FLJ3802842 and rs4631962 in CCND2 were significantly associated with CRC risk in the Taiwanese population. The previously unreported rs1338565 was associated with a significant increased risk of CRC. In addition, we also genotyped tumor tissue and paired adjacent normal tissues of these 705 CRC cases to search for LOH, as well as risk-associated and protective alleles. LOH analysis revealed preferential retention of three SNPs, rs12657484, rs3802842, and rs4444235, in tumor tissues. rs4444235 has been recently reported to be a cis-acting regulator of BMP4 gene; in this study, the C allele was preferentially retained in tumor tissues (p = 0.0023). rs4631962 and rs10795668 contribute to CRC risk in the Taiwanese and East Asian populations, and the newly identified rs1338565 was specifically associated with CRC, supporting the ethnic diversity of CRC-susceptibility SNPs. LOH analysis suggested that the three CRC risk variants, rs12657484, rs3802842, and rs4444235, exhibited somatic allele-specific imbalance and might be critical during neoplastic progression.Given the significant racial and ethnic diversity in genetic variation, we are intrigued to find out whether the single nucleotide polymorphisms (SNPs) identified in genome-wide association studies of colorectal cancer (CRC) susceptibility in East Asian populations are also relevant to the population of Taiwan. Moreover, loss of heterozygosity (LOH) may provide insight into how variants alter CRC risk and how regulatory elements control gene expression. To investigate the racial and ethnic diversity of CRC-susceptibility genetic variants and their relevance to the Taiwanese population, we genotyped 705 CRC cases and 1,802 healthy controls (Taiwan Biobank) for fifteen previously reported East Asian CRC-susceptibility SNPs and four novel genetic variants identified by whole-exome sequencing. We found that rs10795668 in  Colorectal cancer (CRC) affects 1.23 million people worldwide and causes 0.6 million deaths annually; it is becoming the most frequently diagnosed cancer in developed countries There is a direct association between sporadic tumor occurrence and susceptibility variants carried by an individual. Two percent of the European population carries multiple inherited low-risk alleles that increase the rate of CRC incidence about four-fold Neoplastic progression is often associated with accumulation of somatic-cell genetic changes as the tumor progresses 2≥0.8) with the original SNPs.GWAS identify SNPs that tag linkage disequilibrium (LD) blocks in the genome, thus capturing a large proportion of common genetic variations. Fifteen SNPs associated with CRC in the East Asian populations  were evaluated in a Taiwanese population In this study, the tumor and adjacent non-tumor tissues of 705 colorectal cancer patients in Taiwan were collected and genotyped in an attempt to replicate GWAS findings and evaluate the possibility of the allele-specific imbalance in tumor tissues. We found a novel SNP (rs1338565) and two published SNPs (rs10795668 and rs464631962) associated with CRC risk and three SNPs  showing statistical significance in allele specific retention in tumors.http://taiwanview.twbiobank.org.tw/taiwanview/search.do). Written informed consent was obtained from all patients and the study was approved by Institutional Review Board of Taipei Veterans General Hospital, Taipei, Taiwan. The genomic DNA of paired tumor and adjacent normal tissues were extracted using QIAamp Mini Kit according to the manufacturer’s protocols (Qiagen).The study included a population-based series of 705 paired CRC tumors and adjacent normal tissues collected since 2006 at Taipei Veterans General Hospitals. All tissue samples of FFPE were diagnosed by experienced pathologists. The tumor tissues consist of 50% or more tumor cells for collecting DNA. Germline DNA extracted from RNAlater-immersed adjacent normal colonic tissue and corresponding tumor DNA were available. The 1802 control subjects were anonymous healthy blood donors from the Taiwan Biobank (Exome sequence capture was performed following the procedure provided for the Agilent SureSelect Platform (SureSelect Human All Exon V4 kit). The captured library was performed with paired-end 90 base reads on the Illumina HiSeq 2000 platform. The average sequencing depth is more than 100-fold, and the coverage of target region is at least 99%.The adapter sequence in the raw data was removed, and low quality reads were discarded. Sequence reads were aligned to the reference genome (hg19) using the BWA program Nineteen CRC- susceptibility SNPs were genotyped in all samples, and among them, 15 were reported to be directly or indirectly (LD γ2>0.8) associated with CRC susceptibility in East Asians Pearson’s χ2 tests were used to compare the difference of SNP allele and genotype frequencies between cases and well-match controls. Hardy–Weinberg equilibrium of each SNP was tested by goodness-of-fit χ2 test to compare the expected frequency of genotypes in controls. The effects of polymorphisms on the risk of colorectal cancer were expressed as odds ratios (ORs) with 95% confidence intervals (95% CIs), evaluated using unconditional logistic regression analysis. To identify allele-specific imbalance, the genotype of each SNP  was compared in tumor and adjacent non-tumor tissues, and only patients with germline SNP heterozygous calls were used for following analysis. Fisher’s exact test was used for analyzing SNP LOH in cancer tissues. Principal component analysis (PCA) was used to clarify population stratification using 44 unlinked SNPs. Bonferroni and permutation methods were used to adjust p-values in multiple comparisons. All the above statistical analyses were performed using SAS/STAT version 8 software .2≥0.8) between them.Two resources of CRC candidates SNPs were used in this study. One was selected from previous East Asian CRC-susceptibility association article We originally searched a set of genes and mutations in colorectal polyps to assess patients’ subsequent carcinogenic risk. We performed exomic-sequencing experiments on paired tissues from seven CRC patients  and six cases of incidental, non-cancer-related polyps. After differential analysis, we selected 62 CRC carcinogenesis-related gene variations present in tumors and cancer-synchronous polyp but absent in normal tissues and incidental polyps to be verified in 47 pairs of CRC tumor and paired normal adjacent tissues by Sequenom MassArray (data not shown). We identified four candidate SNPs associated with CRC susceptibility in a comparison with 1802 healthy controls from the Taiwan Biobank.We combined 15 reported and 4 newly-identified SNPs to genotype these SNPs in 705 independent CRC pairs of tumor and paired normal adjacent tissues. Although Taiwan Biobank is a database which randomly enrolled samples in the population and serves as good and public controls for other projects. Due to population stratification in these CRC patients and controls, we genotyped 44 frequently used unlinked SNPs  of the mitochondrial ATP synthase ATP5C1 associated with the A allele of rs10795668 would be consistent with maintaining the activities of ATP synthase and cellular respiration and potentially inhibiting tumor progression in colorectal cancer.rs10795668 was reported to be associated with CRC risk and conferred better overall survival CCND2, was identified in East Asians by GWAS 2 = 0.825) with rs4631962, for which the major allele frequency is known for healthy individuals in the Taiwan Biobank CCND2, which encodes cyclin D2, a member of the D-type cyclin family. CCND2 is a critical mediator of cell cycle control (from G1 to S phase) and is overexpressed in a substantial proportion of human colorectal tumors CCND2 is an independent predictor of survival in individuals with CRC PARP11, C12orf5, FGF6, and RAD51AP1are also in close proximity to the SNP; C12orf5 and RAD51AP1 are overexpressed in CRC tissue The new CRC susceptibility locus rs10774214, distally located 150 kb upstream of ZNF239 gene on chromosome 10q11.22. Two neighboring SNPs, rs2230660 and rs2230661, produce missense variations in the exon region of ZNF239 and are in strong LD with rs1338565. The biological rationale for the association between ZNF239 and CRC has not been explored. ZNF239 is a zinc-finger protein that recognizes both DNA and RNA. This dual affinity suggests ZNF239 is involved in transcription and post-transcriptional regulation. As a DNA-binding transcriptional repressor, it represses the IRBP (interphotoreceptor retinoid-binding protein) gene by competing with the CRX (cone-rod homeobox protein) transcriptional activator for DNA binding ZNF239 interactions with lamin A/C and the nuclear matrix may be important for its ability to repress transcription The newly identified rs1338565 is located in an intron of the SMAD7 expression, leading to aberrant TGFβ signaling BMP4 at 14q22 and GREM1 at 15q13 affect TGFβ signaling CRC susceptibility-associated genetic variations have been found to affect gene expression through distant regulatory elements. Our study showed rs4939827 had the highest rate of LOH in CRC patients (36%). rs4939827 reduces 2 = 0.926) with rs647161, which is a newly discovered CRC susceptibility locus in East Asians rs3802842 and rs4444235 have been associated with increased risk of CRC in different populations, but this association was not replicated in our study. rs12657484 showed strong LD , PITX1 is closer to rs12657484 (approximately 134 kb upstream) than rs647161 (approximately 157 kb upstream). The PITX1 gene (encoding paired-like homeodomain 1) has been reported as a tumor suppressor and may be involved in the tumorigenesis of multiple human cancers PITX1 human cancer tissues and cell lines; PITX1 suppresses tumorigenicity by down-regulating the RAS pathway PITX1 was observed in KRAS wild-type CRC tissue, not KRAS-mutant tissue PITX1 may also activate TP53PITX1 expression has been associated with poor survival in CRC patients Rs1265484 is located on chromosome 5q31.1, where a cluster of SNPs are associated with CRC risk C11orf93, with CRC risk was first identified in a GWAS C11orf93 expression by altering the transcription factor binding site POU2AF1, C11orf53, FLJ45803, and LOC120376) and a SNP (rs12296076) identified as polymorphic binding sites for miRNAs in high linkage disequilibrium The association of rs3802842 (11q23), located in the intron of BMP4) gene BMP4 expression. Given that the overexpression of the BMP genes could suppress the Wnt signaling pathway by preventing β-catenin activation and increasing migration and invasion of the mesenchymal phenotype in CRC Previously published GWAS found a significant association between CRC susceptibility and rs4444235 located on chromosome 14q22.2 within the bone morphogenetic protein-4  and paired normal adjacent tissues (N) using Sequenom iPLEX genotyping method and default calling algorithm. Table S1: Allele frequency comparisons of 705 cases and 1,802 controls using 44 unlinked SNPs.(DOCX)Click here for additional data file.


