1A Promoter Research Articles

Assessment of Cdx2 polymorphism in Iranian women with polycystic ovary syndrome

BackgroundWomen’s fertility is affected by polycystic ovarian syndrome (PCOS) as an endocrine disorder with characteristic symptoms such as insulin resistance, polycystic ovaries, menstrual irregularities, and obesity. In polycystic ovarian syndrome, the vitamin D endocrine system is regulated by the vitamin D receptor (VDR) associated with type II diabetes, endocrine dysfunctions, and insulin resistance. Therefore, the current paper deals with the investigation of the connection between Cdx2 VDR gene polymorphism and the biochemical factors in obese PCOS women.Material and methodsIn the current case–control study, 40 obese women without PCOS and 38 obese women with PCOS were enrolled in May–September 2016. Insulin, IGF1, FBS, and HOMA-IR were examined for the participants along with the allelic and genotypic frequency of Cdx2 polymorphism G/A (rs11568820) from Isfahan Fertility and Infertility Center, Iran. The ASM-PCR (multiplex allele-specific PCR) technique was utilized in this regard.ResultsThe age of PCOS women was less (P < 0.001) than the controls. In PCOS women, insulin, FBS, and HOMA-IR serum levels were higher than in the control women (all P values 0.05). For GG, AG, AA,A, and G Cdx2(A/G) genotypic/allelic frequencies were 84.2%, 15.8%, 0%, 7.9%, and 92.1% in cases and 87.5%, 12.5%, 0%, 6.3%, and 93.8% in controls, respectively. HOMA-IR (P = 0.047 and P = 0.033, respectively) and insulin than those with the AG genotype were in PCOS women with the GG Cdx2 genotype. The highest IGF-1 mean value (P = 0.020) was found for the AG genotype in PCOS. In our study, a significant relation was found only between PCOS and FBS, in terms of a logistic regression analysis of Cdx2 and parameters.ConclusionIn the present study, it was indicated that the GG genotype in PCOS subjects was associated with the IGF-1, HOMA-IR, and insulin. Similarly, no association was found between obese PCOS patients and Cdx2 in the 1a promoter area of the VDR gene in our study.

Paternal eNOS deficiency in mice affects glucose homeostasis and liver glycogen in male offspring without inheritance of eNOS deficiency itself

Aims/hypothesisIt was shown that maternal endothelial nitric oxide synthase (eNOS) deficiency causes fatty liver disease and numerically lower fasting glucose in female wild-type offspring, suggesting that parental genetic variants may influence the offspring’s phenotype via epigenetic modifications in the offspring despite the absence of a primary genetic defect. The aim of the current study was to analyse whether paternal eNOS deficiency may cause the same phenotype as seen with maternal eNOS deficiency.MethodsHeterozygous (+/−) male eNOS (Nos3) knockout mice or wild-type male mice were bred with female wild-type mice. The phenotype of wild-type offspring of heterozygous male eNOS knockout mice was compared with offspring from wild-type parents.ResultsGlobal sperm DNA methylation decreased and sperm microRNA pattern altered substantially. Fasting glucose and liver glycogen storage were increased when analysing wild-type male and female offspring of +/− eNOS fathers. Wild-type male but not female offspring of +/− eNOS fathers had increased fasting insulin and increased insulin after glucose load. Analysing candidate genes for liver fat and carbohydrate metabolism revealed that the expression of genes encoding glucocorticoid receptor (Gr; also known as Nr3c1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a; also known as Ppargc1a) was increased while DNA methylation of Gr exon 1A and Pgc1a promoter was decreased in the liver of male wild-type offspring of +/− eNOS fathers. The endocrine pancreas in wild-type offspring was not affected.Conclusions/interpretationOur study suggests that paternal genetic defects such as eNOS deficiency may alter the epigenome of the sperm without transmission of the paternal genetic defect itself. In later life wild-type male offspring of +/− eNOS fathers developed increased fasting insulin and increased insulin after glucose load. These effects are associated with increased Gr and Pgc1a gene expression due to altered methylation of these genes.Graphical abstract

Paternal eNOS Deficiency Affects Glucose Homeostasis and Liver Glycogen Content in Male Offspring Without Inheritance of eNOS Deficiency Itself

