Structural Transitions Mediating Transcription Initiation by T7 RNA Polymerase

AbstractDuring transcription initiation, RNA polymerases appear to retain promoter interactions while transcribing short RNAs that are frequently released from the complex. Upon transition to elongation, the polymerase releases promoter and forms a stable elongation complex. Little is known about the changes in polymerase conformation or polymerase:DNA interactions that occur during this process. To characterize the transitions that occur in the T7 RNA polymerase transcription complex during initiation, we prepared enzymes with Fe-BABE conjugated at 11 different positions. Addition of H2O2 to transcription complexes prepared with these enzymes led to nucleic acid strand scission near the conjugate. Changes in the cleavage sites revealed a series of conformational changes and rearrangements of protein:nucleic acid contacts that mediate progression through the initiation reaction

Visceral adiposity index among young girls with PCOS and its association with phenotypes and metabolic risk

Background: Polycystic Ovarian Syndrome (PCOS) is a growing endocrine-metabolic disease in India. Visceral Adiposity Index (VAI) is a surrogate marker of visceral adipose dysfunction and can be used as a useful predictor of unhealthy PCOS phenotypes in low resource settings. No cut-off has been assessed among Indian population.Methods: Secondary data from 106 diagnosed girls with PCOS and 121 controls was analysed to estimate (i) VAI and BMI among different phenotypes (ii) risk of metabolic disorders using VAI among different phenotypes of PCOS and (iii) compare the overall diagnostic performance (for metabolic syndrome) of VAI, BMI and waist circumference.Results: Majority of the girls in the sample considered for analysis were lean PCOS (61%). Mean VAI among PCOS (3.02) was significantly higher than normal controls (2.81). Classic and Mild Phenotypes had high VAI. A unit increase in VAI score was found associated with 5.23 times higher risk of metabolic syndrome (AOR: 5.23, 95% CI: 2.261-12.086). A higher VAI with cut off value of 2.73 could predict risk of metabolic syndrome among PCOS cases, unlike the cutoff among Caucassian population of 1.67. The cut-off for the non- obese group was even higher i.e. 2.81.Conclusions: Given that Indians are genetically more prone to have excess visceral fat the cut-offs for measuring adiposity also needs to be re-defined. The findings of this small sample throws light on the prevalence of visceral adiposity among lean girls with PCOS emphasizing the need to also screen them for metabolic syndrome, educate them about these complications and motivate them to practice healthy lifestyles

Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions

During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP). Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches

A comprehensive curated resource for follicle stimulating hormone signaling

<p>Abstract</p> <p>Background</p> <p>Follicle stimulating hormone (FSH) is an important hormone responsible for growth, maturation and function of the human reproductive system. FSH regulates the synthesis of steroid hormones such as estrogen and progesterone, proliferation and maturation of follicles in the ovary and spermatogenesis in the testes. FSH is a glycoprotein heterodimer that binds and acts through the FSH receptor, a G-protein coupled receptor. Although online pathway repositories provide information about G-protein coupled receptor mediated signal transduction, the signaling events initiated specifically by FSH are not cataloged in any public database in a detailed fashion.</p> <p>Findings</p> <p>We performed comprehensive curation of the published literature to identify the components of FSH signaling pathway and the molecular interactions that occur upon FSH receptor activation. Our effort yielded 64 reactions comprising 35 enzyme-substrate reactions, 11 molecular association events, 11 activation events and 7 protein translocation events that occur in response to FSH receptor activation. We also cataloged 265 genes, which were differentially expressed upon FSH stimulation in normal human reproductive tissues.</p> <p>Conclusions</p> <p>We anticipate that the information provided in this resource will provide better insights into the physiological role of FSH in reproductive biology, its signaling mediators and aid in further research in this area. The curated FSH pathway data is freely available through NetPath (<url>http://www.netpath.org</url>), a pathway resource developed previously by our group.</p

Targeted genome-wide DNA methylation profiling of ovarian granulosa cells from women with PCOS