Bisphenol A (BPA) is a ubiquitous endocrine disrupting chemical in our daily life, and its health effect in response to prenatal exposure is still controversial. Early-life BPA exposure may impact brain development and contribute to childhood neurological disorders. The aim of the present study was to investigate molecular target genes of neuronal development in trans-placental exposure to BPA.A meta-analysis of three public microarray datasets was performed to screen for differentially expressed genes (DEGs) in exposure to BPA. The candidate genes of neuronal development were identified from gene ontology analysis in a reconstructed neuronal sub-network, and their gene expressions were determined using real-time PCR in 20 umbilical cord blood samples dichotomized into high and low BPA level groups upon the median 16.8 nM.−4 and log2-transformed fold change ≤−1.2) in response to BPA exposure. Fetal cord blood samples had the obviously attenuated gene expression of Sox2 and Pax6 in high BPA group referred to low BPA group. Visualized gene network of Cytoscape analysis showed that Sox2 and Pax6 which were contributed to neural precursor cell proliferation and neuronal differentiation might be down-regulated through sonic hedgehog (Shh), vascular endothelial growth factor A (VEGFA) and Notch signaling.Among 36 neuronal transcripts sorted from DAVID ontology clusters of 457 DEGs using the analysis of Bioconductor limma package, we found two neuronal genes, sex determining region Y-box 2 (Sox2) and paired box 6 (Pax6), had preferentially down-regulated expression (Bonferroni correction p-value <10These results indicated that trans-placental BPA exposure down-regulated gene expression of Sox2 and Pax6 potentially underlying the adverse effect on childhood neuronal development. Bisphenol A (BPA) has a frequent industrial use as a sealant or flux of plastic materials. Its application results in human exposure through the intake of foods and liquids in polycarbonate water bottles, food wraps, plastic bags, baby bottles, protective coatings on food containers, epoxy resin, and dental composites In mammalians, BPA can easily pass through placenta during pregnancy and has a latent effect on postnatal reproductive functions Concerns have been raised about the effect of trans-placental BPA exposure on central nervous system and neuronal development. Early life BPA exposure has been associated with behavior problem such as anxiety, depression, and hyperactivity in children Microarray analysis is an effective way to explore possible mechanisms and has been used to study the molecular pathway of reproductive toxicity of BPA exposure in animal models http://www.ebi.ac.uk/arrayexpress/) (http://www.bioconductor.org) implemented in R (http://cran.r-project.org), and were filtered by choosing probes having a standard deviation >0.15 over all the samples in the analysis. Two different platform chips (Agilent Whole Human Genome Microarray and Affymetrix HG-U133Plus2.0) were pre-processing using RMA algorithm (http://rmaexpress.bmbolstad.com/) prior to merging each other. We selected genes from all platforms based on the NIH Entrez Gene ID and used the median rank score method with the R package CONOR −3  in this study. This study defined 10−4 as the cutoff p-value to make selected genes more specific. Therefore, the genes with expression values in the quadrant of greater or less than log-transformed fold change ±1.2 and p-value less than 10−4  were selected for further network construction and pathway prediction.Three microarray datasets of human cell models in exposure to low-dose BPA were selected from the ArrayExpress database (xpress/) . Data weThis study randomly selected 20 umbilical cord blood samples of 157 healthy pregnant women in a previous birth cohort study The umbilical cord blood samples were centrifuged at 12,000 rpm for 10 min to separate the plasma and corpuscles, and stored at −80°C until analysis. Plasma fraction (500 µl) was mixed with 100 µl of 0.01 M ammonium acetate buffer , 4 ml mixture of n-hexane  and diethyl ether . Then 8.71 µl of 9.187 M perchloric acid  was added into the plasma mixture and centrifuged with 3,000 rpm for 5 minutes. After centrifugation, the organic layer was evaporated to dryness and reconstituted with 100 µl of mobile phase (methanol:water 80∶20 v/v) for BPA determination by a reverse-phase high performance liquid chromatography (HPLC) (D-7000) connected to a UV detector (L-7400) consisting of an autosampler (L-7200), a pump (L-2130) and a degasys (DG-2410) . The QA/QC materials were prepared from a plasma pool in analysis with standard, reagent blank and unknown samples. We performed external calibration using the chromatographic responses of seven standard concentrations in their corresponding solvent. The recovery rates of blanks extended from 96–103%. The relative standard deviation (RSD) among triplicate analyses were 1.99–7.53%, and the recovery percentage was 96.1% with an RSD of 7.53%. The limit of detection (LOD) was 1.75 nM.The buffy coat of umbilical cord blood sample was pretreated using RBC lysis buffer to avoid the interference of red blood cells. RNA isolation was conducted using Trizol reagent with chloroform and isopropanol. The RNA pellet was washed using 75% alcohol and air dried. Total RNA was dissolved in DEPC contained water and stored under −80°C. Total RNA was reverse-transcribed to cDNA using the high capacity cDNA reverse transcription kits  for quantitative real-time analysis. PCR primers  were designed using Primer Express V.3.0 software  according to the mRNA sequence from GenBank. Real-time PCR was performed with the FastStart SYBR Green Master  and was analyzed using the 7300 Real-Time PCR system . The relative level of mRNA expression was analyzed using comparative method by SDS 1.4 software normalized to the endogenous housekeeping gene β-actin.The plug-in system “ClueGO+CluePedia” in the latest version of Cytoscape Software 3.0.2 was used to identify networks and functional pathways of DEGs in response to BPA exposure. ClueGO −4 and ≤−1.2 log2-transformed fold change) in comparison with control respectively responsible for developing neural tube cells and regulating neurogenesis in radial-glial-like neural stem cells Twenty human umbilical cord blood samples were randomly selected from our previous birth cohort This study imported 457 DEGs into Cytoscape plug-in ClueGO+CluePedia to investigate the gene network and functional pathway prediction in response to BPA exposure. The results of Cytoscape analysis presented that totally 959 genes constructed a gene network connecting with the 457 DEGs . The genThe results of ClueGO analysis presented a predicted pathway of Sox2 and Pax6 and their potential interaction genes resulted from BPA exposure in Human exposure to chemicals relevant to disease outcome is difficult to be determined and estimated, especially for the effect on child development. Although some studies have found that BPA affects growth and development of reproductive organ In this study, the step-wise approach including the meta-analysis of microarray DEGs, GO and Bonferroni correction between high and low BPA exposure was used to investigate the neuronal candidate genes Sox2 and Pax6 in exposure to BPA. Additionally, the gene expression of Sox2 and Pax6 were determined in human fetal cord blood to evaluate whether it could be used as biomarkers for childhood neurogenesis deficiency resulting from trans-placental BPA exposure. This study found higher BPA exposure level in human fetal cord blood samples decreased gene expression of Sox2 and Pax6. The visualized network analysis showed that Sox2 and Pax6 were highly involved in the function of neurogenesis. This alternative method to evaluate the impact of xenobiotics exposure on human is more effective than an animal model, and more convenient and time-saving than a cohort study for a long time follow-up.Several but not many studies evaluated the impact of low-dose BPA exposure (<100 nM) on neuronal development. In human embryonic stem cells, BPA exposure  influences mammosphere area and down-regulate the expression of E-cadherin protein in the early differentiation stage of mammary epithelial cells Prenatal BPA exposure impairs murine fetal neocortical development by accelerating neuronal differentiation/migration during the early embryonic stage to affect neuronal plasticity and interferes corticosterone and corticosteroid receptors in the hippocampus This study found that BPA exposure decreased the gene expression of Sox2 and Pax6 in fetal cord blood, and Sox2 and Pax6 were involved in neuronal development according to network analysis. Findings in this study were consistent with a Xenopus laevis model that prenatal BPA exposure decreases the expression of Sox2 and Pax6 and disrupts Notch signaling to inhibit gamma-secretase activity for neurodegenerative abnormalities The visualized network analysis in response to BPA exposure was addressed in Shh pathway plays a role in many processes during embryonic development and remains active in the adult involving in the maintenance of stem cells In general, VEGFA positively regulates Sox2 through Prox1 gene in embryonic eye development and lens differentiation Notch pathway has been recognized as one of the main contributors in regulating neural development and has been proposed as a key mediator in neuroplasticity This study presented an alternative way to investigate the adverse effect of trans-placental xenobiotics exposure on child development in meta-analysis of public microarray datasets for exploring target genes and their potential gene network of neuronal functions. In the network-analysis of BPA exposure, we identified Sox2 and Pax6 as major neuronal transcript candidates and were involved in tetrapyrrole metabolic process. Sub-network analysis identified that Sox2 and Pax6 were highly involved in regulation of neuronal precursor cells and maintenance of stem cells, which supported the importance of Sox2 and Pax6 in neurogenesis. Furthermore, the decreased expression of Sox2 and Pax6 genes were determined in higher BPA level group of fetal umbilical cord blood. The findings suggested that trans-placental BPA exposure might down-regulate Sox2 and Pax6 expression and cause adverse effect on childhood neuronal development. It is necessary to pay attention in maternal BPA exposure during the period of pregnancy and its effect on child development.Figure S1Process of matching probes among different microarray platforms. Microarrays from Affymetrix and Agilent platforms were originally normalized and summarized using RMA and 75th percentile normalization methods, respectively. Probes were combined to take the average measurements for genes with more than two probes. After the combination of genes from both platforms, correlation and median rank score (MRS) were used to select appropriate number of genes for further investigation.(TIF)Click here for additional data file.Table S1457 DEGs in response to BPA exposure and their corresponding ontology clusters.(DOC)Click here for additional data file.Information S1Description text of microarray data processing and differentially expressed analysis.(DOCX)Click here for additional data file.


