Genome-Wide Association Study of Parity in Bangladeshi Women

Human fertility is a complex trait determined by gene-environment interactions in which genetic factors represent a significant component. To better understand inter-individual variability in fertility, we performed one of the first genome-wide association studies (GWAS) of common fertility phenotypes, lifetime number of pregnancies and number of children in a developing country population. The fertility phenotype data and DNA samples were obtained at baseline recruitment from individuals participating in a large prospective cohort study in Bangladesh. GWAS analyses of fertility phenotypes were conducted among 1,686 married women. One SNP on chromosome 4 was non-significantly associated with number of children at P <10^-7 and number of pregnancies at P <10^-6. This SNP is located in a region without a gene within 1 Mb. One SNP on chromosome 6 was non-significantly associated with extreme number of children at P <10^-6. The closest gene to this SNP is HDGFL1, a hepatoma-derived growth factor. When we excluded hormonal contraceptive users, a SNP on chromosome 5 was non-significantly associated at P <10^-5 for number of children and number of pregnancies. This SNP is located near C5orf64, an open reading frame, and ZSWIM6, a zinc ion binding gene. We also estimated the heritability of these phenotypes from our genotype data using GCTA (Genome-wide Complex Trait Analysis) for number of children (h_g ² = 0.149, SE = 0.24, p-value = 0.265) and number of pregnancies (h_g ² = 0.007, SE = 0.22, p-value = 0.487). Our genome-wide association study and heritability estimates of number of pregnancies and number of children in Bangladesh did not confer strong evidence of common variants for parity variation. However, our results suggest that future studies may want to consider the role of 3 notable SNPs in their analysis.     

Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (−)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases

The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter-day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.     

Screening of the c-kit gene missense mutation in invasive ductal carcinoma of breast among north Indian population

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer deaths among females across the world, accounting for 23 % (1.38 million) of total new cancer cases and 14 % (0.45 million) of the total cancer deaths in 2008. c-kit is expressed in mast cell growth factor, cellular migration, proliferation, melanoblasts, haematopoietic progenitors and germ cells. We have designed our study with aim to explore the c-kit gene mutations in invasive ductal carcinoma (IDC) breast. To ascertain the range of mutations in exon 11, 13 and 17 of c-kit gene in 53 cases of IDC breast, we carried out PCR-SSCP followed by DNA sequencing. The mutation frequency of c-kit gene in exon 11, 13 and 17 were 9.43 % (5/53), 1.88 % (1/53) and 3.77 % (2/53), respectively. During our mutational analysis, we have detected five missense mutations in exon 11 (Pro551Leu, Glu562Val, Leu576Phe, His580Tyr and Phe584Leu), one missense mutation in exon 13 (Ser639Pro) and two missense mutations in exon 17 (Arg796Gly and Asn822Ser). It seems that c-kit mutations might participate in breast cancer pathogenesis and may be utilized as predictive marker, since the loss of c-kit positivity is generally linked with different types of breast cancer. Further molecular studies are necessary to validate the association of c-kit gene mutation in IDC breast pathogenesis.     

Modulation of Angiogenic and Inflammatory Response in Glioblastoma by Hypoxia

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy.Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.     

Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.     

What is needed for effective antitumor immunotherapy? Lessons learned using Listeria monocytogenes as a live vector for HPV-associated tumors

As a vaccine vector, Listeria monocytogenes targets the innate immune system, resulting in a cytokine response that enhances antigen-presenting cell function as well as inducing a Th1 profile. It also enhances cell-mediated immunity by targeting antigen delivery in antigen-presenting cells to both the MHC class I pathway of exogenous presentation that activates CD8 T cells and the MHC class II pathway that processes antigen endogenously and presents it to CD4 T cells. In this review, we describe the development of vaccine constructs that target the human papillomavirus 16 (HPV-16) E7 antigen, and we characterize their effects on tumor regression as well as various immune parameters both innate and adaptive. In particular, we describe the effect on tumor angiogenesis, induction of antitumor suppressor factors like CD4⁺CD25⁺ T cells and regulatory cytokines TGF- and IL-10, homing and infiltration of antigen-specific CD8⁺ T cells to the tumor, and also effects of the vaccines on antigen-presenting cells, especially focusing on dendritic cell maturation and ability to influence tumor regression. We believe that the identification of several immune parameters that correlate with antitumor efficacy, and of some that have a negative correlation, may have wider application for other cancer immunotherapeutic approaches.This article is a symposium paper from the second international conference Strategies for Immune Therapy, 29 February–3 March 2004, Würzburg, Germany; summarized by G. Pawelec and C. Gouttefangeas.     

Combinatorial synthesis and antibacterial evaluation of an indexed chalcone library

A 120-membered chalcone library has been designed and prepared from six differently substituted acetophenones (A1-A6) and 20 benzaldehydes (B1-B20). The library was subjected to biological studies targeted against six bacterial strains. For the identification of the most-active member(s) of the library, the so-called indexed or positional-scanning method was applied. Six out of 26 sub-libraries, i.e., AL1-AL6, were synthesized by keeping the acetophenone moiety A fixed and using equimolar quantities of the 20 different benzaldehydes. The remaining 20 sub-libraries BL1-BL20 were prepared by keeping the benzaldehyde B component fixed and varying the six acetophenones (Table 1). The bactericidal activities of the resulting sub-libraries were tested and used as indices to the rows or columns of a two-dimensional matrix. Finally, parallel synthesis of 24 specific members with the highest-expected antibacterial activities, present in two sub-libraries, was carried out. These chalcones were screened again, and the results were exploited for establishing the structure-activity relationship (SAR) and the identification of the lead compound, which turned out to be 1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (A2B2) in terms of activity towards Staphylococcus aureus and Bacillus subtilis (Tables 5-7).     

The central active site arginine in sulfite oxidizing enzymes alters kinetic properties by controlling electron transfer and redox interactions

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated.We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo^VI/V potential lowered by ca. 60–80 mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT^WT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in K_Msulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.