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy of obscure pathophysiologic origins, globally affecting 6-15% women of childbearing age. Emerging evidence on repercussions of environmental insults and changing lifestyles on fecundity and reproductive health have necessitated the study of tissue-specific epigenetic alterations in PCOS development. In semblance to follicular and oocyte defects observed in PCOS ovaries, targeted bisulfite sequencing was performed to generate the methylome signatures of ovarian granulosa cells (GCs) obtained from age-BMI matched women with PCOS (n=3) and healthy, regularly menstruating controls (n=3) using next generation sequencing approach. Paired end sequencing of samples was carried out on Illumina HiSeq 2500 ® platform and data were analyzed using the Bismark tool. Methylation levels of a few selected genes relevant to ovarian function were further validated in GCs obtained from 10 controls and 10 women with PCOS by pyrosequencing.  Relative transcript levels of these genes were assessed by q-RT PCR using Taqman assays. In the methylome analysis, a total of 6486 CpG sites representing 3840 genes associated with pathways such as Wnt signaling, G-protein receptor signaling, angiogenesis, chemokine and cytokine mediated inflammation and integrin signaling showed differential methylation in PCOS. Of these, a total of 2977 CpG sites representing 2063 genes were identified as hypomethylated while 3509 CpG sites in 1777 genes were found to be hypermethylated. Additionally, differential methylation was also noted in several non-coding RNAs regulating vital ovarian functions and which are reported to be dysregulated in PCOS. This data provides compelling evidence in support of epigenetic alterations as etiopathogenic factors associated with ovarian dysfunction in PCOS

Elucidating the impact of obesity on hormonal and metabolic perturbations in polycystic ovary syndrome phenotypes in Indian women.

Polycystic ovary syndrome is a complex endocrinopathy with heterogeneous presentation and multifactorial etiology. We have undertaken this case-control study to compare metabolic and endocrine characteristics in different phenotypic subgroups of women with PCOS and the impact of obesity on them. Women with PCOS (n = 489) were classified into 4 phenotypes according to Rotterdam criteria. Comparisons of clinical, biochemical and hormonal parameters were performed across all phenotypic groups of PCOS and with controls (n = 270) by Welch's ANOVA with subsequent Games-Howell post-hoc test. We found maximum prevalence of normoandrogenic phenotype D, which is milder form of PCOS in terms of insulin resistance, gonadotropin levels and dyslipidemia, followed by phenotype A, in our total study population. After classification of the study group into lean and obese groups, only few insulin and lipid-related traits showed marked differences between phenotypes. Further, we noted that obese women showed adverse metabolic but not androgenic traits compared to lean counterparts in the same phenotype. Metabolic syndrome frequency is increased in hyperandrogenic phenotypes with HDL-C and waist circumference being most predominant contributing factors in total, lean and obese groups. We demonstrate that in our study population there is greater occurrence of phenotype D of PCOS. Our study highlights the importance of clinicians concurrently employing Rotterdam criteria along with obesity status for ascertaining accurate PCOS status and formulating suitable therapeutic intervention

DNA methylome profiling of granulosa cells reveals altered methylation in genes regulating vital ovarian functions in polycystic ovary syndrome

Abstract Background Women with polycystic ovary syndrome (PCOS) manifest a host of ovarian defects like impaired folliculogenesis, anovulation, and poor oocyte quality, which grossly affect their reproductive health. Addressing the putative epigenetic anomalies that tightly regulate these events is of foremost importance in this disorder. We therefore aimed to carry out DNA methylome profiling of cumulus granulosa cells and assess the methylation and transcript expression profiles of a few differentially methylated genes contributing to ovarian defects in PCOS. A total of 20 controls and 20 women with PCOS were selected from a larger cohort of women undergoing IVF, after carefully screening their sera and follicular fluids for hormonal and biochemical parameters. DNA extracted from cumulus granulosa cells of three women each, from control and PCOS groups was subjected to high-throughput, next generation bisulfite sequencing, using the Illumina HiSeq 2500® platform. Remaining samples were used for the validation of methylation status of some identified genes by pyrosequencing, and the transcript expression profiles of these genes were assessed by quantitative real-time PCR. Results In all, 6486 CpG sites representing 3840 genes associated with Wnt signaling, G protein receptor, endothelin/integrin signaling, angiogenesis, chemokine/cytokine-mediated inflammation, etc., showed differential methylation in PCOS. Hypomethylation was noted in 2977 CpGs representing 2063 genes while 2509 CpGs within 1777 genes showed hypermethylation. Methylation differences were also noted in noncoding RNAs regulating several ovarian functions that are dysregulated in PCOS. Few differentially methylated genes such as aldo-keto reductase family 1 member C3, calcium-sensing receptor, resistin, mastermind-like domain 1, growth hormone-releasing hormone receptor and tumor necrosis factor, which predominantly contribute to hyperandrogenism, premature luteolysis, and oocyte development defects, were explored as novel epigenetic candidates in mediating ovarian dysfunction. Methylation profiles of these genes matched with our NGS findings, and their transcript expression patterns correlated with the gene hypo- or hypermethylation status. Conclusion Our findings suggest that the epigenetic dysregulation of genes involved in important processes associated with follicular development may contribute to ovarian defects observed in women with PCOS