Vibrio parahaemolyticus is the main pathogen of food-borne diarrheal in coastal areas. Through the study of pathogen characteristics of 1870 V. parahaemolyticus, the isolation rate of O4:KUT had increased significantly since 2013. In this study, we analyzed virulence, antimicrobial susceptibility, molecular, and epidemiological characteristics of a new serotype named O4:KUT2. O4:KUT2 strains had tlh+tdh+trh–toxRS/new– characteristics and were prevalent during 2013–2015. The 91.5% O4:KUT2 serotype strains were resistant to ampicillin. The growth curves of O4:KUT2 strains were different with O4:K9, O4:K8, and O3:K6 serotype strains. O4:KUT2 strains belonged to ST332 where four strains had a large fragment inserted at recA. Compared the whole genomes of O4:KUT2 strains with O4:K9 strain which also belonged to ST322 isolated from acute diarrhea patients in Zhejiang province in 2012, no different alleles at 2249 loci was found. This finding implied that O4:KUT2 strains might be derived from O4:K9 strains. Clinical presentation of patients positive for V. parahaemolyticus O4:KUT2 were no significant difference with patients positive for O3:K6, although their genetic characteristics were different. The appearance and the increase of proportion of the new serotype O4:KUT2 strains was aware that we should not relax the monitoring of the pathogen spectrum of acute diarrheal patients. Vibrio parahaemolyticus is the main pathogen of food-borne diseases in coastal areas   . The emeV. parahaemolyticus had changed significantly in the composition of serotypes since 2013. The detection rate of O4:KUT serotype had exceeded O3:K6 and became the highest in 2013. In the following years, the rate of O4:KUT remained high. To find out whether O4:KUT is one serotype or contains multiple serotypes, we selected five O4:KUT strains per region per year to sequence their whole genome. According to the analysis of K antigen gene clusters sequences between gmhD and rig  in six general hospitals and one children’s hospital from 2009 to 2017 in Zhejiang province  according to the instructions  culture medium after alkaline peptone water (APW) enrichment to isolate microorganisms. ructions .Vibrio parahaemolyticus strains were serotyped using agglutination tests with 11 O (lipopolysaccharide) and 65 K (capsule) antisera  according to the instructions provided with the reagents. Serotype was defined as a unique combination of O and K. OUT reffered to O untypeable while KUT referred to K untypeable. KUT strains were also test by K72, K73, K74, and K75 antisera .V. parahaemolyticus was extracted by the Yeast/Bact Kit B  and randomly broken by ultrasonic crusher  at length of 350 bp. The whole library was prepared by terminal repair, addition of A-tail, addition of sequencing connector, purification, PCR amplification and HiSeq PE150 sequencing  with a minimum coverage of 120-fold. The sequencing results were assembled by SOAPdenovo software (Version 2.04) and then predicted by GeneMarkS (Version 4.17). Gene Ontology (Version: May 2017), Cluster of Orthologous Group of proteins (COG), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Version: 2016) were used for annotating gene function.Genomic DNA of JACBKD000000000, JACBKE000000000, JACBKF000000000, JACBKG000000000, JACBKH000000000, JACBKI000000000, JACBKJ000000000, JACBKK000000000, JACBKL000000000, and JACBKM000000000.Accession numbers of Whole-genome sequencing (WGS) in NCBI from this study were 2O. Polymerase chain reaction (PCR) were as follows: denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and extension at 72°C for 10 min in ABI Veriti thermal cycler.Primers for K antigen gene cluster specific sequences of KUT2 were designed to identify O4:KUT2 strains from O4:KUT which were listed in 1  were selected for PCR amplification and sequencing. Core genome multilocus sequence typing (cgMLST) including 2254 loci were selected for whole genome analysis (Multilocus sequence typing (MLST) was performed according to the protocols published on the website1 . Seven hanalysis .tlh), hemolysin genes (tdh and trh), orf8 and toxRS/new (GS-PCR). The primers were listed in 2O. The PCR reaction parameters were as following: denaturation at 94°C for 5 min, circulation by 30 cycles with 94°C for 30 s, each gene annealing temperature (vscP), VP1686 (copS), VP1689 (vscK), and VP1694 (vscF), the T3SS2α genes VP1362 (vopB2), VP1339 (vscC2), VP1335 (vscS2), and VP1327 (vopT), and the T3SS2β genes  were detected on 2% agarose gel electrophoresis after four-multiplex PCR  were performed as described previously . StrainsA phylogenetic tree was created by genomes of 16 O4:KUT2 strains and 1 O4:K9 strain sequenced in this study and 842 genomes downloaded from NCBI . The MUSThis study was approved by the Ethics Committees of The First Affiliated Hospital, School of Medicine, Zhejiang University. All participants provided written informed consent.wzz and gmd were different  of V. parahaemolyticus were O4:KUT2 serotype, while 24.5% (49/200) were O3:K6 serotype  and amikacin (17%). The sensitivity rate to piperacillin, ceftazidime, cefotaxime, cefoxitin, gentamicin, ciprofloxacin, and chloramphenicol were 97.5, 99, 93, 98, 99, 98, and 99.5%, respectively.The growth curves of O4:KUT2 strains were different with O4:K9, O4:K8, and O3:K6 serotype strains incubated at 37°C in 3% NaCl of BHI broth with pH 7.4. Under the same nutrition condition, the growth restriction of O4:KUT2 strains affected by the concentration was smaller than O4:K9, O4:K8, and O3:K6 serotype strains, while the concentration of bacteria entering the stable period was higher .recA141 was found which was the allele gene of recA in ST332 at nucleotide position 497, and the inserted genes contained a recA276 allele gene. We named this strain “O4:KUT2-recAin-1.” Two other insertions were detected at the same positon at recA141 by whole genome sequencing of sixteen O4:KUT2 strains , named “O4:KUT2-recAin-2” and “O4:KUT2-recAin-3,” respectively. In the 16 sequenced O4:KUT2 strains, there were one O4:KUT2-recAin-2 strain isolated from Jiangsu province in 2014 and one O4:KUT2-recAin-3 strains isolated from Zhejiang province in 2017, while there were two O4:KUT2-recAin-1, one isolated from Jiangsu province in 2015 and one isolated from Zhejiang province in 2017  in 2015  was also found an insertion at the recA locus  and two O4:KUT2-recAin-3 (HZ17-125 and HZ17-129) starins from diarrheal patients in Hangzhou in 2017. In addition, two strains isolated from marine organisms had the same insertion as O4:KUT2-recAin-3 at recA, but their serotypes were O5:K15  and O4:K42 , not O4:KUT2. No identical insertion was found in other sequenced strains of Vibrionaceae family. Multilocus sequence typing analysis of S020 strain and GIMxtf283-2012.12 strain showed that they were different from O4:KUT2-recAin-3 strains. So we could conclude that O4:KUT2-recAin-3 was not serotype transformed by S020 or GIMxtf283-2012.12 strains.In recA were independent insert events. Further studies will be focused on the molecular mechanism of serum transformation and insertion.Therefore, we speculated that the emergence of O4:KUT2 was the result of seroconversion of O4:K9 and the large fragment insertions at the toxRS sequence detectable by GS-PCR. Although the clinical symptoms of O4:KUT2 patients were lighter than those of O3: K6 group .The datasets generated for this study can be found in the NCBI under Bioproject number The studies involving human participants were reviewed and approved by the Ethics Committees of The First Affiliated Hospital, School of Medicine, Zhejiang University. The patients/participants provided their written informed consent to participate in this study.QZ designed the assays, analyzed the data, and wrote the manuscript. XC designed the study and collected the strains. YL and RW collected the strains, serotyped the strains, detected the virulence-associated genes, and did antimicrobial susceptibility testing. JC collected the strains. YC designed the study. All authors contributed to the article and approved the submitted version.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.


Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common X-linked inherited erythroenzymopathy in Thailand. The clinical and hematological manifestations of G6PD deficiency are variable. Material and Method. A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Mutation analysis was performed by direct DNA sequencing of all coding exons of the G6PD gene. Ninety-one patients (89.2%) were presented with neonatal hyperbilirubinemia and 11 patients (10.8%) were presented with acute hemolytic anemia beyond the neonatal period.  This study aimed to characterize the genotype-phenotype correlation of G6PD mutations in Thai pediatric patients who were followed-up in Phramongkutklao Hospital, a tertiary center in central Thailand.  Molecular analysis of the G6PD gene in 102 G6PD-deficient Thai children identified 12 different mutations. G6PD Viangchan (871G > A) and G6PD Canton (1376G > T) were the first (46.2%) and the second (15.4%) most common identified mutations among both male and female G6PD-deficient individuals, respectively. All affected males were hemizygous for G6PD mutations and had an average G6PD level of 16.7 ± 11.5 (3–76) IU/ml.RBC. Majority of female patients  were heterozygous for G6PD mutations and had an average G6PD level of 133.6 ± 43.4 (9–195) IU/ml.RBC. Two female patients (6.9%) were either homozygous or compound heterozygous for the mutations and had G6PD level in the affected male range (35 and 10 IU/ml.RBC). Only 1 in 27 heterozygous females (3.7%) had G6PD level in the affected male range (9 IU/ml.RBC) which is possibly explained by nonrandom X-chromosome inactivation. The correlation of genotypes, G6PD levels, and clinical phenotypes was not demonstrated in our study in which all of the included G6PD-deficient patients were presented with neonatal hyperbilirubinemia and acute hemolytic anemia, since the genotype-phenotype correlation is normally demonstrated in chronic nonspherocytic hemolytic anemia (CNSHA) G6PD-deficient individuals.  This study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency in Thai children. Our study demonstrated the efficiency of direct DNA sequencing which can identify 12 missense mutations in Thai children. Glucose-6-phosphate dehydrogenase (G6PD) deficiency OMIM 300908) is the most common X-linked inherited erythroenzymopathy in Thailand. The prevalence of G6PD deficiency in Thai populations was 3 to 18% , 3. The 00908 is The clinical and hematologic manifestations of G6PD deficiency vary; in most cases, neonatal hyperbilirubinemia and acute hemolytic anemia precipitated by exogenous oxidative stress such as infections, drugs, and fava beans (favism) are principle clinical presentations. In Thailand, most children affected with G6PD deficiency presented with neonatal hyperbilirubinemia . As for In this study, the genotyping of the G6PD gene and the genotype-phenotype correlation among 102 pediatric patients affected with G6PD deficiency was characterized in Phramongkutklao Hospital, a tertiary care center in central Thailand.To determine the molecular characterization of the G6PD gene, 102 pediatric patients with G6PD deficiency including 73 males (71.6%) and 29 females (28.4%) who were admitted to Pediatrics Department, Phramongkutklao Hospital, between June 2017 and May 2018 were enrolled in our study. The ages ranged from 1 day to 13 years. Clinical manifestations of our patients included neonatal hyperbilirubinemia and acute hemolysis related to oxidative stress. Chronic nonspherocytic hemolytic anemia associated with G6PD deficiency was not identified in this study. The study protocol was approved by the Institutional Review Board of Royal Thai Army, Thailand. The consent was obtained by the study participants prior to study commencement.