Background: It was shown that maternal eNOS deficiency causes fatty liver disease and numerically lower fasting glucose in female wild-type offspring suggesting that parental genetic variants may influence the offspring's phenotype via epigenetic modifications in the offspring despite the absence of a primary genetic defect. This, however, was not shown so far for paternal genes. Methods; Heterozygous male eNOS knockout mice or wild-type male mice were bred with female wild type mice. The phenotype of wild-type offspring of heterozygous male eNOS knockout mice was compared to offspring from wild type parents. Findings: Global sperm DNA methylation decreased and sperm miRNAs pattern altered substantially. Fasting glucose and liver glycogen storage were increased when analyzing wild type male and female offspring of +/- eNOS fathers. The endocrine pancreas in wild-type offspring was not affected. Wild-type male but not female offspring of +/- eNOS fathers had increased fasting insulin and increased insulin after glucose load. Analyzing candidate genes for liver fat- and carbohydrate metabolism revealed that the glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) genes were increased while DNA methylation of GR exon 1A and PGC1a promoter were decreased in the liver of male wild-type offspring of +/- eNOS fathers. Interpretation: Our study suggests that paternal genetic defects may alter the epigenome of the sperm without transmission of the defect itself and alter in later life of the offspring epigenetically gene expression and hence the phenotype without inheriting the defect itself and these alterations might be offspring sex dependent. Funding Information: This study was financially supported by the “Deutsche Forschungsgemeinschaft” to Dr. Berthold Hocher. Declaration of Interests: The authors have no conflict of interest to disclose Ethics Approval Statement: Study design and experimental protocols were conducted according to the local institutional guidelines for the care and use of laboratory animals and approved by the animal welfare ethical committee of the state of Berlin.

NtAIDP1, a novel NtJAZ interacting protein, binds to an AT-rich region to activate the transcription of jasmonate-inducible genes in tobacco

In plants, jasmonate ZIM-domain proteins (JAZs) act as critical regulators, interacting physically with transcription factors (TFs) and other transcriptional regulators to modulate jasmonate (JA)-responsive gene expression and participate in crosstalk with other hormone signalling pathways. Identifying novel JAZ-interacting proteins will provide new insights into JA signalling cascades in plants. Here, we performed yeast two-hybrid screening to identify 70 NtJAZ1-interacting proteins, including an A/T-rich interaction domain containing protein 1 (NtAIDP1) from JA-treated tobacco Bright Yellow-2 (BY-2) cells. NtAIDP1 is localised in the nucleus and interacts with NtJAZ1 via its C-terminal heat shock protein 20 (HSP) domain. Aside from NtJAZ1, NtAIDP1 also interacts with other JA-inducible NtJAZs, including NtJAZ2b, NtJAZ2b.2, NtJAZ5, NtJAZ7, NtJAZ11 and NtJAZ12, but not with NtJAZ3, NtJAZ3b or NtJAZ10, and interacts with NtNINJA, NtDELLA1 and NtDELLA2 in the yeast two-hybrid assay. Furthermore, NtAIDP1 binds to the AT-rich region of the GAG fragment of the putrescine N-methyltransferase 1a (NtPMT1a) promoter and activates the transcriptional activity of the GAG fragment, whereas NtMYC2a interacts with and competitively inhibits the transactivational activity of NtAIDP1 in Arabidopsis mesophyll protoplasts. Overexpression of NtAIDP1 promotes the transcription of NtPDF1.2 and NtJAZ1, but has little effect on the expression of NtPMT1a, quinolinate phosphoribosyltransferase 2 (NtQPT2), and NtMYC2a in tobacco. These results indicate that NtAIDP1 is a new component of the JA signalling pathway and is involved in JA-regulated gene expression.