The Rendu-Osler-Weber disease is a genetic disease which may lead to severe hemorrhage and less frequently to severe organ dysfunction. We report the case of a 22-year-old patient with no personal medical history who was involved in a motorcycle accident and exhibited severe complications related to large arteriovenous pulmonary shunts during his ICU stay. The patient developed an unexplained severe hypoxemia which was attributed to several arteriovenous shunts of the pulmonary vasculature by a contrast study during a transesophageal echocardiographic examination. The course was subsequently complicated by a prolonged coma associated with hemiplegia which was attributed to a massive paradoxical fat embolism in the setting of an untreated femoral fracture. In addition to hemorrhagic complications which may lead to intractable shock, arteriovenous malformations associated with the Rendu-Osler-Weber disease may involve the pulmonary vasculature and result in unexpected complications, such as hypoxemia or severe cerebral fat embolism in high-risk patients. Although epistaxis is usually the first symptom of the Rendu-Osler-Weber disease, the severity of vascular lesions and related organ dysfunctions increases with age , 2. We r2: 99%). An abrupt hypotension occurred (60/35 mmHg) in conjunction with sinus tachycardia (130 bpm), hyperthermia (40°C), and marked oxygen desaturation (SpO2: 81%). Patient's level of consciousness rapidly deteriorated and a petechial rash was noted in the deltopectoral triangle and left eye conjunctiva. There was no evidence of sepsis and blood cultures remained sterile. Biological tests revealed the presence of a coagulopathy  but haemoglobin level was preserved (10.8 g/dL). A vasopressor support was initiated and hypoxemia persisted despite a 100% FiO2 and a PEEP challenge (PaO2/FIO2: 110). Abdominal ultrasound and bedside chest X-ray were unremarkable. Transesophageal echocardiography (TEE) ruled out a traumatic disruption of the aorta and depicted a normal heart. A contrast study excluded a patent foramen ovale but disclosed a massive anatomical shunt of the pulmonary vasculature (2/FIO2: 360). The patient remained comatose with a left hemiplegia. Head CT scan disclosed multiple areas of brain infarction in the right posterior temporal area. Brain magnetic resonance imaging confirmed the presence of ischemic areas located in the right boundaries between the frontal and the parietal areas, associated with a limited infarction confined to the left parietal cortex. The femoral osteosynthesis was performed and as the patient progressively recovered normal consciousness, he was weaned from a ventilator on Day 15. The patient was then discharged from the ICU on Day 23. Further investigations revealed a familial history of the Rendu-Osler-Weber disease, with recurrent severe epistaxis in the patient's father.A 22-year-old motorcyclist without medical history was involved in a violent head-on collision. The patient was initially conscious; he had no motor deficit and no hemodynamic or respiratory compromise. Contrast-enhanced body CT scan ruled out a head trauma but disclosed multiple facial fractures, a mandibular fracture, a rounded opacity in the left pulmonary base consistent with an arteriovenous shunt, a hepatic contusion, a fractured left iliac crest, and a closed fracture of the right femoral diaphysis. The patient was promptly referred to the operating suite where his facial wounds were sutured and a maxillomandibular fixation was placed. The femoral osteosynthesis was postponed due to unstable hemodynamics, and the right lower limb was immobilized with a traction. Hemodynamics were stabilized through the transfusion of red cells and plasma units. The patient was admitted to the ICU with normal blood pressure (120/75 mmHg), body temperature (36.7°C), and blood oxygenation , sustained (negative recruitment maneuvers) hypoxemia under ventilator should raise the diagnosis of an anatomical shunt, mostly intracardiac  or rarely intrapulmonary . TEE conAlthough fat embolism is challenging to recognize in ventilated trauma patients, our patient met all major clinical criteria : petechiOnly 5 to 15% of patients with the Rendu-Osler-Weber disease exhibit anatomical shunt of the pulmonary vasculature . This ge


Weight loss has been shown to significantly improve Adipose tissue (AT) function, however changes in AT gene expression profiles particularly in visceral AT (VAT) have not been systematically studied. Here, we tested the hypothesis that extensive weight loss in response to bariatric surgery (BS) causes AT gene expression changes, which may affect energy and lipid metabolism, inflammation and secretory function of AT. We assessed gene expression changes by whole genome expression chips in AT samples obtained from six morbidly obese individuals, who underwent a two step BS strategy with sleeve gastrectomy as initial and a Roux-en-Y gastric bypass as second step surgery after 12 ± 2 months. Global gene expression differences in VAT and subcutaneous (S)AT were analyzed through the use of genome-scale metabolic model (GEM) for adipocytes. Significantly altered gene expressions were PCR-validated in 16 individuals, which also underwent a two-step surgery intervention. We found increased expression of cell death-inducing DFFA-like effector a (CIDEA), involved in formation of lipid droplets in both fat depots in response to significant weight loss. We observed that expression of the genes associated with metabolic reactions involved in NAD+, glutathione and branched chain amino acid metabolism are significantly increased in AT depots after surgery-induced weight loss. Globally, nearly 1.5 billion adults and 43 million preschool children are overweight (body mass index (BMI) > 25 kg/m2) or obese (BMI > 30 kg/m2)1Obesity is a complex disorder with various contributing genetic, psychological and environmental factors and it is characterized by deposition of an excess amount of fat in the adipose tissue (AT). Obesity has become one of the most serious public health problems of the 21Fat deposition in visceral depots makes obese individuals more prone to complications than subcutaneous fat accumulationIn multiple studies, weight loss has been shown to improve disease risk factors and prevent T2D development in obese individuals7810131417iAdipocytes18092627282930273233343536373839404142iAdipocytes1809 has been validated through the use SAT RNA sequencing data and updated based on the latest proteomics data in Human Protein Atlas (www.proteinatlas.org)4445464748iAdipocytes185026There are already cross-sectional data available for global gene expression profiling in SAT and VAT of obese individuals192021222324iAdipocytes1850. We tested in an unbiased microarray approach the hypotheses that extensive weight loss affects the gene expression profiling of VAT and SAT, and causes alterations in pathways associated with AT energy and lipid metabolism, inflammation, and secretory function (e.g. adipokines). Finally, we validated a subset of our significant findings by quantitative reverse transcription PCR (RT-PCR) methods using SAT and VAT samples obtained from 16 independent individuals that underwent BS.Here, we performed two-step BS with sleeve gastrectomy as first and a Roux-en-Y gastric bypass surgery as second step in 22 severely obese individuals . In ordeIn 22 severely obese individuals, we performed a two step bariatric surgery strategy . Anthropometric and circulating parameters of the participants are shown in Several clinical variables significantly changed and indicated improved health status in these severely obese individuals . Paramet2) and after (26.7 < BMI < 54.6 kg/m2) extensive weight lost, we compared the expression level of genes in AT depots of obese individuals to healthy lean individuals  for hereby, identifying significantly differentially expressed genes before and after the extensive weight loss, and finally detected the direction of the changes in the expression of the genes  biological process (BP) terms to obtain a general perspective on the changes in SAT and VAT likely to result from the weight loss and calculated the gene set statistics using reporter algorithm implemented in the Piano R packageIn SAT, expression of genes including integrin, beta 2 (ITGB2), lipopolysaccharide binding protein (LBP), lymphocyte cytosolic protein 1 (LCP1), matrix metallopeptidase 9 (MMP9), secreted phosphoprotein 1 (SPP1) and Thy-1 cell surface antigen (THY1) associated with the GO Term “immune system process” were significantly decreased after extensive weight loss. Moreover, in VAT, expression of aquaporin 9 (AQP9), complement factor B (CFB), interferon regulatory factor 1 (IRF1), proteoglycan 4 (PRG4) and metalloproteinase inhibitor 1 (TIMP1) associated with the GO Term “immune system process” were significantly decreased after extensive weight loss. This indicated the decreased immune response of the AT depots after weight loss.iAdipocytes1850) in SAT and VAT in response to weight loss, detailed metabolic differences were identified by mapping the significantly differentially expressed genes on the network topology provided by iAdipocytes1850  and patatin-like phospholipase domain-containing protein 2, PNPLA2 (ATGL) were significantly altered by extensive weight loss (p < 0.05). However, after multiple testing the calculated Q-value was higher than 0.05 which we used as a cut of for detecting the statistically significantly changed genes in our study.de novo glutathione synthesis pathways. It is a key metabolite for the reduction of the damage to cellular components caused by reactive oxygen species (ROS), such as H2O2. In order to perform this vital function, cells need to maintain a pool of glutathione in the reduced state (GSH) and this requires an increased pool of glutathione through de novo biosynthesis.We observed that the expression of glutathione s-transferase theta 1 (GSTT1) gene associated with reactions that use glutathione as cofactor is increased in both SAT and VAT after weight loss . GlutathWeight loss may lead to a significant increase in glutathione levels since genes catalyzing reactions that use glutathione as substrate were significantly  upregulated. We first checked the expression of enzymes involved in glutathione synthesis by RT-QPCR and observed that none of these enzymes  were significantly different in SAT and VAT after weight loss. Glutathione may also be regenerated from its oxidized form, GSSG by glutathione reductase (GR) that requires NADPH as a substrate. The resulting NADPH is directly linked to GSH synthesis. Hence, we measured the expression of Nicotinamide nucleotide transhydrogenase (NNT) that catalyzes mitochondrial transmembrane hydride transfer between NADH and NADP+ to generate NADPH in SAT and VAT of the individuals before and after the weight loss by RT-QPCR. We found that expression of the NNT is significantly  increased in the SAT and VAT after extensive weight loss . Taken tde novo and salvage biosynthetic pathways and its utilization. It has been earlier reported that the main sources of NAD+ within the cell are the salvage pathwaysIn addition, expression of nicotinamide nucleotide adenylyltransferase 2, NMNAT2 involved in the NAD+ salvage pathways in SAT as well as changes in the expression NAD+ consuming enzymes in SAT and VAT increased after weight loss . IntracePreviously, it has been shown that AT is the major source of the elevated plasma ROS in obese mice when other tissues including the liver, skeletal muscle, and aorta are examinedMitochondrial dysfunction due to impaired oxidative phosphorylation is a well-established consequence of obesity. Intracellular levels of NAD+ play a key role in the homeostatic control of mitochondrial function of the cell. We hypothesized that increased levels of NAD+ after extensive weight loss could also boost the mitochondrial activity in both fat depots of obese individuals.Previously, we have reported that the expression of the mitochondrial pyruvate carrier 1 (MPC1) and 2 (MPC2) that are essential for several major pathways of carbohydrate, fat, and amino acid metabolismThe branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, are essential amino acids and it has been earlier reported that levels of BCAAs are elevated in plasma of obese individualsWe hypothesized that increased mitochondrial activity of both AT depots after BS, may result in increased catabolism of BCAAs. Hence, we compared the expression of BCAT2 in SAT and VAT of obese individuals before and after weight loss by quantitative RT-PCR analysis  and founAT stores and mobilizes triglycerides and has a major role in regulating whole-body metabolism. Using whole genome expression arrays, we determined SAT and VAT global gene expression changes in six obese individuals undergoing a two-step BS strategy causing extensive weight loss after sleeve gastrectomy (first step surgery). Gene expression profiling of both SAT and VAT in the same individual after significant weight loss allowed us to delineate biological processes most likely related to weight loss. We observed that significant weight loss is associated with significant changes in blood pressure, TGs, HDL-cholesterol, CrP, adiponectin, FPG and FPI. These multiple significant changes in glucose and lipid metabolism as well as AT function in response to weight loss are significant confounding factors, which may either in a concerted mode or as single factors confound the observed gene expression changes. Our study design does therefore not allow drawing conclusions with regard to the specific causative factors of gene expression changes.LDs are an important subcellular organelle that are found in adipocytes and have fundamental roles in metabolism. LDs protect the cell against lipotoxicity by sequestering excess fat and have roles in the development of obesity, T2D and NAFLD. We found that expressions of the SAT enriched gene CIDEA as well as other genes involved in the formation of LDs are increased after extensive weight loss. CIDEA is highly enriched in human SAT compared to other major 32 human tissues in healthy individualsCidea inhibits the brown adipose tissue uncoupling process in rodents and Cidea-null mice are lean and resistant to diet-induced obesityThe expression of CIDEA was also increased in cancer cachexia, a condition associated with extensive weight loss64Alterations in immune pathways were found in obese individuals and the level of inflammation was significantly decreased after extensive weight loss. We found that the expression of genes associated with immune processes were significantly decreased in both SAT and VAT. On the other hand, we detected significant differences in the plasma level of adiponectin and leptin, before and after weight loss but could not detected any significant changes in the expression of these genes in SAT and VAT of the individuals before and after weight loss.nd step Roux-en-Y surgery may have caused the absence of expected gene expression changes in lipolytic genes. Another limitation of our study is that due to the relatively small sample size, a paired sample analysis could not be performed. Therefore further studies are required to confirm our results using larger cohorts and/or different methods (e.g. RNA sequencing) for the quantification of gene expression changes after significant weight loss.The expected changes in the expression of lipolytic genes after the extensive weight loss were also detected in our analysis. However, after multiple testing the calculated Q-value was higher than 0.05 which is used as a cut of for detecting the statistically significantly changed genes in our study. One plausible explanation can be the small sample size used in our study. In addition, we cannot exclude that a body weight maintenance phase two months prior to the 2In order to address obesity in a wider population of affected individuals, less invasive treatments than BS are needed. Our results can be used for understanding the underlying molecular mechanisms of the clinical and physiological effects of BS and designing less invasive and potentially safer interventions to mimic the effects of BS. In summary, we identified previously unrecognized gene expression changes in AT in response to extensive weight loss, which may represent targets for the future development of pharmacological weight loss treatments.2 and the BMI after gastric sleeve resection was 38.6 ± 6.8 kg/m2 , plasma samples, SAT and VAT biopsies were obtained in the context of a two-step bariatric surgery strategy with gastric sleeve resection as the first step and a Roux-en-Y- gastric bypass as second step surgery 12 ± 2 months after sleeve gastrectomy. The baseline BMI in this subgroup was 53.1 ± 10.3 kg/m.8 kg/m2 .nd step surgery and in which AT RNA quality  was sufficient for microarray experiments) at both surgery time points were used for microarray experiments and additional SAT and VAT samples obtained from 16 individuals  were used to validate gene expression changes detected by microarray by RT-QPCR  gene and β-Actin (ACTB) were used as reference in SAT and VAT, respectively. GUSB was reported as the best reference gene in SAT due to its steady mRNA level at different body weights and fat mass across the replicate samplesWe compared gene expression profiles in SAT and VAT of severe obese individuals with that of lean individuals. In parallel, a Singular Value Decomposition (SVD) and a standard clustering analysis were employed to identify gross patterns of the transcriptome. P-values were corrected for multiple testing and analysis of variance (ANOVA) was used to determine significance of differences between ratios at Q-values < 0.05 and log2 fold changes > 0.1.SAT and VAT samples were immediately frozen in liquid nitrogen after explanation. Total RNA was extracted from frozen SAT and VAT obtained from 16 obese male and female individuals underwent BS before and after the surgery using RNeasy Lipid Tissue Mini Kit . Assay-on-demand  probes were used for the quantitative RT-PCR, performed in a 96-well LightCycler 480 system. Expression levels of CIDEA, LPIN1, NMNAT2, MPC1, MPC2, NNT, BCAT1 and BCAT2 were calculated relative to the mRNA expression of GUSB in SAT and ACTB in VAT and calculated with the ΔΔ-Ct method.How to cite this article: Mardinoglu, A. et al. Extensive weight loss reveals distinct gene expression changes in human subcutaneous and visceral adipose tissue. Sci. Rep.5, 14841; doi: 10.1038/srep14841 (2015).