µL PCR reaction mixture contained 100–200 ng genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 0.25 µM of each primer, and 0.2 unit Taq DNA polymerase . The PCR products were visualized on 1% agarose gel electrophoresis. All PCR products were purified and directly sequenced in both directions. The reference sequences were NM_001360016.1 and NP_001346945.1 for G6PD cDNA and G6PD amino acid positions, respectively.After informed consent was obtained from the patients or parents, a total of 102 EDTA blood samples from all individuals were collected. Genomic DNA was extracted from peripheral blood lymphocytes according to the manufacturer's instructions. All 13 coding exons and exon-intron boundaries of the G6PD gene were amplified by PCR using primers as previously described [The activity of G6PD enzyme was performed by the enzyme kinetic method  subsequent to hospital admission and repeated at the steady state approximately 3 months after neonatal jaundice and acute hemolysis. The G6PD enzymatic activity was expressed as international units (IU)/ml.RBC. The normal G6PD enzymatic activity in our laboratory for both males and females was 159–297 and 197–331 IU/ml.RBC, respectively. The G6PD enzymatic activity below the normal range was considered as G6PD deficiency.t-test for data with a parametric distribution. Statistical analysis was performed using IBM SPSS Software, Version 23 , and p value <0.05 was considered to be statistically significant.Baseline values of selected variables were calculated as mean, standard deviation, and range. Continuous variables were compared between the two groups using the unpaired A total of 102 children including 73 males (71.6%) and 29 females (28.4%) were included in our study. Clinical manifestations of our G6PD-deficient children were classified as 91 individuals (89.2%) with neonatal hyperbilirubinemia  and 11 individuals (10.8%) with acute hemolytic anemia  . The majMolecular analysis of the G6PD gene in G6PD-deficient Thai children identified a total of 12 different mutations . Among 1Our study demonstrated the variability of G6PD levels ranging from very low detectable activities to some residual activity . The avep value 0.135) and heterozygous females .Concerning genotype-phenotype correlation study, G6PD level was categorized by each clinical phenotype . In G6PDPlasmodium falciparum. Consequently, the prevalence of G6PD deficiency is high, ranging from 5 to 20% in these regions [G6PD deficiency is seen primarily in populations across the Mediterranean, Africa, Middle East, and Southeast Asia where malaria was endemic, as the deficiency confers selective survival advantage from infection with  regions . In Thai regions . In this regions . Our stu regions , 10 thatTo date, more than 180 mutations are associated with G6PD deficiency , 3, 11. Although novel G6PD mutation was not identified in our study, G6PD Aures and G6PD Valladolid were firstly identified among our Thai children affected with G6PD deficiency. G6PD Aures causing mild G6PD deficiency associated with favism was firstly discovered in Algeria in 1993 . This muConcerning X-linked inheritance, G6PD deficiency occurs more frequently among males than females as in our study for which the majority (71.6%) of patients comprised males . Among hConcerning genotype-phenotype correlation, our data suggest no association between the genotypes, G6PD enzymatic activities, and clinical phenotypes among G6PD-deficient Thai children. The variability of G6PD activity is far from being explained by the type of mutation and is uncorrelated with clinical phenotypes including neonatal jaundice and acute hemolytic anemia which were the only two major clinical features identified in this study. Although G6PD levels remained poorly correlated with the genotype affected with neonatal jaundice and acute hemolytic anemia, it appeared to be better correlated with the genotype presented with chronic nonspherocytic hemolytic anemia (CNSHA) as described in related studies –24. The Although various studies of G6PD mutation have been conducted in Thai populations, DNA sequencing of the G6PD gene was firstly used to comprehensively identify the mutation spectrum among Thai G6PD-deficient children. Polymerase chain reaction and restriction fragment length polymorphisms (PCR-RFLP) have been used to detect common G6PD mutations in Thai populations in central, southern, and northern Thailand , 8, 17. In conclusion, this study characterizes the molecular heterogeneity of G6PD variants causing G6PD deficiency among Thai children. All of the G6PD mutations have been characterized by direct DNA sequencing of G6PD genes. Twelve different G6PD mutations were identified in our study. G6PD Viangchan, G6PD Kaiping, and G6PD Canton were the three most common mutations identified in our study and accounted for more than 75% of our patients. Although genotype-phenotype correlation was not demonstrated in our study, molecular analysis of the G6PD gene remains useful for diagnostic confirmation and carrier detection leading to appropriate genetic counseling for patients and their family members in the future.


IGF2BP2, HMG20A, and HNF1B variants in the susceptibility of Type 2 diabetes mellitus (T2DM), and to identify their association with age, gender, BMI, and smoking and alcohol drinking behavior among the Han Chinese population.Background: The present study aimed to investigate the roles of insulin related gene IGF2BP2, rs7178572 in HMG20A, rs4430796, and rs11651052 in HNF1B were genotyped by using the Agena MassARRAY. Odds ratio (OR) and 95% confidence intervals (CI) were calculated by logistic regression.Methods: About 508 patients with T2DM and 503 healthy controls were enrolled. Rs11927381 and rs7640539 in HMG20A rs7178572  and HNF1B rs11651052  increased the risk of T2DM. Rs7178572, rs4430796, and rs11651052 might be related to the higher T2DM susceptibility not only by itself but also by interacting with age, gender smoking, and alcohol drinking. Rs11927381 also conferred the higher T2DM susceptibility at age ≤ 59 years. Besides, rs7178572-AA (P = 0.032) genotype and rs11651052 GG (P = 0.018) genotype were related to higher glycated hemoglobin and insulin level, respectively.Results: We found that Conclusion: Specifically, we first found that rs11927381, rs7640539, and rs11651052 were associated with risk of T2DM among the Han Chinese population. We also provide evidence that age, gender, BMI, smoking, and drinking status have an interactive effect with these variants on T2DM susceptibility. Type 2 diabetes mellitus (T2DM) is a complex, heterogeneous and chronic metabolic disorder, and is characterized by defects in insulin secretion and/or insulin action leading to hyperglycemia . It is rIGF2BP2, HMG20A, and HNF1B genes might affect the level of insulin and lead to the development of T2DM. IGF2BP2 regulates insulin-like growth factor 2 (IGF2) translation that participates in the growth and insulin signaling pathways [HMG20A is expressed in both human and mouse islets, and the levels of HMG20A are decreased in islets of T2DM donors compared with islets from non-diabetic donors [HNF1B contributes to pancreatic cell formation and controls the specification, growth, and differentiation of the embryonic pancreas [IGF2BP2, HMG20A, and HNF1B polymorphisms and T2DM risk, the study on other polymorphisms in these genes is insufficient [Insulin deficiency is the main characteristic of T2DM. Expression dysregulation of pathways . HMG20A c donors . HNF1B cpancreas . Althougfficient .IGF2BP2 rs11927381 and rs7640539, HMG20A rs7178572, HNF1B rs4430796, and rs11651052 variants with T2DM susceptibility among the Chinese Han population. Given that environment/lifestyle changes can modify the risk of T2DM [In the present study, we aimed to investigate the association of  of T2DM , such asAbout 508 T2DM patients and 503 age- and gender-matched healthy controls were enrolled into the study from the First Affiliated Hospital of Xi’an Jiaotong University and Xizang Minzu University. All recruited subjects were genetically unrelated ethnic Han Chinese. T2DM cases were identified according to 2017 China Guideline for Type 2 Diabetes. T2DM patients were diagnosed as fasting plasma glucose ≥ 7.0 mmol/l and/or postprandial plasma glucose ≥11.1 mmol/l . PatientIGF2BP2, rs7178572 in HMG20A, rs4430796 and rs11651052 in HNF1B were selected according to the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and the 1000 Genomes Project data , with minor allele frequencies (MAFs) >5% and a pairwise tagging r2 of ≥0.8 in Chinese Han population. Genotyping was performed using Agena MassARRAY system  [Peripheral blood samples were obtained from each subject in vacutainers containing disodium-EDTA anticoagulant. Genomic DNA was isolated using the GoldMag DNA Purification Kit  according to the manufacturer’s protocol, and was stored at −20°C until further analysis. Rs11927381 and rs7640539 in  U.S.A.) ,16, and T test, as appropriate. Continuous variables and categorical variables were presented as means ± standard deviation (SD) orabsolute number , respectively. Hardy–Weinberg equilibrium (HWE) for each SNP in the control group was assessed using a goodness-of-fit χ2 test. The frequencies of genotype and allele between healthy controls and T2DM patients were compared with χ2 test. The correlation between selected SNPs and T2DM risk was estimated by odds ratios (OR) and 95% confidence intervals (CI) using logistic regression models, after adjusting for age and sex [P value < 0.05 was considered statistically significant.Statistical analyses were carried out using SPSS version 17.0  and PLINK version 1.0.7. Demographic and clinical data between patients and controls were compared using chi-square test or independent sample  and sex . To expl and sex . FurtherP = 0.841 and P = 0.712, respectively). Demographic and clinical characteristics of participants were listed in About 508 patients with T2DM  and 503 healthy controls  were included. There was no significant differences in the distribution of age and gender between T2DM patients and the healthy controls  were successfully genotyped, and all SNPs were in accordance with HWE , smokers , or alcohol drinkers (homozygote and recessive). For rs4430796 polymorphism, GG genotype was predominantly related to a higher risk of T2DM among subjects with BMI ≤ 24 kg/m2 or drinkers. Besides, rs4430796 also showed a risk-increasing effect among non-drinkers. For rs11651052 variant, BMI, smoking, and alcohol drinking status had interactive effect with selected SNPs on T2DM risk. An association of rs11651052 and T2DM risk was observed in both subjects with BMI > 24 kg/m2  and subjects BMI ≤ 24 kg/m2 (homozygote and additive). In non-smokers, a trend of higher risk of developing T2DM was also found for subjects with A allele, and AA, AA-AG genotypes, and in additive model. Similarly, rs11651052-A allele had a higher the incidence of T2DM in smokers. In drinkers, individuals with rs11651052 AA genotype had 7.65- and 7.49-fold increased risk of developing T2DM than drinkers who carried GG genotype and combined AG-GG, respectively. In non-drinkers, rs11651052 was also associated with T2DM occurrence .Stratified analyses were also carried out to estimate the effect of these polymorphisms with BMI and behavioral factors  on T2DM risk, as shown in HMG20A rs7178572, HNF1B rs11651052, and IGF2BP2 rs7640539, which were conferring risk toward T2DM progression. The accumulated effect of rs7178572-GG, rs11651052-AA, and rs7640539-TA conferred a higher risk for T2DM, as shown in The association between higher order interactions of SNP–SNP and T2DM risk was analyzed by MDR as summarized in IGF2BP2 rs11927381 and rs7640539 with the levels of retinol-binding protein . The association of HMG20A rs7178572 with glycated hemoglobin was identified (P = 0.032). Besides, carriers of HNF1B rs11651052 GG genotype had significantly higher insulin level than AA and AG genotypes (P = 0.018). However, there was no relation of different genotypes with the remaining clinical parameters (P > 0.05).The relation between selected SNPs and different clinical parameters of T2DM patients was investigated, as illustrated in IGF2BP2 rs11927381 and rs7640539, HMG20A rs7178572, HNF1B rs4430796, and rs11651052 with T2DM susceptibility. We found that HMG20A rs7178572 and HNF1B rs11651052 were related to an increased T2DM risk in the overall. Given that T2DM represents a complex disorder