or <350 mg/mmol), a ≥50% reduction from peak values, an improvement or normalization of the serum albumin concentration, and stable serum creatininet-test and the chi-square test were used for two-group comparisons of continuous variables and categorical variables, respectively. A poor event-free renal survival curve was prepared using the Kaplan-Meier method. Cumulative hazard curves were plotted for CR and PR. Poor event-free renal survival and incidence rates of CR or PR in different groups were compared using the log-rank test. Univariate and multivariate Cox regression analyses were performed to analyze the correlation between the clinical parameters and outcomes. For all variables, the proportional hazard assumption was tested and found to be fulfilled. The statistical analysis software Medcalc version 15.6 was used for the survival analysis, and SPSS version 23 was used for Cox regressions and all other statistical analyses. The P value was 2-sided, and P < 0.05 was considered statistically significant.Data are presented as the mean ± standard deviation (SD). Student’s A total of 506 patients with idiopathic MN identified from the consecutive renal biopsy cases from September 11, 2006 to November 25, 2015 at The First Affiliate Hospital of Wenzhou Medical University were included in this retrospective study. Most patients were treated with RAS receptor inhibitors: angiotensin-converting enzyme inhibitors (ACEis) or angiotensin receptor blockers (ARBs), glucocorticoids, cyclophosphamide, calcineurin inhibitors (CNIs) including tacrolimus and cyclosporine and other immunosuppressors. The usual doses of drugs were as follow: (1) RAS receptor inhibitors: double dosage given orally daily; (2) glucocorticoids plus cyclophosphamide: full oral dosage (1 mg/kg/d) of glucocorticoids per day and i.v. cyclophosphamide 1 g/month until reaching an accumulated dose of 6–8 g for 6 months; (3) CNI therapy (tacrolimus or cyclosporine): tacrolimus 0.05–0.075 mg/kg/d given orally in two divided doses 12 hours apart, without prednisone, for 6–12 months or cyclosporine 3.5–5.0 mg/kg/d given orally in two equally divided doses 12 hours apart, with prednisone 0.15 mg/kg/d, for 6 months.Because of the use of glucocorticoids and immunosuppressors, 24 patients experienced infections during the follow-up period: 20 infections were respiratory tract infections, particularly pulmonary infections; 1 was a lower limb skin infection; 1 patient had both a respiratory and lower limb skin infection; 1 patient had tuberculotic hydrothorax; 1 patient had Hepatitis E virus infection; 1 patient was infected with neurosyphilis; and 2 patients died because of serious infections. Five patients had embolic events during the follow-up period including 4 cerebral infarctions and 1 mesenteric arterial embolism. One patient suffered a subarachnoid hemorrhage and 1 patient had an upper gastrointestinal hemorrhage secondary to a duodenal ulcer. Four patients had repetitive renal biopsies. One patient’s twin brother also had nephrotic syndrome. Two patients had renal biopsies done during pregnancy.Of the 506 patients with IMN, 8 were excluded because of missing fluorescent IHC data. There were 73 cases of IgA+ and 425 cases of IgA− in the remaining 498 patients. Overall, 18 patients in the IgA+ group and 138 patients in the IgA− group did not have a follow-up examination after 6 months. Thus, 55 cases of IgA+ and 287 cases of IgA− were included in the outcome and prognosis analyses. The patient selection flow chart is displayed in Fig. P = 0.011, Table Demographics including gender distribution, age at disease onset, and BMI were similar in the IgA+ and IgA− groups Table . The proP = 0.003, Table P < 0.05, Table P = 0.009, Table P = 0.006) and lesions  and PAS staining . Notably, the K-M analysis also revealed that the one, two, and three-year cumulative incidence rates of PR or CR were 25.6%, 67.7%, and 87.2%, respectively, in patients with IgA+, whereas the rates were 17.3%, 47.0%, and 66.4%, respectively in patients with IgA− .To compare the prognostic disease characteristics of the two patient groups, we performed Kaplan-Meier (K-M) analyses to investigate the cumulative poor event-free renal survival and cumulative incidence rates of PR or CR. Patients with longer than 6 months of follow-up duration, including 55 cases of IgA+ and 287 cases of IgA−, were included for the K-M analyses. A poor event was defined as an occurrence of a 50% reduction in eGFR, ESRD, or all-cause mortality. The one-year and 4-year cumulative poor event-free renal survival rates were 100.0% and 47.4%, respectively, in patients with IgA+, and they were 96.9% and 78%, respectively, in patients with IgA− Fig. . AlthouggA− Fig. . The patP = 0.0002) , but good prognoses among the different treatment groups were negative  Fig. . The difive Fig. .Figure 5P = 0.517) as a whole: 3 (5.5%) versus 13 (4.5%) between the IgA+ and IgA− groups (P = 0.758) without treatment; 22 (40.0%) versus 96 (33.1%) between the IgA+ and IgA− groups with RAS receptor inhibitor treatment (P = 0.323); 13 (23.6%) versus 58 (20.0%) between the IgA+ and IgA− groups (P = 0.541) receiving treatment with GC + CTXt; and 14 (25.5%) versus 101 (34.8%) between the IgA+ and IgA− groups (P = 0.176) under CNI treatment  without treatment: general treatment without RAS inhibitors or glucocorticoids or immunosuppressors; (2) RAS receptor inhibitors; (3) glucocorticoids plus cyclophosphamide (GC + CTX); and 4) CNI therapy (tacrolimus or cyclosporine). There were no treatment differences between the IgA+ and IgA− groups  and IMN alone (425 cases), which compared the prognostic, clinical, and histopathological characteristics of the two patient groups. Similar to the findings in the current study, the previous reports have also shown that IgG deposits are mainly in the capillary walls, while IgA deposits are predominantly in the mesangial area in the renal pathology of patients with overlapping MN and IgAN19.Overlapping MN and IgAN is considered to be a rare medical condition. The majority of previous studies on this rare condition were reports of a single case or a case series and focused on a description of the renal pathologyIn the current study, we found similar baseline clinical