An advanced Markov-Chain Monte Carlo approach called Subset Simulation is described in Au and Beck (2001)  Specifications Table•An advanced stochastic approach has been used to simulate extremes of urban microclimate.•The urban boundary layer (UBL) is sensitive to urban surface state as the lower boundary condition.•Urban form and design is highly related to urban boundary layer dynamics.1pf=10−4 , can be effectively broken down into 4 different sampling, including the first level of unconditional direct Monte Carlo simulation following a prescribed probability distribution function (PDF) and 3 subsequent conditional MCMC levels, each sample with a moderate conditional probability of p0=0.1.To quantify the numerical sensitivity of the UCM-SCM framework, we used an advanced Markov-Chain Monte Carlo (MCMC) procedure, known as the Subset Simulations X by calculating the average deviation of conditional sample means to that of the original PDF:i is the conditional (MCMC) level index, N=3 the total conditional levels, E[X] the statistical mean  of the original unconditional distribution in level 0, E[X|Y>yi] the mean value of X at conditional level i, Y the value of monitored model response, and yi the threshold values at exceedance probability of each intermediate level i. The magnitude of PSI quantifies the significance of sensitivity, while the sign of PSI indicates the correlation between monitored output Y and input parameter X, i.e. positive PSI means increasing X will lead to an increase of output Y and negative PSI means increasing X will lead to a decreased Y.For better quantification, a percentage sensitivity index (PSI) devised in zh, virtual potential temperature θv, and specific humidity q in the mixed layer) under different surface greening scenarios with different vegetation fraction fveg is presented in Ws, residual soil water content Wr, and saturated hydraulic conductivity Ks); (2) dimensional parameters ; (3) atmospheric parameters controlling the upper boundary conditions of the UBL . It can be shown that the model predictions of UBL dynamics are in general most sensitive to dimensional parameters, and relatively insensitive to hydrological parameters. This implicates that urban form and design can significantly alter UBL dynamics and the effect of regularly irrigated urban vegetation on the UBL is steady.The error analysis of key input parameters for critical model outputs (UBL height


Vaccination has proven itself as the most effective tool to control and prevent the disease and to facilitate the safe trade of live animals.Viral vaccine manufacturing processes present some specific constraints as compared to other biotech products linked to the cell substrate used and to the viral production.Multiple cell lines are used for productions such as VERO, MDCK, MRC5, BHK, and CHO cells. BHK-21 cells were selected because they are the most commonly used cells for vaccine production.As the feasibility of transferring the BHK-21 cells growth in microcarriers from a conventional bioreactor to single-use bioreactor (SU) was demonstrated, actually was time to scale-up and verify the flexibility and ease of use of these SU bioreactors enable rapid scale-up without any loss in product quality. This document describes a scaled-up production of a bovine viral vaccine.Identification of optimal parameters values, testing at intermediary bioreactors scales (2 liters - 50 liters) and application of the new process settings at industrial bioreactor scale of 200 liters were performed. Taking into account that a critical feature of microcarrier-based cell culture is the homogeneity of cell adhesion to the beads, mixing and aeration performance evaluation remain mandatory to ensure robust scale-up of cell culture processes.The goal of this study was to evaluate the possibility to produce the antigens for animal vaccines manufacturing in a bioreactor SU as alternative to roller bottles without any loss in product quality. Evaluation of COG reduction was done.Selection of appropriate culture conditions can be important to achieve consistent cell culture and virus production across sites and scales. Because characteristics like tank geometry and hardware  are not subject to change during scale-up, the scalability from 2L to 200L in the BIOSTAT® STR bioreactor was an easy strategy for our production process.Several studies were conducted to compare the growth of BHK-21 cells in microcarriers.Identification of optimal parameters values and testing at intermediary bioreactor scales (2 liters - 50 liters). Application of the new process settings at industrial fermentor scale (200 liters)For the scale down models, the cultures were conducted with:- Biostat B plus and Bisotat B (Sartorius-stedim): Univessel® SU 2L biorector and conventional bioreactor with glass vessels of 10L.- BIOSTAT® CultiBag STR 50 L- BIOSTAT® CultiBag STR 200 litersCultures were regularly sampled to monitor the in-process parameters such as final cell concentration and product yield (TCID50/ml titration). In this study, these performances were evaluated.Finally, taking into account that for vaccine formulation microcarriers must be eliminated from the viral suspension, filtration through Sartopure PP2 cartridges (from Sartorius Stedim Biotech ) was performed.The most important process criterion for evaluating the performance of each size of bioreactor is their ability to support the same level of viral production as in a conventional bioreactor. To evaluate this point, antigen production for a bovine vaccine was produced in the different bioreactors.• Comparable results between 2L, 10L, 50L and 200L concerning cell density, viability, productivity and product quality .• Comparable results between 2L, 10L, 50L and 200L: Cell density ⇒ Viability ⇒ Productivity ⇒ Product quality.• Good agitation and easy speed calculation according scale-up.• Bioreactor system allows even.• Equivalent cell growth profiles.• Equivalent cell population homogeneity on microcarriers. Good cell attachment and propagation.Therefore offering a suitable process to produce our bovine vaccine using attachment dependent cells.Next potential goal: Reduction COG• Reducing number of RB needed to inoculate the bioreactor and increasing virus yields.Carme Aulinas, Elisabeth Cortés, Carme Torres and Xell Gratacós for the excellent laboratory work and Sartorius-Stedim biotech for technical support


Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bacterial-type transcription factor (TF), called RosR, and five TFIIB proteins, homologs of eukaryotic TFs, combinatorially regulate over 100 target genes important for the response to extremely high levels of peroxide. These genes include 20 other transcription factors and oxidative damage repair genes. RosR promoter occupancy is surprisingly dynamic, with the pattern of target gene expression during the transition from rapid growth to stress correlating strongly with the pattern of dynamic binding. We conclude that a hierarchical regulatory network orchestrated by TFs of hybrid lineage enables dynamic response and survival under extreme stress in archaea. This raises questions regarding the evolutionary trajectory of gene networks in response to stress. Complex circuits of genes rather than a single gene underlie many important processes such as disease, development, and cellular damage repair. Although the wiring of many of these circuits has been mapped, how circuits operate in real time to carry out their functions is poorly understood. Here we address these questions by investigating the function of a gene circuit that responds to reactive oxygen species damage in archaea, microorganisms that represent the third domain of life. Members of this domain of life are excellent models for investigating the function and evolution of gene circuits. Components of archaeal regulatory machinery driving gene circuits resemble those of both bacteria and eukaryotes. Here we demonstrate that regulatory proteins of hybrid ancestry collaborate to control the expression of over 100 genes whose products repair cellular damage. Among these are other regulatory proteins, setting up a stepwise hierarchical circuit that controls damage repair. Regulation is dynamic, with gene targets showing immediate response to damage and restoring normal cellular functions soon thereafter. This study demonstrates how strong environmental forces such as stress may have shaped the wiring and dynamic function of gene circuits, raising important questions regarding how circuits originated over evolutionary time. All organisms encounter reactive oxygen species (ROS) originating from biotic and abiotic sources. ROS are produced at relatively low levels as natural byproducts of aerobic respiration, Fenton reactions, or other biotic sources e.g. stress-responsive TFs) typically resemble those of bacteria at the amino acid sequence level Relative to the other domains of life, the function of TFs that control the oxidant response in archaea remain understudied. To our knowledge, only a few transcription factors have been characterized to date H. salinarum as a model, both to characterize the genome-wide binding dynamics of an ROS-responsive transcription factor, and to analyze regulatory network function during ROS stress in archaea. This hypersaline adapted archaeal model organism encounters high levels of abiotic oxidants in its natural salt lake environment, where intense solar radiation and desiccation are frequent Here we use the salt-loving archaeon H. salinarum2O2 and paraquat). Genes directly and indirectly controlled by RosR in response to oxidant encode macromolecular repair functions. In the current study, we ask which of these genes are direct targets of RosR regulation. Integrated analysis of genome-wide binding location time course data with gene expression data demonstrates that RosR binds and regulates over 100 target genes. These encode molecular repair functions and a surprisingly high number of other TFs. RosR binds many of these sites in the absence of stress. Upon exposure to H2O2, RosR disengages from DNA at most loci. However, at other