influenced by the interplay between genetic and behavioral factors, we analyzed the effect of age, gender, BMI, smoking, and alcohol drinking on the relationship of these variants with T2DM susceptibility. Our stratified analysis showed that rs7178572, rs4430796, and rs11651052 had higher risk of T2DM occurrence in males and subjects with age ≤ 59 years. In addition, rs11927381 also contributed T2DM susceptibility at age ≤ 59 years. We also found that these genetic variants might increase the risk of T2DM occurrence not only by itself but also by interacting with smoking and alcohol drinking. Besides, rs7178572-AA (P = 0.032) genotype and rs11651052 GG (P = 0.018) genotype might have higher glycated hemoglobin and insulin levels, respectively. To the best of our knowledge, this was the first to explore the effects of the relationships between IGF2BP2 rs11927381, rs7640539 and HNF1B rs11651052 and T2DM susceptibility in the Chinese Han population.We performed a case–control study to investigate the association of IGF2BP2), located on chromosome 3q27, encodes an mRNA-binding protein associated with RNA location, stability, and translation. IGF2BP2, highly expressed in pancreatic islets, is involved in β-cell function by regulating IGF2 post-translational modification [IGF2BP2 variations were also associated with decreased insulin secretion and hyperglycemia [IGF2BP2 were investigated for the relationship with T2DM; however, there were very few studies on rs11927381 and rs7640539. Only one study reported the association between IGF2BP2 rs11927381 and the increased T2DM risk among Slavonic population [Insulin-like growth factor 2 binding protein 2  gene, located in 15q24.3, is a member of high mobility group (HMG) box-containing genes. HMG20A encodes a widely expressed non-histone chromosomal protein controlling gene expression by histone modification [High mobility group 20 A  showed an association with T2DM in European obese subjects [HMG20A, which was related to an increased T2DM susceptibility. However, a previous study showed there was no significant relationship between rs7178572 and the risk of T2DM among Han population in southern China [2. These results are required to validate in larger populations.ntiation . Previousubjects . Our resrn China , such inrn China . Obesityrn China . Smokingrn China . Alcoholrn China . TherefoHNF1B), located on chromosome 17q21.3, encodes a transcription factor that involved in tissue-specific regulation of gene expression and embryonic development of numerous organs [HNF1B gene played the important role in the primary pathophysiology of diabetes. It was involved in the loss of neurogenin-3 (Ngn3)-positive endocrine progenitor cells, pancreatic atrophy, and a reduced insulin sensitivity to endogenous glucose production leading to the reduction of insulin secretion [HNF1B were associated with the susceptibility of T2DM. Rs4430796 (A>G) in intron 2 of HNF1B is the most frequent SNP in Chinese population. Notably, the mutant allele frequency for rs4430796 is quite different between different ethnic groups. The mutant allele G frequency in the study was 0.289, similar to the healthy Han Chinese and Asian, but significantly different from Caucasian (0.47) and African (0.67) [2 and drinkers. These results indicated that gene-behavioral habit interactions might operate in the pathogenesis of T2DM. Rs11651052 (G > A) is another SNP in HNF1B, and no study has analyzed the SNP now. In our study, we first reported that rs11651052-A allele increased 1.26-fold risk of T2DM compared with G allele. Our stratified analysis showed that rs11651052 had a higher T2DM susceptibility in males and subjects with age ≤ 59 years, suggesting the risk association of this polymorphism might be age dependent.Hepatocyte nuclear factor-1β (s organs . HNF1B gecretion . Previoun (0.67) . Previoun (0.67) , which wInevitably, several intrinsic limitations should be considered. First, the subjects were enrolled from the identical hospitals; therefore, the selection bias could not be completely excluded. Second, some clinical characteristics were not analyzed because of missing or uncollected data in controls. Third, explicit mechanisms of these polymorphisms on the development of T2DM are still bewildered and further research is required. Therefore, further well-designed large and prospective studies and functional experiments should be conducted to verify our finding.IGF2BP2, HMG20A, and HNF1B had the risk effect on T2DM occurrence among the Chinese Han population. Specifically, we first found that rs11927381, rs7640539, and rs11651052 were associated with the increased risk of T2DM occurrence. We also provided evidence that age gender, BMI, smoking, and alcohol drinking status had interactive effect with these variants on T2DM susceptibility, suggesting that gene-behavioral habit interactions might play critical roles in the risk of developing T2DM. Our study may increase the understanding of IGF2BP2, HMG20A, and HNF1B variants on the pathogenesis of T2DM.To sum up, our study revealed that variants in


During influenza epidemics, Japanese clinicians routinely conduct rapid influenza diagnostic tests (RIDTs) in patients with influenza-like illness, and patients with positive test results are treated with anti-influenza drugs within 48 h after the onset of illness. We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children , using a test-negative case-control design based on the results of RIDTs in the 2018/19 season. The VE against influenza A(H1N1)pdm and A(H3N2) was analyzed separately using an RIDT kit specifically for detecting A(H1N1)pdm09. The adjusted VE against combined influenza A (H1N1pdm and H3N2) and against A(H1N1)pdm09 was 39%  and 74% , respectively. By contrast, the VE against non-A(H1N1)pdm09 influenza A (presumed to be H3N2) was very low at 7%. The adjusted VE for preventing hospitalization was 56%  against influenza A. The VE against A(H1N1)pdm09 was consistently high in our studies. By contrast, the VE against A(H3N2) was low not only in adults but also in children in the 2018/19 season. In Japan, rapid influenza diagnostic tests (RIDTs) are considered core tools for determining whether or not to start treatment with anti-influenza drugs. During influenza epidemics, Japanese clinicians routinely use RIDTs in the examination of patients with influenza-like illness, and most patients with positive test results are treated with anti-influenza drugs within 48 h after the onset of illness , 2. We hThe purpose of this study was to measure the VE in the 2018/19 season based on the results of RIDTs, including the VE for preventing hospitalization and by vaccine dose. In addition, the VE against influenza A(H1N1)pdm09 was measured using an RIDT kit specifically for detecting A(H1N1)pdm09.To assess VE, we used a TNCC design based on RIDT results –7, 11. CThe vaccine strains in the 2018/19 season were A/Singapore/GP1908/2015 (A(H1N1)pdm09) (A/Michigan/45/2015-like), A/Singapore/INFIMH-16-0019/2016 (A(H3N2)), B/Phuket/3073/2013 (Yamagata lineage), and B/Maryland/15/2016 (Victoria lineage).Nasopharyngeal swabs were obtained from patients. The swab for each test kit was inserted deeply into the back of at least one nostril, left in place for several seconds, rotated to absorb secretions, placed into the medium of each kit, and tested immediately. RIDT kits capable of differentiating between influenza A and influenza B were used in all hospitals, including the ImmunoAce FLU kit , Quick Chaser Flu A, B kit , QuickNavi-Flu kit , Espline Influenza A&B-N kit , and Spotchem FLORA FluAB . All of these kits have high sensitivity (approximately 85%–95%) and specificity (up to 100%) compared to reverse transcription polymerase chain reaction (RT-PCR) .The ImunoAce Flu test is designed to not only detect influenza A or B, but also to detect A(H1N1)pdm09 with the use of an additional test kit . Its sensitivity and specificity for A(H1N1)pdm09 were 97.6%  and 92.6% (95%CI: 82.1%–97.9%) in adults, respectively . Thus, bRIDT-positive patients were enrolled as cases and RIDT-negative patients were enrolled as controls. The information shown in (number of influenza-positives among vaccinated patients × number of influenza-negatives among unvaccinated patients) / (number of influenza-negatives among vaccinated patients × number of influenza-positives among unvaccinated patients).Adjustments to the VE are explained below. The VE for preventing influenza A(H1N1)pdm09 was also analyzed in three hospitals where the ImunoAce Flu and the Linjudge FluA/pdm were used.In the TNCC design, the cases were patients who were hospitalized because of influenza  after diagnosis at an outpatient clinic. Patients hospitalized because of influenza-like illness who were RIDT-negative served as the control group. The VE for preventing influenza hospitalization was estimated based on the vaccine coverage rate of both hospitalized patient groups.In Japan, a single dose is recommended for children ≥13 years old, but a two-dose regimen is recommended for all children aged 6 months to 12 years old . Doses op < 0.05 was considered statistically significant for all analyses.Statistical analyses were performed using the SPSS 25.0 software program  and the Ekuseru-Toukei 2015 for Windows software program . p-values were also Bonferroni corrected for the dose analysis .To analyze the VE for both preventing influenza and preventing hospitalization, binary logistic regression methods were chosen. Confounding factors, such as age (0–15 years old), comorbidity (yes or no), colder or warmer area , and month of onset, were entered in the analysis by the forced entry method. For some analyses, the time tested after onset  was also entered. These factors for adjustment have been the same since our 2013/14 study [This study was approved by the Keio University Ethics Committee . Eligible patients and their guardians were informed about the study objectives and methods verbally or via posters in outpatient clinics. We recorded the necessary information using a standardized questionnaire sheet. The requirement for obtaining written consent was waived by the Keio University Institutional Review Board because testing patients with an RIDT is a standard practice, and all questions regarding this study are essential in daily practice.The change in the number of RIDT-positive or RIDT-negative patients is shown in A total of 4243 children 6 months to 15 years old were enrolled in the 2018/19 season . ChildreOf the children with influenza A, 97% (1920/1987) had received an RIDT within 48 h of onset. Ninety-seven percent (1994/2055) of the children with influenza A were treated with antivirals  . The oveThe adjusted VE for preventing influenza A illness was 39%  . The adjOf the 238 cases in 3 hospitals, 36 (10 vaccinated and 26 unvaccinated) cases were A(H1N1)pdm09-positive, 71 (44 vaccinated and 27 unvaccinated) cases were A(H1N1)pdm09-negative (defined as A(H3N2)), and 131 cases were influenza-negative. The adjusted VE for preventing influenza A was 37%  by ImmunoAce FLU, and the adjusted VE for preventing influenza A(H1N1)pdm09 was 74%  by Linjudge FluA/pdm. The VE in preventing influenza A(H3N2) was 7%  .There were 205 hospitalized children , and 3929 were not hospitalized  after diagnosis at outpatient clinics . The adjp-value <0.05) and in 3 to 5 year olds  . There was no significant difference between the one- and two-dose regimens.The number of enrollees among influenza-positive and influenza-negative children by age group is shown in In the 2018/19 season, a relatively low VE against influenza A  was shown in this study, when the ratio of isolated strains for A(H1N1)pdm09 and A(H3N2) was approximately 40% vs. 60% in Japan . AlthougAlthough we previously reported low VEs against influenza A infection during influenza epidemics mostly caused by the A(H3N2) subtype, such as 37% in 2014/15 (99% of influenza A strains identified were A(H3N2)) , the degIn the 2018/19 season, the antigenicity of most A(H3N2) strains isolated in Japan was similar to the vaccine strain A/Singapore/INFIMH-16-0019/2016 (A(H3N2)) (genetic clade 3C.2a1) . HoweverThe VE against A(H1N1)pdm09 was