Evidence for association of vasopressin receptor 1A promoter region repeat with childhood onset aggression

ObjectiveChildhood onset aggression can cause major suffering to affected families and is associated with many negative outcomes in the child's later life, including poor academic performance, adolescent delinquency, drug abuse, depression and antisocial personality disorder. Currently available prevention and intervention strategies have limited efficacy, but a better understanding of underlying genetic and neurobiological factors can lead to more effective prevention and treatment strategies, through genetic screening programs and novel therapies. MethodThis study examined the RS1 (n = 299 aggression, n = 192 controls) and RS3 (n = 291 aggression, n = 189 controls) microsatellite repeats within the promoter region of the vasopressin receptor 1A gene (AVPR1A) and their association with extreme childhood aggression, as assessed by the Child Behavior Checklist (CBCL), as well as the Teacher Report Form (TRF) and Youth Self Report (YSR). Binary logistic regression was used to model the relationship between microsatellite length and childhood aggression. Age and sex were used as covariates. ResultsLogistic regression revealed a nominally significant association between one specific RS3 repeat and non-aggressive status. No association was found for any of the RS1 repeats. In a separate model, grouping repeats into short and long, carriers of long RS3 repeats were nominally significantly associated with non-aggressive status. ConclusionsThese findings suggest a role for AVPR1A and its RS3 microsatellite in extreme childhood aggression and could lead to a better understanding of the biological pathways of aggressive behavior. However, independent replication and further research into the functionality of studied genetic variants is required.

Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 μg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300 was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes.

BnaA02.NIP6;1a encodes a boron transporter required for plant development under boron deficiency in Brassica napus

Boron (B) is an essential micronutrient for the plant normal growth. In Arabidopsis, NIP6;1 is a boric acid channel required for the proper distribution of boric acid, especially in the nodal regions of shoots. BnaA02.NIP6;1a, a homologous gene of AtNIP6;1 in Brassica napus, was reported to play a key role in B transport activity. However, little is known about the other functions of BnaA02.NIP6;1a in Brassica napus. In this study, we found that BnaA02.NIP6; 1a was localized in both plasma membrane and cytoplasm, which was different from that in Arabidopsis. The transgenic Arabidopsis plant containing a BnaA02.NIP6;1a promoter driven GUS reporter gene displayed strong GUS activity in roots, stems, leaves, especially in buds and open flowers, which are different from the expression pattern from its homologous gene in Arabidopsis. Silencing BnaA02.NIP6;1a repressed vegetative growth under B-deficient condition in Brassica napus. More importantly, knockdown of BnaA02.NIP6;1a in rapeseed resulted in the reduction of boron accumulation in the flower under boron deficiency and lead to severe sterility, which has not yet been reported before. Furthermore, nip6;1 mutant in Arabidopsis only showed the loss of apical dominance phenotype under boron deficiency at reproductive stage, whereas BnaA02.NIP6;1 RNAi lines exhibited large amounts of abnormal development of the inflorescence as compared with the wild type under boron limitation. Taken together, our results demonstrate that BnaA02.NIP6;1a encodes a boron transporter required for plant development under boron deficiency in Brassica napus, which shows its novel and diverse function in rapeseed compared with model plant Arabidopsis.

Identification of RASSF1A promoter hypermethylation as a biomarker for hepatocellular carcinoma

BackgroundRAS association domain family protein 1A (RASSF1A) promoter hypermethylation is suggested to be linked to hepatocellular carcinoma (HCC), but the results remained controversial.MethodsWe evaluated how RASSF1A promoter hypermethylation affects HCC risk and its clinicopathological characteristics through meta-analysis. Data on DNA methylation in HCC and relevant clinical data were also collected based on The Cancer Genome Atlas (TCGA) database to investigate the prognostic role of RASSF1A promoter hypermethylation in HCC.ResultsForty-four articles involving 4777 individuals were enrolled in the pooled analyses. The RASSF1A promoter methylation rate was notably higher in the HCC cases than the non-tumor cases and healthy individuals, and was significantly related to hepatitis B virus (HBV) infection-positivity and large tumor size. Kaplan–Meier survival analysis revealed that HCC cases with RASSF1A promoter hypermethylation had worse outcomes. Receiver operating characteristic curves confirmed that RASSF1A promoter methylation may be a marker of HCC-related prognoses.ConclusionsRASSF1A promoter hypermethylation is a promising biomarker for the diagnosis of HCC from tissue and peripheral blood, and is an emerging therapeutic target against HCC.

Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p&amp;lt;0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p&amp;lt;0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system

Limbic system associated membrane protein (Lsamp) is a neural adhesion protein which has been recently found to be differentially expressed between serotonergic neuron subtypes. We have previously shown elevated serotonin (5-HT) turnover rate in Lsamp-deficient mice. The purpose of the current study was to elucidate the role of Lsamp in serotonergic neurotransmission.Chronic (18 days) administration of serotonin reuptake inhibitor (SSRI) escitalopram (10 mg/kg) significantly increased general activity in wild-type mice in the open field and protected exploration in Lsamp−/− mice in the elevated-plus maze. An important psychopathology-related endophenotype, elevated 5-HT turnover in the brain of Lsamp-deficient mice, was reproduced in the saline group. Escitalopram restored the elevated 5-HT turnover of Lsamp-deficient mice to a level comparable with their wild-type littermates, suggesting that high 5-HT turnover in mutants is mediated by the increased activity of serotonin transporter (SERT protein encoded by Slc6a4 gene). The baseline level of Slc6a4 transcript was not changed in Lsamp-deficient mice, however, our immunohistochemical analysis showed partial co-expression of Lsamp with both SERT and Tph2 proteins in raphe. Overactivity of SERT in Lsamp−/− mice is further supported by significant elevation of Maoa transcript and increase of DOPAC, another Mao A product, specifically in the raphe. Again, elevation of DOPAC was reduced to the level of wild-type by chronic SSRI treatment. The activity of Lsamp gene promoters varied in 5-HT producing nuclei: both Lsamp 1a and 1b promoters were active in the dorsal raphe; most of the expression in the median raphe was from 1b promoter, whereas Lsamp 1a promoter was almost exclusively active in the caudal subgroup of raphe nuclei. We suggest that Lsamp may have an impact on the integrity of serotonergic synapses, which is possibly the neurochemical basis of the anxiety- and sociability-related phenotype in Lsamp-deficient mice.

SUN-115 Distinct DNA Methylation Signature in Neuroendocrine Tumors of Different Primary Sites and Hereditary Predisposition

ObjectiveThere is scant data of the genome-wide methylome alterations in neuroendocrine tumors (NET). Thus, the goal of this study was to compare the DNA methylation signature of NETs with respect to various primary sites and inherited genetic predisposition syndromes including von Hippel-Lindau (VHL) and multiple endocrine neoplasia type 1 (MEN1).MethodsGenome-wide DNA methylation analysis of 96 NETs (primary and metastatic) was performed by using the Illumina Infinium EPIC Array. Principal component analysis (PCA) and unsupervised clustering analyses were performed to identify distinct methylome signatures. The methylation status of genetic drivers such as APC were assessed by primary site.ResultsA total of 835,424 CpGs methylation sites were quantified. Hypermethylated CpG sites were detected more frequently in sporadic vs. MEN1-related vs. VHL-related NETs, respectively (p < 0.001 for all comparisons), while hypomethylated CpGs sites were more common in VHL-related NETs vs. sporadic and MEN1-related NETs (p<0.001 for both comparisons).Small-intestinal NETs (SINETs) had the most differences at CpGs with the highest number of hyper- and hypomethylated CpG sites, followed by duodenal NETs (DNETs) and pancreatic NETs (PNETs, p<0.001 for all comparisons). PCA showed distinct clustering of SINETs and three NETs of unknown primary. Sporadic, VHL-related and MEN1-related PNETs formed distinct groups on PCA. VHL-related NETs clustered separately showing pronounced CpG hypomethylation, while sporadic and MEN1-related NETs clustered together showing relative CpG hypermethylation. In a subgroup analysis, MEN1-related SINETs, DNETs and gastric NETs had distinct methylome signatures, respectively, with complete separation by PCA and unsupervised hierarchical clustering. Furthermore, we found CpG hypermethylation in the APC (adenomatous polyposis coli) gene, specifically in the 1A promoter, with higher methylation levels in gastric- and DNETs vs. SINETs, PNETs and NETs of unknown primary (p < 0.001 for all comparisons).ConclusionVarious primary NET sites and genetically predisposed MEN1-related NETs have distinct DNA CpG methylation signatures. The methylome signatures identified in this study may be useful for non-invasive molecular characterization of NETs, through DNA methylation profiling of biopsy samples or circulating tumor DNA.