features in the overlapping IMN and IgAN groups and the IMN only group. Although the current study showed a statistically significantly higher pathological stage in the IgA+ group than in the IgA− group, the increment was only approximately 14% (1.88 vs. 1.65) and therefore may represent a minimal clinical significance.To the best of our knowledge, the current study is the first comprehensive comparison of the renal pathological features of overlapping IMN and IgAN versus IMN only and to define the unique renal histopathology associated with overlapping IMN and IgAN. Compared with the IMN only group, the renal histopathology of the overlapping IMN and IgAN groups not only exhibited significantly increased mesangial cell proliferation and matrix expansion, which are characteristic of the renal histopathology of IgAN but also showed increased inflammatory cell infiltration and higher proportions of arteriole hyalinosis and lesions. However, the proportion of segmental sclerosis was significantly lower in the overlapping IMN and IgAN group compared with the IMN alone group.20. Although the cumulative poor event-free renal survival rate was lower in the patients with overlapping IMN and IgAN than in the patients with IMN alone, the difference was not statistically significant. A previous case series study showed a similar renal prognosis in overlapping MN and IgAN versus MN only12. Stokes et al. also indicated that the combination of MN and IgAN may not lead to a poorer clinical outcome compared with MN alone12. Similarly, Nishida et al. reported a pediatric case of coexisting MN and IgAN and suggested that overlapping MN and IgAN might not lead to deleterious outcomes21. The physiological and cellular mechanisms underlying the overlapping IMN and IgAN-associated increase in disease remission remains unclear.The lower chance of segmental sclerosis in the patients with overlapping IMN and IgAN may explain the high incidence of PR or CR in these patients. In the current study, we performed K-M analysis to discover that the one, two, and three-year cumulative incidence rates of PR or CR were significantly higher in the patients with overlapping IMN and IgAN than in the patients with IMN only. Consistently, the multivariate Cox model analysis also revealed that increased IgA expression was significantly associated with the incidence of PR or CR. PR and CR have been recommended as surrogate endpoints to evaluate the outcomes of patients with IMNTo avoid the effects of different treatments between overlapping IMN and IgAN and IMN only, we took treatment into account. We found that there was a better prognosis with drug treatment than without treatment, and the prognosis rank differed between different drug treatments.16. The proportions of patients with comorbidities, such as edema, hypertension, diabetes, infection, embolism, nephrotic syndrome, and/or microscopic hematuria, were similar between the IgA+ and IgA− groups. In other words, the clinical symptoms were comparable between the IgA+ and IgA− groups, and therefore the treatments were also comparable between the IgA+ and IgA− groups. Therefore, we can evaluate IgA’s deposition independent of the different treatments.However, despite the different drug treatments, poor prognosis displayed no significant statistical difference between overlapping IMN and IgAN and IMN only. We speculate that the reason there is no difference is because the different treatments in our research were chosen according to the clinical symptoms, such as persistent nephrotic syndrome, rather than pathological features such as the presence or absence of IgA deposition, which is also recommended by the KDIGO clinical practice guidelines for glomerulonephritis of IMN19. Miyazaki et al. reported a case of the development of IgAN 14 years after the diagnosis of MN22. Genetic factors may contribute to the development of overlapping IgAN and MN. Familial overlapping of IgAN and MN has been reported previously24.The etiology of overlapping IMN and IgAN is unlikely to be due to chance alone. IMN and IgAN could develop simultaneously, or either IgAN or IMN could be the primary event and subsequently stimulate the other nephropathy26. The correlation between high interstitial fibrosis and the incidence of PR or CR may be explained by the interstitial fibrosis-mediated nonspecific reduction in urine secretion. We speculate that increased interstitial fibrosis may result in the blockage of capillary loops and the reduction in urinary protein excretion, therefore leading to a false sense of a better prognosis.The current study also found that increased age at biopsy and high baseline SCr and serum UA levels significantly increased the risk of a 50% reduction in eGFR, ESRD, or all-cause mortality. Similarly, recent studies have also demonstrated that persistent nephrotic range proteinuria and elevated creatinine levels are associated with poor outcomes in patients with IMNThe main limitation of the current study is that the follow-up duration of patients varied greatly, which results in big variations in the renal survival analysis. We are currently continuing to follow up with the patients and will further investigate the prognostic features of overlapping IMN and IgAN. The M-type phospholipase A2 receptor (PLA2R) has mostly been studied in IMN since 2009. However, because our patient selection occurred between September 11, 2006 and November 25, 2015 in this research and the commercial detection of PLA2R in the clinic was not mature during this time in our district, PLA2R was not a consideration in our study.This retrospective observational study found that, in addition to presenting the IgAN-characteristic renal histopathology, such as significantly increased mesangial cell proliferation and matrix expansion, patients with overlapping IMN and IgAN also exhibited unique features, such as increased inflammatory cell infiltration, higher proportions of arteriole hyalinosis and lesions, but lower proportions of segmental sclerosis compared with patients with IMN only. In addition, overlapping IMN and IgAN appears to correlate with an increased incidence of disease remission and thus may not lead to a poorer prognosis compared with IMN only.Supplement infromation