consistently high in our studies. We previously reported high VEs against A(H1N1)pdm09, such as 77% in the 2013/14 season, using RIDT kits specifically for detecting A(H1N1)pdm09, and the VE increased with age: from 67% in the 1- to 2-year-old group, to 84% in the 3- to 5-year-old group, and 90% in the 6- to 12-year-old group .Because we used RIDTs, digital immunoassays, or even rapid nucleic acid amplification tests, we were unable to diagnose influenza A subtypes separately, except when RT-PCR was performed. We believe that the combination of ImunoAce Flu and Linjudge FluA/pdm is highly useful for distinguishing between influenza A subtypes compared with RT-PCR, although the sensitivity and specificity of the combination of tests is lower than with RT-PCR . This coUsing RT-PCR, high VE against A(H1N1)pdm09 in children was reported worldwide in the 2018/19 season. In the US, where most of the isolated influenza type A strains were A(H1N1)pdm09, the VE against A(H1N1)pdm09 in children 6 months to 17 years old was 62% . In contIn the present study, the VE for children 6 to 11 months old was shown to be the highest among all age groups, although the VE in this age group has not been statistically significant in our previous studies. It is generally thought that, in children 6 to 11 months old, the VE may be lower than in older groups because the vaccine-induced antibody response is immature . HoweverAlthough a meta-analysis showed no convincing evidence that the influenza vaccine reduces mortality, hospitalization rates, or serious complications in children , we repoAnother observation was that only a two-dose regimen was effective against influenza A in children 3 to 5 years old. In our combined analysis of five seasons (2013/14–2017/18), both one- and two-dose regimens were effective against influenza A in all seasons , 25, 26.The major limitation of our past studies has been that our diagnostic tools are RIDTs, not RT-PCR. The sensitivity of RIDTs was recently reported to be as low as 42.6% for influenza A and 33.2% for influenza B in adult patients, leading to a deep distrust of RIDTs in the world medical community , althougIn contrast, in Japan, during the 2009 H1N1 pandemic, the sensitivity and specificity of the RIDT  was determined to be 80.0% and 97.1% in adults and children, compared with RT-PCR . MoreoveIn conclusion, during the mixed influenza A subtype epidemic in the 2018/19 season, the VE against influenza A was low (39%), but the VE against influenza A(H1N1)pdm09 was high (74%). By contrast, the VE against influenza A(H3N2) was very low (7%). This very low VE against A(H3N2) was a serious problem not only in adults but also in children and was attributed to mutations in the egg-adapted vaccine strain of A/Singapore/INFIMH-16-0019/2016 (A(H3N2)) as observed or reported previously , 32. Non


HMGCR rs17244841,APOE rs7412 and rs429357, and SLCO1B1 rs2306283 and rs11045818 were genotyped using an Applied Biosystems DNA sequencing method (ABI3730×1). We found that atorvastatin reduced total cholesterol and low-density lipoprotein (LDL) more significantly  in patients with wild genotype than variant alleles APOE rs7412C > T and SLCO1B1 rs2306283A > G. Furthermore, the ALT level was elevated significantly  by 27% in patients with heterozygous SLCO1B1 rs11045818 G/A genotype, while it was not elevated among wild genotype carriers. Additionally, atorvastatin reduced total cholesterol more significantly  in patients with SLCO1B1 rs2306283A and rs11045818G haplotypes and increased ALT levels by 27%  in patients with SLCO1B1 rs2306283G and rs11045818A haplotypes. In conclusion, it was found in this study that APOE rs7412, SLCO1B1 rs2306283, and rs11045818 genotypes can be considered as potential genetic biomarkers of atorvastatin response among DM2 patients of Jordanian Arabic origin. Further clinical studies with larger sample numbers are needed to confirm these findings.Atorvastatin is commonly used among type 2 diabetic (DM2) patients at the University of Jordan Hospital to prevent cardiovascular complication. However, we noticed that there is a wide inter-individual variation in the efficacy and toxicity of atorvastatin. This study aimed to find out the effects of major genetic variants in 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), Apolipoprotein E (APOE), and Solute Carrier Organic Anion (SLCO1B1) genes on atorvastatin response among DM2 patients. A sample of 139 DM2 patients on 20 mg of atorvastatin was included in this study. The lipid and glycemic profile and the levels of hepatic enzymes alanine aminotransferase (ALT) and aspartate transaminase were recorded before and after 3 months of atorvastatin treatment. Additionally, the genetic variants  Atorvastatin is a strong, long-acting inhibitor of 3-hydroxy glutaryl co-enzyme A reductase (HMGCR), which is the rate-limiting step in the biosynthesis of cholesterol . AtorvasHMGCR, APOE, and SLCO1B1 genes influence statin response  × 100® Genomic DNA purification kit (USA) according to the manufacturer’s instructions. The concentration (ng/µL) and the purity (A260/A280) of DNA were measured using an ultraviolet NanoDrop spectrophotometer. The accepted purity ratio of the extracted DNA was within 1.8 ± 0.1 [EDTA tubes were used to collect 5 mL venous blood samples. DNA was isolated from the whole blood using a Wizard.8 ± 0.1 .HMGCR rs17244841, APOE rs7412 and rs429357, and SLCO1B1 rs2306283 and rs11045818 genetic variants were amplified using polymerase chain reaction (PCR). The PCR mixture was prepared in a total volume of 50 µL containing 50 ng of isolated DNA, 10 pmole of each forward and reverse primer  for sequencing the amplified nucleotide sequences of the targeted g method . The samg method . The DNAdatabase .SLCO1B1 rs2306283 genotypes. The homozygous genotype was not detected among the DM2 patients, for HMGCR rs17244841, APO rs7412, and SLCO1B1 rs11045818 genotypes, so a student t-test was used to compare the mean values between the “wild” and heterozygous genotypes. One-tailed t-test was used to compare between the 2 groups since we hypothesized that these genetic variants decrease atorvastatin response. A difference was considered significant when the p-value was less than 0.05. Deviation of the observed genotype frequency from Hardy–Weinberg equilibrium was determined using a chi-squared (X2) test.Statistical analyses were performed using the Statistical Package for Social Sciences  version 23 for Windows. The lipid profile data of the volunteers were normally distributed according to the Kolmogorov–Smirnov test. A one-way analysis of variance (ANOVA) test and Tukey’s HSD post-hoc test were used to analyze the differences in atorvastatin response among different groups of The mean age of the patients was 55 ± 6 years, 57% (79 patients) were male, and 43% (60 patients) were female. The average weight of the patients was 86 ± 14.3 kg. Their lipid and glycemic profiles from before and after 3 months of taking atorvastatin are shown in HMGCR rs17244841A/A, and 10 carried heterozygous A/T, while only one patient had the homozygous HMGCR rs17244841 T/T genotype. The HMGCR rs17244841 A allele frequency was 0.96, while the T allele frequency was 0.04. Regarding the APOE rs7412 T > C genetic variant, 127 patients carried the wild T/T genotype, while 12 patients carried the heterozygous T/C genotype. The APOE rs7412 T allele frequency was 0.96, while the A allele frequency was 0.04.There were 128 volunteers who were genotyped with wild SLCO1B1 rs2306283 A > G variant, 33 patients carried the wild A/A genotype, 64 carried the A/G heterozygous genotype, and 42 carried the G/G homozygous genotype. The SLCO1B1 rs2306283 A allele frequency was 0.47, while the G allele frequency was 0.53. Regarding the SLCO1B1 rs11045818 G > A genetic variant, we found that 108 volunteers carried the wild G/G genotype, 29 carried the heterozygous G/A genotype, and two carried the homozygous A/A genotype. The G allele frequency was 0.88, while the A allele frequency was 0.12. None of the observed genotype frequencies showed a deviation from Hardy–Weinberg equilibrium .For the HMGCR rs17244841 genotype did not show a significant association  with atorvastatin response in terms of lipid profiles, glycemic profiles, and hepatic enzymes  among wild-type (C/C) carriers than heterozygous APOE rs7412 C/T genotype carriers  by 9% and 16% among patients with heterozygous SLCO1B1 rs2306283 A/G and SLCO1B1 rs2306283 G/G genotypes, respectively, while it was not elevated among wild carriers  by 27% among patients with the heterozygous (G/A) genotype than the wild (G/G) SLCO1B1 rs11045818 genotype  among DM2 patients with SLCO1B1 rs2306283 A and rs11045818 G haplotype (29%) than patients with SLCO1B1 rs2306283 G and rs11045818 G (15.5%) haplotype. Furthermore, ALT levels were increased by 27% among patients with SLCO1B1 rs2306283 G and rs11045818 A haplotype. This elevation is statistically significant  in comparison with altered ALT levels among diabetic patients with SLCO1B1 rs2306283 G and rs11045818 G, and those with SLCO1B1 rs2306283 G and rs11045818 A haplotypes after atorvastatin therapy.The frequency of reatment . AtorvasAPOE rs7412 T and SLCO1B1 rs2306283 G alleles significantly reduced the atorvastatin response, with DM2 patients carrying these alleles having significantly higher lipid levels than patients with wild alleles. Additionally, SLCO1B1 rs2306283 G and rs11045818 A variants were associated with a significant elevation in ALT levels after atorvastatin treatment. Hence, these variants might play a role in the risk of atorvastatin-induced hepatotoxicity. These findings suggest that APOE rs7412 and SLCO1B1 rs2306283 and rs11045818 genetic variants are potential genetic biomarkers for atorvastatin response among DM2 patients of Jordanian Arabic origin. Further clinical studies are needed to confirm the findings of this study with a larger sample size.There is wide inter-individual variation in the atorvastatin response among DM2 patients attending the University of Jordan Hospital. Variation in atorvastatin response can be due to multiple factors, such as patient adherence, health status, and drug–drug interaction . Howeverrs2070744, rs1799983, and rs61722009 genetic variants on atorvastatin response among Jordanian DM2 patients [CYP7A1 and ABCG8 genetic variants on atorvastatin response among DM2 Jordanian patients [APOE rs7412, SLCO1B1 rs2306283, and rs11045818 variants significantly affected atorvastatin response among DM2 patients of Jordanian Arabic origin for the first time.This study is not the first to investigate the influence of genetic variants on atorvastatin response among DM2 patients attending the University of Jordan Hospital. Abdullah et al. (2020) studied the effect of endothelial nitric oxide synthase patients . They fopatients . HoweverHMGCR rs17244841 genetic variant affects statin response [HMGCR rs17244841 variant between Jordanians and other ethnic groups. Additionally, this is the first study to investigate the influence of the HMGCR rs17244841 variant on statin response among DM2 patients. The diabetic disease causes physiological and biochemical alterations that can mask the effects of genetic variants on drug response [HMGCR rs17244841 genetic variant has level 3 clinical evidence, which indicates that not enough clinical results support the clinical implementation of HMGCR rs17244841 variant in statin therapy [HMGCR rs17244841 variant does not have a significant clinical impact on atorvastatin response among DM2 patients.Some studies showed that the response ,21, but response . Accordi therapy . The resAPOE gene affect ApoE function and hence cholesterol transport and statin response [APOE rs7412 T allele decreased the atorvastatin response, and APOE rs7412 C/T genotype DM2 patients had a significantly lower response to atorvastatin. This result is in agreement with the report by Lagos et al. that the APOE rs7412 variant reduced statin response among Chilean subjects [APOE rs7412 T allele increases statin response [APOE rs7412 variant on atorvastatin response.ApoE plays a major role as a carrier and transporter of cholesterol in the blood and has a