Frequency Spectrum of Chemical Fluctuation: A Probe of Reaction Mechanism and Dynamics

Even in the steady-state, the number of biomolecules in living cells fluctuates dynamically, and the frequency spectrum of this chemical fluctuation carries valuable information about the dynamics of the reactions creating these biomolecules. Recent advances in single-cell techniques enable direct monitoring of the time-traces of the protein number in each cell; however, it is not yet clear how the stochastic dynamics of these time-traces is related to the reaction mechanism and dynamics. Here, we derive a rigorous relation between the frequency-spectrum of the product number fluctuation and the reaction mechanism and dynamics, starting from a generalized master equation. This relation enables us to analyze the time-traces of the protein number and extract information about dynamics of mRNA number and transcriptional regulation, which cannot be directly observed by current experimental techniques. We demonstrate our frequency spectrum analysis of protein number fluctuation, using the gene network model of luciferase expression under the control of the Bmal 1a promoter in mouse fibroblast cells. We also discuss how the dynamic heterogeneity of transcription and translation rates affects the frequency-spectra of the mRNA and protein number.

Uridine diphosphate glucuronosyl transferase 1A (UGT1A1) promoter polymorphism in young patients with sickle cell anaemia: report of the first cohort study from Nigeria

Background(TA) n repeat sequence (rs8175347) of UGT1A1 gene promoter polymorphism is associated with serum bilirubin levels and gallstones among different sickle cell anaemia (SCA) populations. There are no data on UGT1A1 polymorphisms and their impact on Nigerian SCA patients. In this study, we determined the distribution of the UGT1A1 (TA) n genotypes among a group of young Nigerian SCA patients and healthy controls. In addition, the influence of UGT1A1 (TA) n genotypes on the laboratory and clinical events among the patients was determined.MethodsThe distribution of the UGT1A1 (TA) n genotypes among 101 young Nigerian SCA patients and 64 normal appropriate controls were determined and studied. The UGT1A1 (TA) n genotypes were further classified into subgroups and used to differentiate the clinical events and laboratory parameters of the patients.ResultsFour (TA) n alleles:(TA)5, 6, 7, and 8 were found. These were associated with 10 genotypes: TA5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8, 8/8. The normal (wild-type)-(TA) 6/6), low- (TA) 7/7, 7/8, 8/8), intermediate- (TA) 5/7, 5/8, 6/7, 6/8), and high-activity (TA) 5/5, 5/6,) genotypes were found in 24.8, 24.8, 41.5, and 8.9% patients and 20.3, 15.6, 61, and 3.1% controls respectively. The general genotype distribution of the patients and control group were not significantly different. There were significant differences in serum bilirubin and lactate dehydrogenase (LDH) of the patients when differentiated by the UGT1A1 (TA) n genotypes (p<0.05). Asymptomatic gallstones were found in 5.9% of patients and were significantly of the low-activity genotypes sub-group 5 (20%) vs 1(1.3%) p = 0.0033. Although, bilirubin and fetal hemoglobin (HbF) of patients with gallstones were significantly different from those without gallstone, only the serum bilirubin was associated with UGT1A1 (TA) n genotypes on multivariate analysis (p < 0.0001).ConclusionThis study highlights the contribution of UGT1A1 polymorphisms, a non-globin genetic factor, to the laboratory and clinical manifestations of young Nigerian SCA patients for the first time. It also shows that children with co-inheritance of low UGT1A1 (TA) n affinity genotypes may be at risk of gallstone, hence the need to follow them up.

Frequency spectrum of chemical fluctuation: Aprobe of reaction mechanism and dynamics.

Even in the steady-state, the number of biomolecules in living cells fluctuates dynamically, and the frequency spectrum of this chemical fluctuation carries valuable information about the dynamics of the reactions creating these biomolecules. Recent advances in single-cell techniques enable direct monitoring of the time-traces of the protein number in each cell; however, it is not yet clear how the stochastic dynamics of these time-traces is related to the reaction mechanism and dynamics. Here, we derive a rigorous relation between the frequency-spectrum of the product number fluctuation and the reaction mechanism and dynamics, starting from a generalized master equation. This relation enables us to analyze the time-traces of the protein number and extract information about dynamics of mRNA number and transcriptional regulation, which cannot be directly observed by current experimental techniques. We demonstrate our frequency spectrum analysis of protein number fluctuation, using the gene network model of luciferase expression under the control of the Bmal 1a promoter in mouse fibroblast cells. We also discuss how the dynamic heterogeneity of transcription and translation rates affects the frequency-spectra of the mRNA and protein number.