loci, RosR-DNA binding is dynamic following peroxide exposure, with locus-specific differences in TF occupancy over time. RosR binding is mediated via a 20-bp palindromic cis-regulatory binding sequence. Integration of data generated here in the context of other existing systems biology datasets reveals extensive combinatorial binding of RosR with multiple Tfb proteins throughout the regulon. We conclude that RosR is a master regulator of a hierarchy of TFs that performs global, dynamic physiological readjustment in response to oxidative stress.However, this network was computationally inferred from gene expression data. To experimentally characterize TFs with putative involvement in this network, our previous work identified the winged helix-turn-helix DNA-binding TF RosR. This TF dynamically regulates expression of more than 300 genes in response to oxidative stress in 2O2 over time (see 2O2 (2O2 and remained low for the duration of the time course. (2) At other sites (90 genes), RosR binding was initially lost in the presence of H2O2, but binding recovered within 60 minutes . RosR binding ability in the absence of stress (clusters 1 and 4 at the 0 time point) was previously validated by ChIP-qPCR Previous work demonstrated that the RosR transcription factor is required for the differential expression of genes in response to ROS time see . A total, which was detected nearby 37 of RosR-bound loci , but not detectable elsewhere in the genome  was fused to GFP. Promoter activity was assayed in the ΔrosR vs parent strain in the absence of stress, when RosR binding activity was evident in ChIP-chip experiments for these promoters. Ptrh4 activity is significantly higher in the ΔrosR strain relative to the parent and the empty vector background control  this cis-regulatory sequence carries significant importance in the function of the RosR regulatory network.To validate the function of this computationally predicted binding site experimentally, the native genomic promoter (TATA box and putative cis-regulatory sequence) of  control . This su control . Togethehsp4 and hsp5, peroxidase perA), translation , DNA replication, cell growth and division, and transcription  are downregulated upon ROS exposure, whereas stress response genes  are upregulated . Relative to the isogenic parent strain, both TF knockout strains are significantly impaired for growth in response to oxidative stress induced by addition of H2O2 to the cultures , a peak association is detected at distances of 65–75 bp  and TFIIB (Tfb)]. In contrast, most repressor TFs inhibit gene expression by binding downstream of the PIC 65–75 bp . This reckground ; and (c)p<5.68×10−3; see p-value calculation and p<2.05×10−4 for TfbB; p<2.16×10−7 for TfbG). In contrast, predictions from the model did not match experimental observations regarding combinatorial control by RosR-TfbD and RosR-TfbF pairs ”; bF pairs . Of all H. salinarum RosR is a bifunctional regulator that directly controls a large hierarchy of transcription factors in combination with Tfb proteins to enable extreme oxidative stress survival. The majority of these sites are bound in the absence of stress, with RosR released from DNA in the presence of oxidant. A subset of loci exhibits the opposite binding pattern. We show that RosR binds to a ∼20 bp imperfect palindrome cis-regulatory sequence and directly activates or represses genes encoding functions in transcription, macromolecular repair and central cellular physiology. We demonstrate that RosR regulates genes encoding TFs that are also required for oxidative stress survival. Such regulation is conducted in concert with Tfb proteins. We conclude that RosR plays an important role in a large transcriptional network that enables a rapid response to extreme oxidative stress followed by re-establishment of homeostasis.Data and analyses presented here suggest that H. salinarumThe function of gene products in the RosR regulon reported here reflects the observations from our previous work The integrated genome-wide analysis presented here suggests hypotheses for the RosR biochemical mechanism. Dynamic TF-DNA binding analysis suggests a differential preference in RosR promoter occupancy, as some promoters are re-bound while homeostasis is restored, whereas a small subset of other sites are bound only in the presence of peroxide , suggest2O2 and re-occupied by RosR within 60 minutes of oxidant exposure  may enable RosR to re-bind. For example, ΔperA mutants experience high intracellular H2O2 concentrations during mid-log phase growth, whereas H2O2 is cleared from the H. salinarum parent strain within the time frame tested here In contrast to RosR targets in Cluster 3, a significant fraction of sites are bound in the absence of H2O2 challenge than the parent strain  and binds together with only one other Tfb protein at its target promoters H. salinarum iron regulators Idr1 and Idr2 only regulate one other TF each via TF-TF network interactions.Integrated analysis of several genome-wide binding location and gene expression datasets for TFIIB homologs Escherichia coli, where SoxS regulates at least four other TF-coding genes . Δura3, the parent strain, and transcription factor deletion strain derivatives thereof, were grown in CM supplemented with 0.05 mg/mL uracil to complement the auxotrophy. In-frame gene deletion strains  were constructed using the pop-in/pop-out gene deletion strategy described previously ura3ΔrosR, referred to throughout as ΔrosR for brevity, was constructed previously H. salinarum strains harboring plasmids were cultured in CM supplemented with 20 µg/mL mevinolin for plasmid maintenance. H2O2 was added to mid-logarithmic phase cultures to 25 mM or at inoculation at 5 or 6 mM to test oxidative stress response as displayed in the figures.Strains of H. salinarum harboring VNG0258H::myc  by virtue of the myc epitope tag as described previously H. salinarum genome was tiled every 30 bp in triplicate. Randomly selected regions of the genome were spotted in quadruplicate. The custom tiling microarray design can be accessed via Agilent Technologies AMADID 026819, Gene Expression Omnibus (GEO) platform accession GPL18848. Dye swaps were conducted to correct for bias in incorporation. Seven biological replicate experiments were conducted for VNG0258::myc in the absence of H2O2 and three in the presence of H2O2. These experiments yielded a total of at least 18 replicate intensity data points per 30-bp genomic region per condition. DNA fragments were directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously H. salinarum genome  in at least one time point. Composite p-values for multiple binding peaks nearby the same gene within a given time point were calculated using Fisher's combined probability test Resultant slides were scanned and processed with Feature Extraction software (Agilent). Raw probe intensities were first normalized within each array using density-weighted loess 2O2 at 0, 10, 20, and 60 min; GEO accession GSE33980). These correlations are referred to throughout as “GE-ChIP correlations”. GE-ChIP correlations were calculated separately for each of the ΔrosR deletion and isogenic parent backgrounds as an additional metric for the impact of RosR binding on gene expression. Significance of the difference in GE-ChIP correlations between the parent and ΔrosR strains was calculated using Student's t-test. Genes with strong GE-ChIP correlations (Cs≥0.6) were interpreted as directly activated by RosR, whereas anticorrelations (Cs≤−0.6) were interpreted as repressed. Statistical overrepresentation in archaeal clusters of orthologous genes (arCOG) functional categories Time course profiles of processed ChIP-chip binding data were grouped using Spearman correlated complete linkage hierarchical clustering to identify various dynamic binding patterns. To determine the dynamic relationship between binding and gene expression, each gene in each dynamic binding cluster was correlated to expression data under the same culturing conditions as ChIP-chip from a previous study p<0.01 were selected using the R bioconductor MeDiChI package rosR). Significance of these correlations was computed from by comparing 10,000-fold resampled data to actual data  samples were prepared over the time course described above and subjected to quantitative real-time PCR analysis (qPCR) using SYBR green as previously described H. salinarum Δura3 parent, TF deletion strains Δura3ΔVNG0194 and Δura3Δhrg (deletion of VNG0917G), and the complementation strains . Growth rates were calculated from the slope of the log2 transformed data during logarithmic growth. Reported in the figures are ratios of the growth rates of each strain under H2O2 stress relative to the same strain's growth rate without stress. All growth data are provided in H. salinarum genome sequence 250 bp upstream and downstream of each of the 189 ChIP-chip binding loci  in the absence of stress in 50 mL CM. Samples were collected, washed and fixed as previously described ura3 harboring the empty vector (i.e. GFP-encoding gene with no promoter) or vector containing GFP-encoding gene driven by the strong constitutive Pfdx promoter To validate the predicted cis-regulatory binding sequence, a 200-bp region containing the putative cis-sequence and TATA box of S1 TablePrimers and archaeal strains used in this study.(XLS)Click here for additional data file.S2 TableExpression data and associated RosR binding sites for each gene. Contains binding location coordinates, coordinate-to-gene distances, and binding peak p-values for each gene at each time point.(XLS)Click here for additional data file.S3 TableDetailed annotations for genes nearby RosR binding sites.(XLS)Click here for additional data file.S4 TableDistances between RosR binding locations, each of the five TFB binding locations and associated gene identifiers.(XLS)Click here for additional data file.S5 TableRosR cis-regulatory binding motif sequences and genome coordinates.(XLSX)Click here for additional data file.S6 TableRaw growth data for all TF deletion strains grown under oxidative stress conditions.(XLSX)Click here for additional data file.