role in the metabolism and elimination of cholesterol. Functional genetic variants in the response . We showsubjects . Howeverresponse . TherefoAPO rs7412 genotype. Besides, atorvastatin decreased TC and LDL by only 7.5% and 10%, respectively, among patients with heterozygous APO rs7412 genotype. Accordingly, DM2 patients with wild APO rs7412 genotype responded, while patients with heterozygous genotype did not respond, to atorvastatin therapy. Therefore, DM2 patients with heterozygous APO rs7412 genotype are at higher risk of treatment failure with atorvastatin, which may lead to cardiovascular complications.According to the ACC/AHA guideline classification of statin intensity , 20 mg aSLCO1B1 rs2306283 G variant decreases the capacity of SLCO1B1 transporter [SLCO1B1 rs2306283 G allele, and the anti-hypercholesterolemic effect is decreased. This could explain the reduced atorvastatin response among DM2 patients with the SLCO1B1 rs2306283 G genotype.The major site of action of statins is the liver. Statins enter hepatic cells through SLCO1B1. It is reported that the SLCO1B1 genetic variants are strongly associated with statin-induced myopathy [SLCO1B1 rs2306283 G and rs11045818 A alleles have a significantly higher blood level of ALT enzyme, although the mean of the elevated ALT level was within the normal ALT range (<30 IU/L). Elevated ALT levels might indicate a higher risk of hepatotoxicity [SLCO1B1 genetic variants are associated with drug-induced hepatotoxicity [Some studies showed that myopathy . We fountoxicity ,32.SLCO1B1 rs2306283 G and rs11045818 A variants can affect ALT level is still not clear. Although SLCO1B1 rs11045818 A is a synonymous variant, it might be in a linkage disequilibrium with other functional variants that have a clinical influence on atorvastatin response. Using Haploview software [SLCO1B1 rs11045818 and the functionally non-synonymous SLCO1B1 rs2306283. The level of clinical evidence of SLCO1B1 rs2306283 and rs11045818 on the PharmGKB website is 3. Since we found a significant association between these variants and atorvastatin response, we recommend investigating these variants’ effects on atorvastatin response among different ethnic groups with a larger sample size.The mechanism of how software , we founThere are some limitations to this study. First, the sample size was relatively small, and these findings should be confirmed using a larger sample size. Second, other factors affecting statin response, such as drug–drug interactions, drug–food interactions, and patient adherence, were not included in this study. Third, genetic variants of cytochrome P450s, which metabolize statins, were not studied. Lastly, the patients were on adjusted doses of anti-diabetic metformin therapy, which affects the lipid and glycemic profiles of DM2 patients.APOE rs7412, SLCO1B1 rs2306283, and rs11045818 genotypes have an influence on atorvastatin response. Therefore, they can be considered as potential genetic biomarkers of atorvastatin therapy among DM2 patients of Jordanian Arabic origin. Further clinical studies with larger sample numbers are needed to confirm these findings.In conclusion,


AXUD1, we found that multipotency factor SP5 directly promotes neural plate border identity, while inhibiting premature expression of specification genes. Our results highlight the importance of repressive interactions in the neural crest gene regulatory network and illustrate how genes activated by the same upstream signal become temporally segregated during progressive fate restriction.The process of cell fate commitment involves sequential changes in the gene expression profiles of embryonic progenitors. This is exemplified in the development of the neural crest, a migratory stem cell population derived from the ectoderm of vertebrate embryos. During neural crest formation, cells transition through distinct transcriptional states in a stepwise manner. The mechanisms underpinning these shifts in cell identity are still poorly understood. Here we employ enhancer analysis to identify a genetic sub-circuit that controls developmental transitions in the nascent neural crest. This sub-circuit links Wnt target genes in an incoherent feedforward loop that controls the sequential activation of genes in the neural crest lineage. By examining the cis-regulatory apparatus of Wnt effector gene  SP5 and AXUD1) control the temporal activation of neural crest genes. While SP5 drives the expression of genes expressed at gastrula stages, AXUD1 promotes neural crest genes transcribed during neurulation. We found that these regulators are linked by inhibitory interactions that prevent overlap between transcriptional states. Our results shed light on how the architecture of gene regulatory circuits underlies the timing of developmental transitions during neural crest development.The neural crest is a migratory stem cell population that plays a crucial role in the development of vertebrate embryos. These cells contribute to multiple tissues and organs, including the craniofacial skeleton, the peripheral nervous system, and the pigmentation of the skin. Neural crest formation requires sequential expression of sets of genes that gradually change the identity of progenitor cells. Here we characterize a gene circuit that plays an important role in these developmental transitions. We show that a pair of transcription factors downstream of the Wingless (Wnt) signaling pathway ( PAX7, MSX1, ZIC1, TFAP2A) [SNAI2, FOXD3, and SOX10. Neural crest specifier genes, in turn, activate the effector genes that endow these cells with their unique properties, such as multipotency and migratory capabilities [The neural crest is a migratory stem cell population that gives rise to multiple components of the vertebrate body plan, including the peripheral nervous system and the craniofacial skeleton . The for TFAP2A) . These fbilities .The neural crest GRN is one of the most comprehensive regulatory networks described for a vertebrate cell type, and has served as a platform for investigating molecular mechanisms involved in cell fate commitment and vertebrate evolution –8. Yet, AXUD1  as a critical component of the neural crest GRN [FOXD3 and SOXE factors are only expressed following the start of AXUD1 expression at Hamburger and Hamilton stage 7 (HH7) [AXUD1 is hypothesized to be downstream of canonical Wnts, it does not accurately recapitulate the activity of this signaling system, as Wnt ligands are already present in the early gastrula [AXUD1 transcription only starts during neurulation. This discrepancy suggests a complex mode of regulation that may involve integration of numerous inputs. Identifying how upstream factors control AXUD1 expression may reveal new mechanisms responsible for the emergence of neural crest identity.Recently, we have identified Wnt target gene rest GRN . This trrest GRN ,15, spec 7 (HH7) . This ingastrula , but AXUAXUD1 locus with chromatin conformation capture, we identified a tissue-specific enhancer that interacts with the promoter of this gene in neural crest cells. Dissection of this enhancer allowed for the delineation of a sub-circuit that operates downstream of Wnt signaling to control the temporal expression of neural crest genes. AXUD1 is activated by Wnt signaling and neural plate border genes TFAP2A, MSX1, and ZIC1. Crucially, direct repression by Wnt effector gene SP5 restricts the timing of AXUD1 expression to prevent premature expression of specification genes. We assembled these interactions in a sub-circuit that links Wnt-target genes in an incoherent feedforward circuit, which controls the sequential activation of genes in the neural crest lineage. Our results highlight the importance of repression in the neural crest GRN and demonstrate how the interactions between Wnt target genes drive progressive fate restriction.Here we describe a regulatory sub-circuit that controls the onset of neural crest formation in vertebrate embryos. By surveying the AXUD1 is first detected in the neural crest lineage at (HH7), before the onset of specification markers FOXD3, ETS1 and SOX9 [In situ hybridization in avian embryos shows robust expression of this transcription factor at HH8-9  in neural crest cells isolated from embryos at these developmental stages. Dorsal neural folds from the cranial region of HH8-9 embryos were micro-dissected with iridectomy scissors and dissociated into a single-cell suspension. Cells were fixed with formaldehyde, and the crosslinked chromatin was digested with restriction enzyme NCOI , H3K27Ac association and TFAP2A occupancy in neural crest cells [AXUD1 promoter (elements 9–10) .To identify active enhancers of st cells . TFAP2A st cells . This ants 9–10) . To tests at HH9 , whereass at HH9  were unas at HH9 . Activitsibility . HistoloAxud1E1 activity, we next examined transgenic embryos at different stages of neural crest development. We detected specific signal from the Axud1E1 reporter construct in both pre-migratory (in situ hybridization (FISH) for AXUD1 and eGFP in transgenic embryos showed that prior to migration, the enhancer drives reporter expression only in neural crest cells that express AXUD1  spanned 500bp, and was highly active in AXUD1+ cells , located at the 5' region of Axud1E1, displayed similar but weaker activity  that is in close proximity to a shadow regulatory element.To understand the regulation of onstruct . We founonstruct . The firlls Figs  and S2B.d TFAP2A . The secity Figs  and S2D.ity Figs  and Axudhromatin , we hypoy silent . Next, wud1E-500 , whereasxud1E-SE  in neuraAXUD1 as a downstream target of canonical Wnt signaling, acting as a secondary effector of the pathway during neural crest formation [Axud1E1-500. To define if the enhancer responds to Wnt signaling, we disrupted the pathway in transgenic avian embryos transfected with the Axud1E1-500 reporter construct. For these experiments, we transfected embryos with a control morpholino on the left side, and morpholinos targeting components of the Wnt signaling pathway on the right side . In situ hybridization showed that LEF1 is strongly enriched in the neural crest during specification, whereas we were unable to detect discernable expression patterns for TCF7 and TCF7L2 in the head of avian embryos  failed to interact with the enhancer  or on neural folds (HH9) transfected with a Wnt dominant-negative construct [Axud1E1-SE, which is not accessible , which were mutated by replacing the endogenous sequence with a 100bp segment of the coding sequence of eGFP   , was abl1E1-300) . Next, w1E1-300) . To asse1E1-300)  for tran1E1-300) , loss of1E1-300) . These fMSX1 and ZIC1) and the multipotency factor SP5 in the regulation of AXUD1, we performed loss-of-function experiments for these transcription factors. Gastrula-stage embryos (HH4) were electroporated with control and targeted morpholinos and incubated until HH9. MSX1 and ZIC1 morphants exhibited a reduction in reporter expression  [Axud1E1 (To confirm that SP5 directly regulates CUT&RUN) . This teCUT&RUN)   directly activates a target gene (AXUD1) but also promotes the expression of a repressor (SP5) of said gene. We propose that the sub-circuit relies on repressive interactions between Wnt target genes to promote temporally distinct phases of gene expression. The inhibitory interaction between SP5 and AXUD1 prevents these factors and their downstream targets from being expressed at the same time. Since SP5 activates neural plate border genes [Comparative analyses of developmental GRNs show that these genetic programs are composed of collections of sub-circuits, which are responsible for executing well-defined tasks . Here we-circuit  is strucuit . NonethSNAI2 [These results highlight the importance of repressive interactions in the neural crest GRN. A significant shortcoming of the current version of the network is that it is built primarily of positive regulatory links; only a small number of studies have focused on transcriptional repressors, like SNAI2 . This isSNAI2 , or to pSNAI2  will be All experiments were performed in accordance with Memorandum of Understanding and Agreement 16274–1, which was approved by the Institutional Biosafety Committee of Cornell