SAT-199 A Hypomethylated GATA6 Gene Body May Function as an Active Enhancer in Endometriotic Cells

[Background]Endometriosis is an estrogen-dependent, inflammatory disease characterized by the presence of endometrium-like tissue primarily on the pelvic peritoneum and ovaries. There is accumulating evidence suggesting epigenetic aberrations play a role in the pathogenesis and/or the pathophysiology in endometriosis. A number of aberrant gene expressions have been demonstrated in endometriosis, and these aberrations may be related to epigenetic aberrations. Our Genome-wide DNA methylation analysis showed a GATA6 gene body highly hypomethylated in endometrial cells. From our observation that GATA6 was highly expressed in endometriotic cells, we hypothesized the presence of cis-acting element within the gene body.[Objective]We examined a potential function of hypomethylated GATA6 gene body as a cis-acting element in endometriotic cells.[Patients]The Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of cycling premenopausal women who had uterine leiomyoma. These patients had received no hormonal treatment before tissues were removed. [Methods]Stromal cells were prepared from endometriotic and endometrial tissues. GATA6 mRNA expressions was examined using RT-PCR. Immunocytochemical analysis was performed using anti-GATA6 antibody. The potential function of hypomethylated gene body as a cis-acting element was evaluated by ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies and eRNA expression analysis.[Results]We hypothesized the presence of CpG hypomethylation, which is responsible for increased gene expression in endometriotic cells. 1) Using endometrial cells as control, differentially methylated CpGs endometriotic cells were extracted. 2) We finally extracted a stretch of 14 CpGs within GATA6 gene body. 3) GATA6 mRNAexpression in endometriotic cells was 50-fold higher than that in endometrial cells. The expression depended on 1a promoter. 4) Wild type GATA6 expression was observed in endometriotic cells, but not in endometrial cells. 5) Immunocytochemistry showed positive staining of GATA6 in endometriotic cell nuclei. 6) ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies predicted the presence of an active enhancer within the gene body sequence in endometriotic cells. 7) Supporting this observation, putative eRNA was transcribed from the region.[Conclusion]Our results suggest that the gene body sequence of GATA6 may function as an active enhancer under the control of DNA methylation in endometriotic cells. This is the first implication showing a link between an aberrant DNA methylation of cis element and gene expression in endometriosis.

Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

Vascular aging has a strong relationship with cardiovascular disease. Fos-related antigen 1 (Fra-1), also referred to as Fos-like antigen 1, is a transcription factor and has been reported to be involved in many pathologic processes. Here, we demonstrate that Fra-1 plays a critical role in angiotensin II (Ang II)-induced vascular senescence. Fra-1 expression is increased significantly in Ang II-induced rat aortic endothelial cell (RAEC) senescence and the arteries from Ang II-infused mice. Interestingly, silencing Fra-1 blocks Ang II-induced senescence phenotypes in RAECs, including decreased senescence-associated β-galactosidase staining, and mitigated proliferation suppression and senescence-associated secretory phenotype. Further, knocking down Fra-1 inhibits vascular aging phenotypes in an Ang II-infused mice model. The up-regulated Fra-1 also exists in human atherosclerotic plaques and Ang II-induced vascular smooth muscle cells as well as in replicated senescence RAECs. Mechanistic studies reveal that Fra-1 preferentially associates with c-Jun and binds to the cyclin-dependent kinase inhibitor 1a (p21) and cyclin-dependent kinase inhibitor 2a (p16) promoter region, leading to elevated gene expression, which causes senescence-related phenotypes. In conclusion, our results identify that Fra-1 plays a novel and key role in promoting vascular aging by directly binding and transcriptionally activating p21 and p16 signaling, suggesting intervention of Fra-1 is a potential strategy for preventing aging-associated cardiovascular disorders.-Yang, D., Xiao, C., Long, F., Wu, W., Huang, M., Qu, L., Liu, X., Zhu, Y. Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

MicroRNA-96 expression induced by low-dose cisplatin or doxorubicin regulates chemosensitivity, cell death and proliferation in gastric cancer SGC7901 cells by targeting FOXO1.

