Variants in EMX2 and PTEN do not contribute to risk of endometriosis

Endometriosis has a genetic component, and significant linkage has been found to a region on chromosome 10q. Two candidate genes, EMX2 and PTEN, implicated in both endometriosis and endometrial cancer, lie on chromosome 10q. We hypothesized that variation in EMX2 and/or PTEN could contribute to the risk of endometriosis and may account for some of the linkage signal on 10q. We genotyped single nucleotide polymorphisms (SNPs) in a case-control design to evaluate association between endometriosis and common variations in these two genes. The genotyping and statistical analysis were based on samples collected from Australian volunteers. The cases were 768 unrelated women with surgically confirmed endometriosis selected from affected sister pair (ASP) families participating in the Australian Genes behind Endometriosis Study. The controls were 768 female participants in twin studies who, based on screening questions, did not have a diagnosis of endometriosis. Genotypes of 22 SNPs in the EMX2 gene and 15 SNPs in the PTEN gene were the main outcome measures. Statistical analysis provided measures of linkage disequilibrium and association. Permutation testing showed no globally significant association between any SNPs or haplotypes and endometriosis for either gene. It is unlikely that the EMX2 or PTEN gene variants investigated contribute to risk for initiation and/or development of endometriosis.

KRAS variation and risk of endometriosis

Endometriosis is a common gynaecological disease with symptoms of pelvic pain and infertility which affects 7-10% of women in their reproductive years. Activation of an oncogenic allele of Kirsten rat sarcoma viral oncogene homologue (KRAS) in the reproductive tract of mice resulted in the development of endometriosis. We hypothesized that variation in KRAS may influence risk of endometriosis in humans. Thirty tagSNPs spanning a region of 60.7 kb across the KRAS locus were genotyped using iPLEX chemistry on a MALDI-TOF MassARRAY platform in 959 endometriosis cases and 959 unrelated controls, and data were analysed for association with endometriosis. Genotypes were obtained for most individuals with a mean completion rate of 99.1%. We identified six haplotype blocks across the KRAS locus in our sample. There were no significant differences between cases and controls in the frequencies of individual single-nucleotide polymorphisms (SNPs) or haplotypes. We also developed a rapid method to screen for 11 common KRAS and BRAF mutations on the Sequenom MassARRAY system. The assay detected all mutations previously identified by direct sequencing in a panel of positive controls. No germline variants for KRAS or BRAF were detected. Our results demonstrate that any risk of endometriosis in women because of common variation in KRAS must be very small.

Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26

Endometriosis is a common gynecological disease that affects up to 10% of women in their reproductive years. It causes pelvic pain, severe dysmenorrhea, and subfertility. The disease is defined as the presence of tissue resembling endometrium in sites outside the uterus. Its cause remains uncertain despite >50 years of hypothesis-driven research, and thus the therapeutic options are limited. Disease predisposition is inherited as a complex genetic trait, which provides an alternative route to understanding the disease. We seek to identify susceptibility loci, using a positional-cloning approach that starts with linkage analysis to identify genomic regions likely to harbor these genes. We conducted a linkage study of 1,176 families (931 from an Australian group and 245 from a U.K. group), each with at least two members--mainly affected sister pairs--with surgically diagnosed disease. We have identified a region of significant linkage on chromosome 10q26 (maximum LOD score [MLS] of 3.09; genomewide P = .047) and another region of suggestive linkage on chromosome 20p13 (MLS = 2.09). Minor peaks (with MLS > 1.0) were found on chromosomes 2, 6, 7, 8, 12, 14, 15, and 17. This is the first report of linkage to a major locus for endometriosis. The findings will facilitate discovery of novel positional genetic variants that influence the risk of developing this debilitating disease. Greater understanding of the aberrant cellular and molecular mechanisms involved in the etiology and pathophysiology of endometriosis should lead to better diagnostic methods and targeted treatments.