University.Fertilized Leghorn White chicken eggs were obtained from the Department of Animal Science, University of Connecticut. Eggs were incubated at 37°C until embryos reached the desired developmental stages. Embryos were collected and cultured according to the EC protocol , and stain situ hybridization, embryos were fixed in phosphate buffer saline (PBS) containing 4% paraformaldehyde (PFA) for 2 hours at RT. Following fixation, embryos were dissected, washed with PBST, dehydrated and stored in methanol at −20°C. Whole-mount in situ hybridization was performed as previously described [in situ hybridization, we used the Tyramide TSA system from Perking Elmer  as previously described [For escribed . For douescribed .AXUD1 locus with minimal overlap. Amplification efficiency (slope and intercept) of primers pairs (fragment primers plus constant primer) was verified via qPCR of 10x dilutions of digested/ligated control library [GAPDH) as described in [Chromosome Conformation Capture (3C) libraries were generated based on previously published protocols –49 with  library . Interacribed in   and the total RNA was isolated following manufacturer’s protocol. cDNA was then synthesized using SuperScript III Reverse Transcriptase and Random Primers  according to the kit’s protocol. RT-PCR was performed using Power Sybr Green PCR master mix  in an ABI viia7 RT-PCR machine. eRNA levels at the HPRT1 and expressed as a fold change compared to the control sample. The qPCR primer sequences are listed in To quantify changes in gene expression caused by SP5 loss-of-function or gain-of-function, we microdissected single neural folds from control and targeted sides of the embryo, which were subsequently lysed in lysis buffer from Power SYBR Green Cells-to-CT Kit. RNA extraction and cDNA preparation were performed according to the kit's protocol). RT-PCR was performed using Power Sybr Green PCR master mix  in an ABI viia7 RT-PCR machine. Ct values of all genes were normalized to reference gene ex ovo electroporation, as previously described [Enhancer plasmids, morpholinos and expression vectors were transfected in chick embryos at HH4 by escribed . ConstruAxud1E1-2500bp:mCherry, were dissected and dissociated in Accumax . 500 cells per sample were analyzed and plotted in categorical scatter plots using Seaborn Python library. ZIC1, MSX1 and SP5 binding sites in Axud1E1-300 were identified by scanning this sequence using the JASPAR database of transcription factor binding profiles, with a minimal relative profile score threshold of 80% [Putative enhancers  defined d of 80% . Mutant For each experiment, chromatin was isolated from 20 cranial neural folds dissected from HH8-9 embryos or 10 anterior neural plate border regions from HH5 embryos. Immunoprecipitation was performed as described , using tFor whole-mount immunohistochemistry, embryos were collected at appropriate developmental stages and fixed in 4% PFA-PB for 20 mins at RT. Following fixation, embryos were dissected from the filter paper and washed in TBS containing 0.1% Triton and 1% DMSO (TBTD). Embryos were blocked at RT for 2h in TBTD supplemented with 10% donkey serum and incubated in primary antibody  diluted in blocking solution, overnight at 4°C. Following the primary antibody incubation, embryos were washed, blocked for 30mins at RT, and stained with appropriate secondary antibodies (Molecular Probes) for 2h at RT. Following the secondary antibody step, the embryos were washed, stained with DAPI and post-fixed with 4% PFA for 1h prior to imaging. Whole-mount images were collected using an upright Zeiss Axio Imager fluorescent microscope.All loss-of-function assays were performed with double-sided injections of morpholinos and their respective controls to allow single-embryo internal controls. Wnt knockdown was performed by the combined inhibition of WNT 1 and 4 by morpholinos at 1.25uM each, while CTNNB1, SP5, MSX1, ZIC1 and TFAP2A morpholinos were used at 1.5uM . EmbryosSP5 expression constructs were assembled by insertion of the full-length cDNA sequence of avian SP5 in a pCI-H2B-RFP backbone [SP5 expression construct was electroporated as described above, and paired single neural folds  were dissected and processed individually for RT-PCR.For overexpression assays the backbone . The codAxud1E1-500 [crispor.tefor.net) and cloned downstream of the U6 promoter in the cU6.3 vector  for 30 min. After dissociation, cells were resuspended in HANKS solution supplemented with 0.5% BSA. Control and target GFP+ cell suspensions (150–300 cells) were sorted into 50 μl of lysis buffer from the Power SYBR Green Cells-to-CT Kit  using a BD AriaFusion cell sorter. Samples were processed following the manufacturer’s protocol. Axud1E1-500 and Axud1E1-SE expression levels were determined using RT-PCR as described above. cU6.3 and pCAGG-nls-Cas9-nls-GFP vectors were a gift from Dr. Marianne Bronner.We employed a CRISPR-Cas9 system optimized for chick embryos to disrupt the activity of d1E1-500 . Two gRN3 vector . To asseAxud1E1 accessibility, we performed ATAC-qPCR in embryos transfected with a TFAP2A morpholino. HH4 embryos were bilaterally injected with control and targeted morpholinos, as described previously. Embryos were incubated at 37°C until HH9, when dorsal neural folds were surgically dissected. Paired single neural folds were individually processed for DNA tagmentation following the ATAC protocol described in [To examine the control of ribed in . For eacNeural folds were dissected from HH7-8 embryos (n = 20 per CUT&RUN experiment). Cells were dissociated in Accumax for 20min at RT under mild agitation. CUT&RUN experiments were carried out as previously described . BrieflyCUT&RUN libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit  following the manufacturer’s protocol. Fragment analysis was performed with ABI 3730xl DNA Analyzer to perform quality control for the libraries. Equimolar concentrations of the libraries were pooled using the KAPA Library Quantification Kit—ROX Low  and sequenced with paired-end 37bp reads on an Illumina NextSeq500 instrument.Paired-end sequencing reads from the CUT&RUN libraries were trimmed using Cutadapt . Reads wS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Fig(A) Immunohistochemistry for the endogenous TFAP2B protein (magenta) in transverse sections of an HH9 transgenic embryo transfected with Axud1E1:eGFP (turquoise) (A). Reporter expression indicates enhancer activity (A’) in TFAP2B+ cells located at the dorsal neural folds (A”). (B) Immunohistochemistry (transverse sections) for HNK1 marker (magenta) in an HH10 transgenic embryo transfected with Axud1E1:eGFP (turquoise) (B). Arrows indicate specific activity of Axud1E1 (B’) in migratory HNK1+ cells (B”). HH, Hamburger and Hamilton; Scale bars represent 50μm.(TIF)Click here for additional data file.S2 Fig(A-A’) Extended Axud1E1 reporter activity in late migratory neural crest cells is due to GFP stability. (A) Transient transgenesis expression pattern of Tfap2aE1:mChe (magenta) and Axud1E1:GFP depicting Axud1E1 reporter activity in late migratory neural crest cells (arrows). (A’) Same embryo presented in (A) after double fluorescent in situ hybridization targeting mcherry and gfp indicated reduced expression of Axud1E1 in late migratory cells (arrows). (B) Double fluorescent in situ hybridization for AXUD1 and eGFP in transgenic embryos shows colocalization (arrows) of the endogenous gene (magenta) and the enhancer reporter  (B’). (C) Representative ChIP-qPCR experiment for the active chromatin mark H3K27ac and the neural crest pioneer factor TFAP2A indicates that Axud1E1 is an active neural crest enhancer. (D) Double fluorescent in situ hybridization for AXUD1 and eGFP in transgenic embryos shows colocalization (arrows) of the endogenous gene (magenta) and the enhancer reporter  (D’). (E-F) Transverse sections of HH8 (E) and HH10 (F) embryos transfected with constructs Axud1E1-500 (magenta) and Axud1E1-SE (turquoise). Axud1E1-500 and Axud1E1-SE are expressed in the same pre-migratory (arrowheads in D) and migratory neural crest cells (arrowheads in E). (G) Quantification (RT-PCR) of eRNA depicting enrichment of Axud1E1-500 in neural crest cells. While Axud1E1 is actively transcribed, shadow element Axud1E1-SE is silent in dissected neural folds. Error bars represent ± SEM. Statistical significance determined via an unpaired t-test. HH, Hamburger and Hamilton. Scale bars represent 100μm  and 50μm (E-F). ***p < 0.001.(TIF)Click here for additional data file.S3 Fig(A) LEF1 colocalizes with the neural crest cell marker TFAP2B. Double immunohistochemistry (transverse section) for LEF1 and TFAP2B (A). A’ and A” show higher magnification of the area indicated in A.(TIF)Click here for additional data file.S4 Fig(A-B) Double fluorescent in situ hybridization for AXUD1 and eGFP in transgenic embryos shows colocalization (arrows) of the endogenous gene (magenta) and the enhancer reporter (turquoise) for Axud1E1-mutAB (A-A’) and Axud1E1-300 (B-B’). HH, Hamburger and Hamilton; Scale bars represent 100μm.(TIF)Click here for additional data file.S5 Fig(A) Western blot for SP5 and TFAP2A in embryos bilaterally transfected with SP5 and TFAP2A morpholinos, respectively. (B) Quantification of relative number of Caspase-3 positive cells at the dorsal neural tube in embryos transfected with WNT1/4, SP5 and TFAP2A morpholinos. Error bars represent ± SEM. The statistical significance was determined via an unpaired t-test. (C) Mutation of TFAP2A binding sites resulted in no change of Axud1E1-300 activity. Embryos were transfected with wild-type enhancer (Axud1E1-300) on the left side and the TFAP2A mutant construct on the right side. (D) TFAP2A promotes Axud1E1 accessibility. Quantification of chromatin accessibility (ATAC-qPCR) of embryos transfected with TFAP2A morpholino shows loss of Axud1E1 accessibility following knockdown of the pioneer factor. The FOXD3 enhancer (Foxd3NC1), which is not bound by TFAP2A, is unaffected by TFAP2A loss-of-function. Error bars represent ± SEM. The statistical significance was determined via unpaired t-test. HH, Hamburger and Hamilton; Scale bars represent 100μm (C); **p < 0.01.(TIF)Click here for additional data file.S6 Fig(A-C)SP5 is transiently expressed in the neural crest lineage. Whole mount in situ hybridization (A-C) and transverse sections (A’-C’) show expression of SP5 in the neural plate border (A and A’) and dorsal neural folds (B and B’). During neural crest specification, SP5 is excluded from the dorsal neural tube (C and arrow in C’). (D)SP5 expression levels in the neural crest lineage. Data from RNA-seq analysis shows rapid decrease of SP5 mRNA levels during neural crest development from stages HH6 to HH16. (E-G)SP5 expression colocalizes with the neural crest markers PAX7 (arrows in E) and TFAP2B (F) in early neural crest cells. Colocalization of SP5 and TFAP2B is lost in migratory/late neural crest cells . G’ and G” present a magnification of the area highlighted in G. HH, Hamburger and Hamilton; Scale bars represent 500μm (A-C), 100μm (A’-C’); 200μm (E-G).(TIF)Click here for additional data file.S7 Fig(A) Chromatin immunoprecipitation for CTNNB1 shows temporal changes in the regulation of SP5 and AXUD1 by Wnt signaling. Association of CTNNB1 with an SP5 enhancer (Sp5E) decreases during neural crest specification. Conversely, binding of the Wnt effector to Axud1E1 significantly increases from HH5 to HH9. Error bars represent ± SEM. The statistical significance was determined via an unpaired t-test. (B) Fragment size distribution of the two replicates of SP5 CUT&RUN read pairs. Motif enrichment analysis via HOMER for regions occupied by SP5 shows enrichment for GC boxes, similar to other Sp/Klf Zn2+-finger transcription factors. pValue indicates significance of motif occurrence as reported by HOMER. (C) Pairwise Pearson correlation of SP5 and H3K27Ac CUT&RUN replicates. (D) CUT&RUN profiles of SP5 and H3K27Ac at the cis-regulatory elements Tfap2aE1, Msx1E1 and Zic1E1. The three elements are robustly active in the neural crest lineage. HH, Hamburger and Hamilton; Scale bars represent 200μm (D); ***p < 0.001.(TIF)Click here for additional data file.