Biomarkers that drift differentially with age between normal and premalignant tissues, such as Barrett’s esophagus (BE), have the potential to improve the assessment of a patient’s cancer risk by providing quantitative information about how long a patient has lived with the precursor . In the case of BE, which is a metaplastic precursor to esophageal adenocarcinoma (EAC), such biomarkers would be particularly useful because EAC risk may change with BE dwell time and it is generally not known how long a patient has lived with BE when a patient is first diagnosed with this condition. In this study we first describe a statistical analysis of DNA methylation data  derived from tissue samples from 50 BE patients to identify and validate a set of 67 CpG dinucleotides in 51 CpG islands that undergo age-related methylomic drift. Next, we describe how this information can be used to estimate a patient’s BE dwell time. We introduce a Bayesian model that incorporates longitudinal methylomic drift rates, patient age, and methylation data from individually paired BE and normal squamous tissue samples to estimate patient-specific BE onset times. Our application of the model to 30 sporadic BE patients’ methylomic profiles first exposes a wide heterogeneity in patient-specific BE onset times. Furthermore, independent application of this method to a cohort of 22 familial BE (FBE) patients reveals significantly earlier mean BE onset times. Our analysis supports the conjecture that differential methylomic drift occurs in BE  and hence allows quantitative estimation of the time that a BE patient has lived with BE. Barrett’s Esophagus (BE) is a metaplastic precursor to esophageal adenocarcinoma (EAC). When a patient is diagnosed with BE, it is generally not known how long he/she has had this condition because BE is asymptomatic. While the question of how long a premalignant tissue or lesion has been resident in an organ (dwell time) may not be of importance for cases where curative interventions are readily available (such as adenomas in the colon), for BE, curative interventions are either costly or carry patient risks. Knowledge of a precursor’s dwell time may therefore be advantageous in determining the cancer risk due to the stepwise accumulation of critical mutations in the precursor. In this study, we create a molecular clock model that infers patient-specific BE onsets from DNA methylation data. We show that there is considerable variation in the predicted BE onset times which translates, using mathematical modeling of EAC, into large variation in individual EAC risks. We make the case that, notwithstanding other known risk factors such as chronological age, gender, reflux status, etc., knowledge of biological tissue age can provide valuable patient-specific risk information when a patient is first diagnosed with BE. There is great interest in the molecular characterization of precancerous fields and lesions  in the breast) to quantify their neoplastic potential, although it is generally not known how long such lesions (or fields) have sojourned in a patient when they are discovered. This point is of particular importance in the case of Barrett’s esophagus (BE), a variable-length metaplastic precursor of esophageal adenocarcinoma (EAC) that has been shown to undergo a stepwise progression to cancer involving multiple rate-limiting events –3. In spInter-individual variability in the EAC progression risk may depend on the duration of how long a patient has lived with BE (BE dwell time). In a large population-based study in Northern Ireland, Bhat et al.  found a Recently identified age-related changes in DNA-methylation have led to the notion of a biological tissue age which, although highly correlated with chronological age, may differ significantly from it , 14. It BE onset is a useful event-marker of cancer risk. In the following we describe the data and methodologies that support this conclusion.To infer patient-specific BE onset times from the measured methylation levels of identified CpGs that drift differentially between BE and SQ tissues, we use a Bayesian model that accounts for (CpG-specific) random effects in drift rates, measurement error, and a patient-specific BE onset time. Furthermore, to gain insights into how the age of BE onset may influence EAC risk, we used a recently developed mathematical model for EAC incidence to compute standardized lifetime risks for the individuals in data set D2 given their predicted BE onset times , 16. Addminfi package, which includes background level corrections, color adjustments and Subset-quantile Within Array Normalization (SWAN) normalization. SWAN is specifically designed for HumanMethylation450 array data to account for systemic differences between the Infinium I and Infinium II probe designs , we apply the ergodic theorem and approximate the posterior probability by the fraction of MCMC samples satisfying γk > γl. The prior Pr[H0] is computed similarly except we sample onset times si for the two groups of patients being compared directly from the uniform prior distributions si ∼ Uniform.Thus, we are interested in testing hypotheses ative H1 . To comphttps://github.com/yosoykit/BE_Clock_Model.The methods outlined in this section are implemented by the Bayesian BE clock model. All necessary tools to employ this model via the Gibbs sampler are available in documented R code at N2 = 30 patients) with the BE clock set of 67 CpGs. See si, i = 1, .., N2. For these 30 patients, median MCMC estimates for BE onset ages ranged from 2.0 to 59.0 years of age, with a median of 33.6 years of age. The model also estimates CpG specific drift rates bi,j, j = 1, .., 67 for the BE clock set and a measurement standard deviation parameter, σBEi for each individual i  derived from data set DV  the DV prior introduces no bias  because it was not used for the BE clock CpG marker set selection.The BE onset age estimates for the patients in D2 were obtained using prior To investigate the sensitivity of the estimated BE onset ages on the number of CpGs used in the MCMC algorithm we randomly subsampled smaller sets (n = 5 and 20) from the full set of 67 identified BE clock CpGs. We found our estimates of BE onsets to be robust in terms of the number of BE clock CpGs needed to discriminate among patients of similar chronological age who reveal rather distinct (early versus late) BE onset estimates  with FBE, with age range 39–84 at time of biopsy (mean age = 62.7). To quantify the aggregation of BE and EAC in families, Chak et al. performed a study with 411 patients with BE and/or its associated cancers, and found that familial BE (FBE) can be determined in 7.3% of patients, comprising 9.5% of EAC cases . One hypH0: γ3 > γ2  model after BE initiation  by using tissue age biomarker data to inform the modeling of the neoplastic progression to EAC. Such an integrated perspective for cancer risk management has recently been suggested by Li and colleagues . To thiss for each patient into As a demonstration, we used this model to compute the patient-specific risk of developing EAC by age 88 assuming a standardized 1950 birth cohort, allowing for gender-specific model parameters, by inputting the BE onset age estimate A fundamental problem in predicting the risk of esophageal adenocarcinoma (EAC) in patients with BE continues to be the difficulty in assessing the neoplastic potential of BE, which is considered the premalignant field in which EAC arises. Several lines of evidence and theoretical considerations support the notion that both BE segment length and the duration of how long BE has been present in a patient  are important determinants of EAC risk in addition to environmental and genetic risk factors , 37, 38.Unfortunately, a clinical determination of when a patient first developed BE is presently not possible because BE is mainly asymptomatic (over 90% of EAC cases do not present with a prior history of BE ). For thFinally, we tested our clock model using methylation array data from 22 familial BE patients (set D3). Patients from both groups D2 and D3 have similar age distribution see . HoweverGiven the theoretical implications of our proposed model of BE initiation and progression to EAC, we propose that once a patient’s BE onset has been estimated from his/her methylomic drift profile, his/her risk of developing EAC can be estimated more precisely. We have used a previously validated multistage clonal expansion model for EAC incidence which explicitly considers the uncertainty of the timing of BE onset in the general population and describes, conditional on when BE develops, the stochastic process of neoplastic progression from metaplastic to dysplastic tissue to cancer , 16. TheS1 TextExplicit distributions used in the Markov Chain Monte Carlo (MCMC) inference and an analysis for robustness of imputing normal squamous M-values is provided. For the multistage clonal expansion for EAC (MSCE-EAC) model, we derive the equation for EAC risk given a patient’s BE onset age from the backward Kolmogorov equations corresponding to the multi-type branching process .(PDF)Click here for additional data file.S1 Figν(t), followed by a ‘two-hit’ tumor initiation process with Poisson initiation rates μ0, μ1, which leads to the stochastic appearance of premalignant progenitor cells in the tissue. Premalignant cells undergo a first clonal expansion described by a birth-death-migration process with cell division rate αP, cell death-or-differentiation rate βP, and malignant transformation rate μ2. Malignant cells, in turn, undergo a second clonal expansion by a birth-death-detection process with cell division and death rates αM and βM, respectively, allowing for stochastic growth and possibly extinction of the malignant tumor. Clinical detection occurs through a size-based detection process with parameter ρ. TSG, tumor suppressor gene [Normal squamous epithelium may transform to BE with an exponentially distributed onset time with rate sor gene .(TIFF)Click here for additional data file.S2 FigComparison of the posterior distributions of BE onsets for two 84 year old BE patients (pt. 21 and pt. 28) in study D2 using the identified set of 67 BE clock CpGs (thin solid line). To test the relative robustness of the estimated mean BE onsets, we also generated random subsamples (without replacement) of size 5 and 20 from the 67 clock CpGs. Shown are the distributions of the median BE onset estimates using MCMC (5K cycles) for n = 5 CpGs (thick solid lines) and n = 20 CpGs (dashed line).(TIF)Click here for additional data file.S3 Figbj rates in DV colored in red) that hide an interesting correlation. Rather than homogenous drift, the correlation between longitudinal drift rates in D1 and DV  suggests the presence of heterogeneity in marker-specific drift rates. Ultimately, there was minimal effect conferred on posterior parameter estimates due to “winner’s curse” bias inherent in the D1 drift rates calculated during BE clock marker selection versus validation DV drift rates when used as two candidate priors in the MCMC Click here for additional data file.S4 FigFor the unmasked (grey boxplots) and masked (purple boxplots) implementations of inferring BE onset ages, we found that using an imputation of the intercept and drift rates of SQ tissue values across the D2 patients rather than exact matched SQ values is a robust approach (see (TIFF)Click here for additional data file.S5 FigAcross patients in data sets D2 (blue points) and D3 (black points), there is high correlation between the median MCMC posterior estimates for BE onset age and the corresponding EAC risk before age 88 as predicted by the multistage clonal expansion model  that uti(TIFF)Click here for additional data file.S1 TablePatient-specific information  for 10 serially sampled BE patients (D1), 10 serially sampled patients in an independent validation cohort (DV), 30 cross-sectional BE patients (D2), and 22 familial BE (FBE) patients (D3). Age at biopsy, sex, and whether a matched normal squamous (SQ) tissue sample was obtained at time of biopsy is recorded for all patients. Also, median MCMC estimates  for BE onset times are provided for cross-sectional patients.(XLSX)Click here for additional data file.S2 TableFor 67 total CpGs in BE clock set, columns of this table (in order) correspond to CpG name, gene location of CpG (IGR: intergenic region), chromosome location of CpG, CpG island type location (“‘OpenSea” indicates that the CpG does not lie in a CpG island), CpG island name, whether the CpG is on a promoter region, the CpG-specific population rate from linear regression over 30 D2 samples, and the drift rates for each CpG derived from the D1 and DV patients, respectively. The DV prior drift rates were used as prior information in the BE clock model.(XLSX)Click here for additional data file.