Exclusion of angiotensinogen gene in molecular basis of human hypertension: Sibpair linkage and association analyses in Australian anglo-caucasians

Linkage with essential hypertension has been claimed for a microsatellite marker near the angiotensinogen gene (AGT; chromosome 1q42), as has association for the AGT variants M235T, G(-6)A and A(-20)C. To more rigorously evaluate AGT as a candidate gene for hypertension we performed sibpair analysis with multiple microsatellite markers surrounding this locus and using more sophisticated analysis programs. We also performed an association study of the AGT variants in unrelated subjects with a strong family history (two affected parents). For the linkage study, single and multiplex polymerase chain reaction (PCRs) and automated genescan analysis were conducted on DNA from 175 Australian Anglo-Celtic Caucasian hypertensives for the following markers: D1S2880-(2.1 cM)-D1S213-(2.8 cM)-D1S251-(6.5 cM)-AGT-(2.0 cM) -D1S235. Statistical evaluation of genotype data by nonparametric methods resulted in the following scores: Single-point analysis - SPLINK, P > 0.18; APM method, P > 0.25; ASPEX, MLOD < 0.28; SIB-PAIR, P > 0. 24; Multipoint analysis - MAPMAKER/SIBS, MLOD < 0.24; GENEHUNTER, P > 0.35. Exclusion scores of Lod -4.1 to -5.1 were obtained for these markers using MAPMAKER/SIBS for a lambda(s) of 1.6. The association study of G(-6)A, A(-20)C and M235T variants in 111 hypertensives with strong family history and 190 normotensives with no family history showed significant linkage disequilibrium between particular haplotypes, but we could find no association with hypertension. The present study therefore excludes AGT in the etiology of hypertension, at least in the population of Australian Anglo-Celtic Caucasians studied.

Complete cytogenetic response and major molecular response as surrogate outcomes for overall survival in first-line treatment of chronic myelogenous leukemia: A case study for technology appraisal on the basis of surrogate outcomes evidence

Objectives In 2012, the National Institute for Health and Care Excellence assessed dasatinib, nilotinib, and standard-dose imatinib as first-line treatment of chronic phase chronic myelogenous leukemia (CML). Licensing of these alternative treatments was based on randomized controlled trials assessing complete cytogenetic response (CCyR) and major molecular response (MMR) at 12 months as primary end points. We use this case study to illustrate the validation of CCyR and MMR as surrogate outcomes for overall survival in CML and how this evidence was used to inform National Institute for Health and Care Excellence’s recommendation on the public funding of these first-line treatments for CML. Methods We undertook a systematic review and meta-analysis to quantify the association between CCyR and MMR at 12 months and overall survival in patients with chronic phase CML. We estimated life expectancy by extrapolating long-term survival from the weighted overall survival stratified according to the achievement of CCyR and MMR. Results Five studies provided data on the observational association between CCyR or MMR and overall survival. Based on the pooled association between CCyR and MMR and overall survival, our modeling showed comparable predicted mean duration of survival (21–23 years) following first-line treatment with imatinib, dasatinib, or nilotinib. Conclusions This case study illustrates the consideration of surrogate outcome evidence in health technology assessment. Although it is often recommended that the acceptance of surrogate outcomes be based on randomized controlled trial data demonstrating an association between the treatment effect on both the surrogate outcome and the final outcome, this case study shows that policymakers may be willing to accept a lower level of evidence (i.e., observational association).

Combined effect of double antireflection coating and reversible molecular doping on performance of few-layer graphene/n-silicon Schottky barrier solar cells

Few-layer graphene films were grown by chemical vapor deposition and transferred onto n-type crystalline silicon wafers to fabricate graphene/n-silicon Schottky barrier solar cells. In order to increase the power conversion efficiency of such cells the graphene films were doped with nitric acid vapor and an antireflection treatment was implemented to reduce the sunlight reflection on the top of the device. The doping process increased the work function of the graphene film and had a beneficial effect on its conductivity. The deposition of a double antireflection coating led to an external quantum efficiency up to 90% across the visible and near infrared region, the highest ever reported for this type of devices. The combined effect of graphene doping and antireflection treatment allowed to reach a power conversion efficiency of 8.5% exceeding the pristine (undoped and uncoated) device performance by a factor of 4. The optical properties of the antireflection coating were found to be not affected by the exposure to nitric acid vapor and to remain stable over time.

Molecular characterisation of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here we characterised Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vat-vatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic SPATE secreted by UPEC during infection.