Matching samples were collected from n = 114 non-fasting women between 12–25 weeks gestation in a clinical trial in Newcastle, Australia. Samples were analysed by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to quantify total 25(OH)D and its analytes and examined using Bland-Altman plots, Pearson correlation (r), intraclass correlation coefficient and Cohen’s Kappa test. Serum total 25(OH)D ranged from 33.8–169.8 nmol/L and plasma ranged from 28.6–211.2 nmol/L. There was a significant difference for total 25(OH)D based on sample type  5.36, 9.90, p ≤ 0.001). The mean difference between serum and plasma concentrations was statistically significant for 25(OH)D3  and Epi-25(OH)D3 . Of 114 participants, 28% were classified as vitamin D deficient (<50 nmol/L) or insufficient (<75 nmol/L) based on plasma sample and 36% based on serum sample. Nineteen (16.7%) participants changed vitamin D status category based on sample type. 25-hydroxyvitamin D quantification using LC-MS/MS methodology differed significantly between serum and plasma, yielding a higher value in plasma; this influenced vitamin D status based on accepted cut-points, which may have implications in clinical and research settings.Measurement of vitamin D status has significant use in clinical and research settings, including during pregnancy. We aimed to assess the agreement of total 25-hydroxyvitamin D (25(OH)D) concentration, and its three analytes (25-hydroxyvitamin D The circulating concentration of 25-hydroxyvitamin D (25(OH)D) is considered the accepted clinical biomarker of vitamin D status . With in2, 25(OH)D3, and Epi-25(OH)D3 separately [Testing is most commonly conducted through measurement of 25(OH)D via assay and quantification of 25(OH)D is routinely outsourced to be completed in external laboratories for clinical and research requirements. Previous studies have examined vitamin D in serum vs. plasma in small sample sizes, using assay techniques, and found serum and plasma to be mostly agreeable ,5,6,7, bparately ,11,12,13n = 25) and Abu Kassim et al. (n = 10) did not find a significant difference between EDTA plasma, heparin plasma and serum for 25(OH)D2 and 25(OH)D3 concentrations in healthy adults [3 or total 25(OH)D were not examined. Albarhani et al. examined the usefulness of diluted plasma for quantification of 25(OH)D3 and Epi-25(OH)D3 compared to serum in umbilical cord blood samples (n = 20), and although their findings demonstrated close agreement for 25(OH)D3 in serum and plasma across two independent laboratories (r = 0.983) issues with analytical sensitivity in regards to limits of detection (LoD) for Epi-25(OH)D3 quantification highlighted issues with the use of diluted plasma instead of serum in other analytes of vitamin D [2 and total vitamin D were not reported. In a study of 13 healthy adults, Mena-Bravo et al. found that plasma and serum provided similar levels for 24,25(OH)D3, 25(OH)D3 and cholecalciferol (D3), while significantly higher concentrations of 1,25(OH)D3 were detected in plasma versus serum [Four previous studies have compared sample type for 25(OH)D quantification using LC-MS/MS; however, the samples were collected from non-pregnant populations of small sample sizes. Zhang et al. Dus serum . Howeverus serum .n = 88) and non-pregnant women (n = 20) and found differences in vitamin D metabolism across a range of analytes in pregnancy, as well as across gestation [It is noted that none of the previous studies examined comparability of total 25(OH)D serum and plasma concentrations, and no study has examined changes in clinical vitamin D status based on sample type during pregnancy. The effect of the sample type on resulting concentrations of 25(OH)D and its analytes during pregnancy needs to be identified clearly to ascertain the suitability of using serum and plasma interchangeably in clinical and research settings. Pregnancy is associated with various hormonal and physiological changes in the body, and with vitamin D also acting as a hormone, adaptive changes of vitamin D homeostasis and metabolite concentrations in pregnancy may have implications for the systemic circulation of total 25(OH)D and its analytes . Vitaminestation . WhetherThe primary aim of this study was to investigate the comparability of vitamin D quantification using LC-MS/MS between serum and plasma samples collected during pregnancy in a large well-defined cohort of women enrolled in a clinical asthma trial. The secondary aim was to examine the proportion of women who change vitamin D category based on sample type.Serum and plasma samples were collected from pregnant women who were enrolled in a clinical trial of asthma management during pregnancy, conducted in Newcastle, Australia. Details of the trial are previously described . Briefly2, 25(OH)D3 and Epi-25(OH)D3. The LoD for both 25(OH)D3 and Epi-25(OH)D3 was 2.0 nmol/L, and 3.0 nmol/L for 25(OH)D2. For blood samples with values below the LoD, a level equal to the detection limit was used, then divided by the square root of 2 D3 and 2.12 nmol/L for 25(OH)D2) [A non-fasting peripheral blood sample was collected by venepuncture (by a research nurse trained in phlebotomy) into a 6ml EDTA (1.8 mg/mL) plasma tube and 6mL plain serum tube at enrolment (between 12–25 weeks gestation), and processed within 60 min. All samples were centrifuged (3000 rpm) at 4 °C for ten minutes, aliquoted into Eppendorf tubes, and stored at −80 °C. All samples were collected between 2017–2019 and analysed in 2019. Samples were transported by courier on dry ice to the Centre for Microscopy, Characterisation & Analysis in Western Australia, for quantification of vitamin D via LC-MS/MS; this laboratory is certified by the Vitamin D Standardisation Program (VDSP) . LC-MS/M5(OH)D2) . Vitamin5(OH)D2) .2, 25(OH)D3, epi-25(OH)D3 and detect proportional bias [2, 25(OH)D3 and Epi-25(OH)D3 were examined using the paired t-test with 95% confidence intervals (CI). A p-value < 0.05 was considered statistically significant. The mean, range, standard deviation (SD) and coefficient of variation (CV) were reported for each analyte by sample type. The proportion of participants that were vitamin D sufficient, insufficient and deficient were determined based on sample type, as well as any participants that changed category based on the use of plasma compared to serum for total 25(OH)D quantification. Cohen’s Kappa test was used to explore interrater reliability between participants vitamin D status and sample type. Statistics were computed with STATA IC v15.1 , and Microsoft Excel .The Shapiro Wilks test was used to determine normality of the data. Bland-Altman plots were used to compare the difference between plasma and serum concentrations of total 25(OH)D, 25(OH)D2 and 11.8% (27/228) samples were below the LoD for Epi-25(OH)D3  samples were below the LoD for 25(OH)D25(OH)D3 . The mea25(OH)D3 .2 = 0.903), 25(OH)D3 (r2 = 0.910) and Epi-25(OH)D3 (r2 = 0.862); correlation analyses were not conducted for 25OHD2 given the high percentage of samples below the LoD.p = < 0.001) for total 25(OH)D, 7.38 nmol/L  for 25(OH)D3, and 0.39 nmol/L  for Epi-25(OH)D3 D3 .p ≤ 0.001) with 83.3% agreement found. In total, 16.7% (n = 19) of participants changed vitamin D status category based on which sample type was used. Of the n = 19 that changed vitamin D status based on serum vs plasma samples; n = 12 participants changed from insufficient (<75 nmol/L) to sufficient (≥75 nmol/L), n = 3 changed from deficient (<50 nmol/L) to insufficient (50- <75 nmol/L), n = 1 changed from insufficient to deficient and n = 3 changed from sufficient to insufficient.In regards to vitamin D status based on sample type, for total 25(OH)D; results are shown in This study is the first to examine the comparability of vitamin D quantification by LC-MS/MS in serum and plasma samples collected during pregnancy, and the first to examine the impact of sample type on vitamin D adequacy. Despite a relatively high Pearson and intraclass correlation between serum and plasma concentrations, examination of the concentration bias revealed a significant difference in the mean concentrations of total 25(OH)D in serum vs. plasma. Furthermore, we found 16.7% of participants changed category based on sample type, with 13% changing from deficient to insufficient or insufficient to sufficient with the use of plasma instead of serum.These results are of concern in a clinical context, especially for pregnant women, as even small differences for patients close to cut-off points for vitamin D deficiency or insufficiency may result in misclassification of their vitamin D status, and may influence subsequent treatment decisions. The current national Australian pregnancy guidelines state that vitamin D supplementation may be considered for women with vitamin D levels <50 nmol/L, highlighting the impact this may have on recommended maternal supplementation, based on the vitamin D test result obtained . Cut-poiDue to our large sample size, we were able to use Kappa statistic to explore participant’s vitamin D status category based on sample type ,31. Our Assessing the comparability of sample types for results is important for future research and to assist in the VDSP’s goal of standardising vitamin D analysis. These results provide evidence of an important difference in vitamin D quantification, and assessment of vitamin D status, during pregnancy, based on sample type. These results are applicable to LC-MS/MS methods; whether there are differences with nonchromatographic methods based on radioimmunoassay’s, such as antibody assays, is unknown. Whether the results would be altered with different anticoagulants used is unknown and requires further investigation. Although serum and plasma are commonly used blood specimen types; they are not equivalent biological matrices . Serum a


We determined whether plasma concentrations of the receptor for advanced glycation end products (RAGE) and the soluble (s) form of RAGE (sRAGE) in healthy individuals and patients with type 2 diabetes (T2D) modulate vascular remodeling. Healthy individuals and patients with T2D were divided into two age groups: young = <35 years old or middle-aged (36–64 years old) and stratified based on normal glucose tolerance (NGT), impaired (IGT), and T2D. Plasma titers of sRAGE, the RAGE ligands, AGEs, S100B, S100A1, S100A6, and the apoptotic marker Fas ligand Fas(L) were measured by enzyme-linked immunosorbent assay (ELISA). The apoptotic potential of the above RAGE ligands and sRAGE were assessed in cultured adult rat aortic smooth muscle cells (ASMC). In NGT individuals, aging increased the circulating levels of AGEs and S100B and decreased sRAGE, S100A1 and S100A6. Middle-aged patients with T2D presented higher levels of circulating S100B, AGEs and FasL, but lower levels of sRAGE, S100A1 and S100A6 than individuals with NGT or IGT. Treatment of ASMC with either AGEs or S100B at concentrations detected in T2D patients increased markers of inflammation and apoptosis. Responses attenuated by concomitant administration of sRAGE. In middle-aged patients with T2D, lower circulating plasma levels of sRAGE may limit decoy and exogenous trapping of deleterious pro-apoptotic/pro-inflammatory RAGE ligands AGEs and S100B, increasing the risk for diabetic complications. N-carboxymethyl-lysine (CML), on circulating proteins such as albumin [Diabetes mellitus can result from insulin deficiency Type 1-T1D) or insulin resistance Type 2-T2D) and constitutes a worldwide epidemic [-T1D or i-T2D and  albumin . Studies albumin ,19.RAGE is a membrane-bound protein that is structurally composed of three extracellular Ig-like domains, a single transmembrane helix, and a short intracellular domain . Ligand 1c [Cross-sectional human population studies report that circulating sRAGE is a potential