MicroRNA-96 (miR-96) is transcriptionally associated with the induction of chemoresistance following chemotherapy by targeting to FOXO1 mRNA at one of two predicted binding sites in its 3'-untranslated region sequence. The upregulation of miR-96 is associated with a high risk of chemoresistance. Nevertheless, the mechanism by which miR-96 is upregulated remains largely undefined. In the present study, the gastric cancer SGC7901 cell line was treated with different doses of the chemotherapeutic agents cisplatin and doxorubicin. miR-96 expression was analyzed by reverse transcription-quantitative polymerase chain reaction at different time points. Western blot and chromatin immunoprecipitation were performed to analyze the expression levels of the target gene. The effects of miR-96 on chemosensitivity were assessed by a carboxyfluorescein succinimidyl ester/propidium iodide labeling assay, and its effects on proliferation were assessed by Cell Counting Kit-8 or EdU staining assays. The results demonstrated that treatment with a low dose of either chemotherapeutic agent induced miR-96 expression. Upregulation of miR-96 caused the post-transcriptional repression of FOXO1 expression. Decreases in FOXO1 protein levels led to a decrease in the transcriptional activity of the cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21) promoter region, and thus the expression of p21 was downregulated in a tumor protein p53-independent manner. As a result, induction of miR-96 expression caused chemoresistance and promoted proliferation in SGC7901 cells. Taken together, the results of the present study revealed that treatment with cisplatin or doxorubicin could induce expression of miR-96 at certain doses. Upregulation of miR-96 is partially associated with chemoresistance and miR-96 can also promote cell proliferation by repressing p21.

Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway.

Hepatitis B virus core protein (HBc) is expressed preferentially in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). HBc can function as an oncogene arising from its gene regulatory properties, but how it contributes functionally to hepatocarcinogenesis remains unclear. In this study, we determined the molecular and functional roles of HBc during HBV-associated hepatocellular tumorigenesis. HBc increased tumor formation of hepatoma cells. Moreover, expression of HBc specifically promoted proliferation of hepatoma cells in vitro. Mechanistic investigations revealed that these effects were caused by activation of the Src/PI3K/Akt pathway through proximal switch from inactive Src to the active form of the kinase by HBc. HBc-mediated sarcoma (Src) kinase activation was associated with down-regulation of C-terminal Src kinase (Csk). In addition, HBc enhances Src expression by activation of alternative Src 1A promoter in an Sp1 transcription factor-dependent manner. Proliferation induced by stable HBc expression was associated with increased G1-S cell cycle progression mediated by Src kinase activation. HBc-induced cellular proliferation and tumor formation were reversed by administration of the Src inhibitor saracatinib. Together, our findings suggest that HBc promotes tumorigenesis of hepatoma cells by enhancing the expression of total Src and the active form of the kinase and subsequently activates Src/PI3K/Akt signaling pathway, revealing novel insights into the underlying mechanisms of HBV-associated hepatocarcinogenesis.-Liu, W., Guo, T.-F., Jing, Z.-T., Yang, Z., Liu, L., Yang, Y.-P., Lin, X., Tong, Q.-Y. Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway.

Relationship between RAS Association Domain Family Protein 1A Promoter Methylation and the Clinicopathological Characteristics in Patients with Ovarian Cancer: A Systematic Meta-Analysis

Background: To investigate the relationship between RAS association domain family protein 1A (RASSF1A) promoter methylation and the clinical features, and the survival of ovarian cancer patients. Methods: A comprehensive literature search was conducted in the PubMed, Embase, EBSCO, and Cochrane Library databases. The overall ORs with their 95% CIs were calculated in this meta-analysis. Results: Finally 17 relevant publications with 1,108 ovarian cancer samples were available for the current meta-analysis. RASSF1A promoter methylation had a significantly higher level in ovarian cancer than in low malignant potential (LMP) tumors. No significant relationship was observed between RASSF1A promoter methylation and the clinicopathological characteristics in ovarian cancer. Two studies reported that RASSF1A promoter methylation was not correlated with the survival of patients with ovarian cancer. Conclusions: Our findings suggest that the use of RASSF1A promoter methylation could distinguish ovarian cancer and LMP tumors. ­RASSF1A promoter methylation may not be correlated with the clinical features and the survival of ovarian cancer patients. More studies with large sample sizes are essential in the future.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study.

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome-wide analysis of DNA methylation in colorectal tumours from 36 never-smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never-smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10-5 ) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never-smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking-associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.