Oxazolidine-2-thiones: A molecular modeling study

Two oxazolidine-2-thiones, thio-analogs of linezolid, were synthesized and their antibacterial properties evaluated. Unlike oxazolidinones, the thio-analogs did not inhibit the growth of Gram positive bacteria. A molecular modeling study has been carried out to aid understanding of this unexpected finding.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell−cell adhesion. PECAM-1 has been shown to mediate cell−cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.

Can current force fields reproduce ring puckering in 2-O-sulfo-α-l-iduronic acid? A molecular dynamics simulation study

The monosaccharide 2-O-sulfo-α-l-iduronic acid (IdoA2S) is one of the major components of glycosaminoglycans. The ability of molecular mechanics force fields to reproduce ring-puckering conformational equilibrium is important for the successful prediction of the free energies of interaction of these carbohydrates with proteins. Here we report unconstrained molecular dynamics simulations of IdoA2S monosaccharide that were carried out to investigate the ability of commonly used force fields to reproduce its ring conformational flexibility in aqueous solution. In particular, the distribution of ring conformer populations of IdoA2S was determined. The GROMOS96 force field with the SPC/E water potential can predict successfully the dominant skew-boat to chair conformational transition of the IdoA2S monosaccharide in aqueous solution. On the other hand, the GLYCAM06 force field with the TIP3P water potential sampled transitional conformations between the boat and chair forms. Simulations using the GROMOS96 force field showed no pseudorotational equilibrium fluctuations and hence no inter-conversion between the boat and twist boat ring conformers. Calculations of theoretical proton NMR coupling constants showed that the GROMOS96 force field can predict the skew-boat to chair conformational ratio in good agreement with the experiment, whereas GLYCAM06 shows worse agreement. The omega rotamer distribution about the C5–C6 bond was predicted by both force fields to have torsions around 10°, 190°, and 360°.

Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

Molecular dynamics simulation of the phosphorylation-induced conformational changes of a tau peptide fragment

Aggregation of the microtubule associated protein tau (MAPT) within neurons of the brain is the leading cause of tauopathies such as Alzheimer's disease. MAPT is a phospho-protein that is selectively phosphorylated by a number of kinases in vivo to perform its biological function. However, it may become pathogenically hyperphosphorylated, causing aggregation into paired helical filaments and neurofibrillary tangles. The phosphorylation induced conformational change on a peptide of MAPT (htau225−250) was investigated by performing molecular dynamics simulations with different phosphorylation patterns of the peptide (pThr231 and/or pSer235) in different simulation conditions to determine the effect of ionic strength and phosphate charge. All phosphorylation patterns were found to disrupt a nascent terminal β-sheet pattern (226VAVVR230 and 244QTAPVP249), replacing it with a range of structures. The double pThr231/pSer235 phosphorylation pattern at experimental ionic strength resulted in the best agreement with NMR structural characterization, with the observation of a transient α-helix (239AKSRLQT245). PPII helical conformations were only found sporadically throughout the simulations. Proteins 2014; 82:1907–1923. © 2014 Wiley Periodicals, Inc.

Diaryl dihydropyrazole-3-carboxamides with significant in vivo antiobesity activity related to CB1 receptor antagonism: Synthesis, biological evaluation, and molecular modeling in the homology model

A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compoundsthe bisulfate salt of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (−)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(−)-3-(4-chlorophenyl)-N-methyl-N‘-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.

Molecular modeling of Bt Cry1Ac (DI–DII)–ASAL (Allium sativum lectin)–fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI–DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI–DII domains of Cry1Ac and lectin has been identified using protein–protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI–DII–lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI–DII–DIII) protein. Molecular mechanics/Poisson–Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein–protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic–aromatic, aromatic–sulphur, cation–pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac–APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac–APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

Molecular and biochemical characterisation of transgenic banana lines containing iron uptake and storage enhancing genes

This thesis investigated the basis for availability of iron (Fe) and zinc (Zn) content in different banana fruits grown in Uganda and Australia. Rather than micronutrient content levels in different banana cultivar, genotype and environment interactions explained much of the differences. Such information should provide important insights for future developments in the biofortification of banana. Bananas consumed in Uganda did not contain sufficient levels of Fe and Zn that meet the nutrient requirements for vulnerable groups.