biomarker of several pathologies, although conflicting data exists in the context of diabetes and cardiovascular diseases . Decreas1c . Interes1c . Alterna1c ,40,41. F1c . The divThese findings identify sRAGE to be a marker of diabetic complications. However, none of these studies address a key gap in our current knowledge—quantifying RAGE ligand levels, specifically the S100 proteins, in parallel with plasma sRAGE expression. Since diabetes may differentially impact the mechanisms controlling RAGE ligand generation and clearance, determining sRAGE levels in parallel with circulating RAGE ligands could be useful for assessing diabetic risk factors. In this study, we aimed to quantify total sRAGE, the RAGE ligands AGEs, S100B, S100A1 and S100A6 in a young, lean, healthy human reference group and middle-aged individuals stratified according to glucose tolerance status (GTS). We hypothesized that impaired glucose tolerance (IGT) and T2D reduces circulating sRAGE in comparison to a lean, healthy reference group. To validate our findings, we used a rodent abdominal aortic vascular smooth muscle cell (ASMC) culture model to determine whether circulating concentration of sRAGE acts as a decoy for cytoprotection against high glucose and/or RAGE ligand inflammatory/apoptotic actions.The plasma level of the decoy sRAGE, that bind excess RAGE ligands, was decreased in middle-aged individuals with NGT-B, IGT and T2D compared to younger individuals with NGT-A A. ComparIn cultured adult rodent abdominal ASMC, 24 h exposure to AGEs at a concentration (50 μg/mL) seen in the plasma of T2D increased the expression of S100B and RAGE and reduced S100A1 and S100A6 expression A. The adTo determine whether the RAGE ligands AGEs, S100B, S100A1, and S100A6 directly induce ASMC early and late apoptosis, ASMC were treated for 24 h with either vehicle (PBS), sRAGE (1.5 ng/mL), S100B (100 pg/mL), AGEs (50 μg/mL), S100A6 (1.5 ng/mL-concentration observed in the plasma of young NGT-A), or S100A1 (10 ng/mL-concentration observed in young NGT-A) A–C. TreaTo define the cellular mechanisms by which RAGE ligation leads to apoptosis, we evaluated the cleavage of the initiator death receptor pathway caspase-8 and the executioner mitochondria-dependent caspase-3. A co-immunoprecipitation strategy followed by Western blot analysis demonstrated heterodimerization of caspase-8 and RAGE in ASMC cell lysates treated with vehicle (PBS), HG, AGEs, S100B, or S100A6 A. As repThe MAPK signaling pathway plays an important role in diabetic vascular damage ,22. TherTo our knowledge, the present study is the first to report circulating concentrations of sRAGE with the RAGE ligands AGEs, S100B, S100A1 and S100A6 in the context of young, healthy human individuals and middle-aged individuals who are either healthy, have impaired glucose tolerance, or T2D. Our primary findings show young individuals present the highest concentration of sRAGE, anti-apoptotic S100A1, and S100A6 and the lowest concentration of pro-apoptotic AGEs and S100B when compared to middle-aged individuals with NGT, IGT or T2D. These findings are consistent with previous reports showing lower sRAGE expression in patients with obesity and impaired glucose tolerance ,35,36,37Our data are, admittedly, limited by the number of individuals per group and not being age-matched across all groups, but do show that chronological age plays an essential role in sRAGE concentration. Secondly, we acknowledge that this study was completed at a single site with one demographic population (Greek). Finally, our data is limited by the measurement of only total sRAGE using the R&D ELISA kit. Total sRAGE levels decrease with advancing age. Total sRAGE consists of two circulating soluble isoforms: esRAGE derived from alternative splicing and cRAGE generated by proteolytic cleavage of RAGE by ADAM10 and/or membrane-bound RAGE proteolysis (MMPs). esRAGE does not vary during aging and eventually represents the predominant circulating sRAGE isoform in the elderly . ConversInterestingly, in middle-aged individuals, circulating sRAGE in states of impaired glucose tolerance and T2D remains comparable to NGT. Conflicting results have been reported regarding circulating sRAGE and T2D ,35,36,37Our study also reveals that aging also decreases the concentration of the cytoprotective RAGE ligands S100A1 and S100A6 with a further decrease in states of IGT and T2D whereas pro-apoptotic/inflammatory circulating AGEs and S100B accumulate in T2D. Additionally, inverse correlations between sRAGE and S100B in T2D, and S100A6 and AGEs in IGT characterize middle age states of IGT and T2D by a shift from cytoprotective  to pro-apoptotic/pro-inflammatory  RAGE ligands. However, the juxtaposition of the T2D phenotype against a young, healthy phenotype demonstrates the degree to which circulating sRAGE, S100A1, S100A6, AGES, and S100B in states of IGT, and advanced age deviate from optimum health. We have demonstrated here that dysregulation of sRAGE and ligands of RAGE is associated with different phenotypes, and therefore are likely under parallel regulation. The progressive decrease of sRAGE and cytoprotective S100A1 and S100A6 with aging may result in the accumulation of AGEs and S100B, fueling signaling pathways favoring the diabetic state.It is well established that the binding of RAGE with ligands drives a cascade of signaling that converges at NF-κB, promoting inflammatory and apoptotic processes ,22. RAGERAGE is a pattern-recognition receptor that predominately binds to endogenous molecules that are generated or released during cellular, physiological, or pathological stresses ,11,12,13A large number of middle-aged (36–65 years) Greek diabetic patients with an initial diagnosis different from insulin-dependent diabetes mellitus (IDDM) (type 1 diabetes) were evaluated at the outpatient Diabetic Clinic of Attikon University General Hospital in Athens, Greece, between June 2009 and May 2012 after giving their informed consent. Patients with infection, liver disease, or pregnancy were excluded from the study. Upon enrollment into the study, height and weight measurements were collected from each patient. After overnight fasting, data from a 75-g oral glucose tolerance test (OGTT) was collected from each patient. In parallel, blood samples were obtained through peripheral venipuncture at 0 and 120 min to measure plasma glucose, serum insulin, and serum C-peptide concentrations. Patients with a known history of diabetes or a fasting plasma glucose ≥126 mg/dL did not undergo an OGTT and were excluded from the study. A total of 30 patients were enrolled in the study and classified according to the criteria by the American Diabetes Association into 10 individuals with normal glucose tolerance (NGT), 10 with impaired glucose tolerance (IGT) and 10 with type 2 diabetes (T2D). Ten healthy control individuals (<35 years of age) were used as a reference group. All subjects had no family history of diabetes mellitus, demonstrated normal blood glucose levels, and provided informed consent.The institutional review board of “Attikon” University General Hospital approved this study during the 19th meeting 11 March 2009. All patients gave their written informed consent.Serum cholesterol , and high-density lipoprotein (HDL)), triglycerides, aspartate transaminase (AST), alanine aminotransferase (ALT), and gamma glutamyl transferase (γ-GT) were assessed using a Cobas 8000 analyzer . Plasma glucose was measured using the glucose oxidase method . Serum C-peptide and insulin levels were quantified using the respective radio-immunoassays from Millipore .Total plasma sRAGE in humans was determined by a commercial enzyme-linked immunosorbent assay (ELISA) . S100B , AGEs , and FasL  levels were determined by enzyme immunometric assay according to manufacturer instructions. For the human S100A1 ELISA, a 96-well plate was coated with a rabbit anti-S100A1 antibody  and incubated overnight at 4 °C temperature. Recombinant S100A1  serving as a standard, and experimental samples were added and incubated overnight at 4 °C. Sheep anti-human S100A1  was added, followed by incubation for 3 h at room temperature. Donkey anti-sheep antibody  was added and further incubated for 2 h at room temperature. TMB substrate  was added followed by incubation for 20 min at room temperature in the dark. The reaction was stopped by adding stop solution, and the color was read at 450 nm with a correction at 570 nm. For the S100A6 ELISA, a 96 well plate was coated with sheep anti-S100A6  overnight at 4 °C then washed and blocked overnight. Aliquots of recombinant S100A6  serving as a standard and experimental samples were added and incubated overnight at 4 °C. Chicken anti-S100A6  solution was added followed by a 3 h incubation at room temperature. Goat anti-chicken HRP  was added and the samples were incubated for an additional 2 h. TMB substrate was added followed by a 20 min incubation for 20 min at room temperature. The reaction was stopped by the addition of stop solution and the color was read at 450 nm with a correction at 570 nm.Primary cultures of vascular smooth muscle cells (SMC) were initiated by enzymatic dissociation from the abdominal aortas (A) of eight-week-old Sprague Dawley rats, as previously described . CultureN-terminal kinase (JNK), total JNK , cleaved and total caspase-3  and caspase-8  antibodies.Cell lysates were prepared from frozen powder, and immunoprecipitation studies and Western blotting were performed as described previously ,46,50. It) values for the housekeeping gene 18S and the gene of interest in each sample were determined. For each gene of interest, the mean fold-change is shown relative to vehicle/control.RNA was extracted from primary cultures of abdominal aortic vascular smooth muscle cells using Trizol  and reverse transcribed using QuantiTeck kit . Real time quantitative (RT)-PCR was performed according to the instructions of the manufacturer (Qiagen) and as previously described ,50. In bTM caspase-3 Assay kit #2 .Cellular apoptosis was determined by flow cytometric analysis using the FITC Annexin V-Apoptosis detection kit . Caspase-3 activity was measured using Enzchekp values lower than 0.05 were considered statistically significant.The study sample characteristics are given as percentages for categorical variables, mean and standard error of the mean (SEM) for continuous variables with normal distribution, and median (25th and 75th percentiles) for continuous variables without normal distribution. The Kolmogorov-Smirnov test was used to test for normal distribution of data. Group comparisons for these variables were performed using Fisher’s exact test, t-test, or ANOVA with Dunnett’s multiple comparison test (for two or more groups) and Kruskal-Wallis test with Dunn’s multiple comparison test for more than two groups, respectively. Correlations were assessed as partial Spearman correlation coefficients adjusting for the variables indicated. In conclusion, younger individuals exhibit high circulating levels of the decoy sRAGE. In middle-aged individuals, particularly those with T2D, decreased plasma sRAGE may limit decoy and exogenous trapping of deleterious RAGE ligands AGEs and S100B, increasing the risk for diabetic complications. Our in vitro results with ASMC support pro-inflammatory and proapoptotic effects of AGEs and S100B at concentrations observed in the plasma of T2D. Taken together, these studies suggest that clinically measuring sRAGE and RAGE associated ligands, namely AGEs and S100 proteins, may serve as a feasible diagnostic and/or prognostic marker to assess the risk of developing diabetes and diabetes-induced complications as individuals age. Importantly, prospective clinical trials will have to define the impact of sRAGE and RAGE ligands as biomarkers of cardiovascular risk in T2D.