Limb girdle muscular dystrophies are a large group of both dominantly and recessively inherited muscle diseases. LGMD1D is caused by mutated DNAJB6 and the molecular pathogenesis is mediated by defective chaperonal function leading to impaired handling of misfolded proteins which normally would be degraded. Here we aim to clarify muscle pathology of LGMD1D in order to facilitate diagnostic accuracy.After following six Finnish LGMD1D families, we analysed 21 muscle biopsies obtained from 15 patients at different time points after the onset of symptoms. All biopsies were obtained from the lower limb muscles and processed for routine histochemistry, extensive immunohistochemistry and electron microscopy.Histopathological findings were myopathic or dystrophic combined with rimmed vacuolar pathology, and small myofibrillar aggregates. These myofibrillar inclusions contained abnormal accumulation of a number of proteins such as myotilin, αB-crystallin and desmin on immunohistochemistry, and showed extensive myofibrillar disorganization with excess of Z-disk material on ultrastructure. Later in the disease process the rimmed vacuolar pathology dominated with rare cases of pronounced larger pleomorphic myofibrillar aggregates. The rimmed vacuoles were reactive for several markers of defect autophagy such as ubiquitin, TDP-43, p62 and SMI-31.Since DNAJB6 is known to interact with members of the chaperone assisted selective autophagy complex (CASA), including BAG3 – a known myofibrillar myopathy causing gene, the molecular muscle pathology is apparently mediated through impaired functions of CASA and possibly other complexes needed for the maintenance of the Z-disk and sarcomeric structures. The corresponding findings on histopathology offer clues for the diagnosis. Limb girdle muscular dystrophy (LGMD) 1D is an autosomal dominant myopathy usually with late onset of weakness at the age of 30–60 and slow progression, although more aggressive forms can occur . TypicalMyofibrillar myopathies (MFMs) are a group of myopathies that share common morphological findings but have different molecular etiologies , 7. MFMsDNAJB6 [We have previously reported preliminary data on muscle biopsy findings in patients with p.F93L mutation in DNAJB6 , 10 and DNAJB6  with theDNAJB6 mutation p. F93L. Pedigrees of the families FF1–6 are presented in Fig. 15 patients from six unrelated Finnish families were studied, four of them had two and one patient three biopsies, with an age range from 47 to 78 years. Patient data and their clinical findings are presented in Table All together 21 muscle biopsy samples were collected from four anatomically different muscles , gluteus (G), soleus (S) and gastrocnemius medialis (GM)). Fresh-frozen 10 μm cryostat sections of muscle tissue were stained with routine histochemical techniques , 13. FroAntibodies applied for immunohistochemical stainings are detailed in Table Ultrastructural studies were performed on ten patients. Muscle specimens were processed for semithin and ultrathin sections according to standard methods  and examClinical summary of the patients is presented in Table On light microscopy general myopathic or dystrophic features of variable severity depending on the stage of the disease were observed in all cases: fibrosis and fatty replacement of the muscle tissue, fiber size variation with atrophic and occasional hypertrophic fibers and central nucleation Fig. , b. TherOn immunohistochemistry moderate or strong expression of several proteins including myotilin, desmin, alphaB-crystallin, proteasome and SMI310 were shown in the myofibrillar lesions with aggregates Fig. , c, d T, and dysOn electron microscopy the myofibrillar lesions showed severe structural sarcomere disarrangement associated with prominent excess and dispersion of disintegrated Z-disk material Fig. . Other fDNAJB6 mutated LGMD1D shows distinct findings that should provide clues for a correct diagnostic approach even in a sporadic patient without known family history. On light microscopy the most common finding is the presence of early stage myofibrillar disintegration with small myofibrillar aggregates and rimmed vacuolated fibers. On immunohistochemistry many proteins are found in the aggregates, and the rimmed vacuoles are reactive for several proteins associated with autophagy. The light microscopy findings are in general similar to MFMs. However, the myofibrillar aggregates are commonly distributed in scattered fibers and are smaller than in a typical MFM. Only in one biopsy large and pleomorphic aggregates in groups of fibers unevenly distributed across the fascicles more characteristic of a typical MFM were seen. In addition, rubbed-out fibers commonly found in MFMs such as desminopathies and alphaB-crystallinopathies are not a common feature in LGMD1D.The muscle pathology in our study of DNAJB6 mutant biopsies. However, the endomysial inflammatory cells were few in number without larger infiltrates and no invasion in non-necrotic fibers was observed. In addition, there was no significant expression of MHC HLA Class I, thus clearly preventing any confusion with sporadic inclusion body myositis (s-IBM) despite the rimmed vacuolation. In addition, no significant numbers of cytochrome oxidase-negative muscle fibers or ragged red fibers were present. Clinically, the involvement of hamstrings was always more severe than the quadriceps and without the finger flexor weakness as with s-IBM.Secondary inflammatory changes that often may occur in genetic myopathies were observed in approximately half of the The ultrastructural findings in myofibrillar myopathies may show some differences in the genetically different myopathies . CompareThe autophagic degenerative component of the pathology in LGMD1D with the basic features of rimmed vacuoles on light microscopy and disorganized myofibrillar structures, Z-line dispersion, and dilatation of sarcoplasmic reticulum into vacuolar formations on ultrastructure are not particularly different compared to the similar pathology of the other myofibrillar myopathies or s-IBM. However, eosinophilic inclusions in the rimmed vacuoles that occasionally may be observed in s-IBM were rare in the LGMD1D samples.DNAJB6 gene [DNAJB6 is expressed in many tissues [DNAJB6 and LGMD1D disease.LGMD1D is caused by mutations in JB6 gene . DNAJB6 JB6 gene . DNAJB6  tissues  with its tissues . Our pre tissues . In this tissues . The domExceptionally, LGMD1B has been reported to cause myofibrillar changes similar to LGMD1D, however without rimmed vacuolar pathology .Histopathology of LGMD1D is different from the other more prevalent autosomal dominant limb-girdle muscular dystrophies; markedly different from the usually nonspecific pathology in LGMD1B and LGMD1C and also distinct from the more closely related LGMD1A myotilinopathy.Authors take full responsibility of the data, the analyses and the interpretation. All patients have given their informed consent prior to their inclusion in the study. All investigations have been performed in accordance of with Declaration of Helsinki and its amendments. This study has been approved by the ethical committee of Tampere University Hospital. The authors declare that they have no conflicts of interest.