Polycystic ovary syndrome in Indian women: a mass spectrometry based serum metabolomics approach

Introduction

Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrinological disorder with uncertain pathogenesis and is very common in women of reproductive age. There are few reports of utilizing metabolomics approach to understand the complex pathophysiology of PCOS. However, excluding one previous NMR-based metabolomics study, none of the study was conducted in Indian population.

Objective

The study aims to compare the serum metabolomic profile of PCOS women with controls from the Eastern region of India.

Methods

PCOS women (n?=?35) and healthy control women (n?=?30) undergoing tubal ligation were recruited for this study. Serum metabolic profiles were generated using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS). Multivariate statistical analysis was applied to spectral data obtained from both the LC-MS/MS and GC/MS.

Results

Nine metabolites were identified to be most significantly dysregulated in sera of PCOS women; however, few other identified metabolites were also altered but with lesser significance. Amongst these metabolites, riboflavin, sucrose, adenine and N-acetyl glycine, phosphoric acid and cortisol were down-regulated, whereas, thymine, cystathionine, and phenylalanine were up-regulated in PCOS when compared with controls. The observed changes in metabolite expression suggested alterations in aminoacyl-tRNA biosynthesis, metabolism of nitrogen, alanine-aspartate-glutamate, galactose, glycine-serine-threonine, and pyrimidine-purine among several metabolic pathways possibly implicated in these PCOS women.

Conclusion

The altered metabolites identified in PCOS women of Eastern Indian population, provide insight into current perceptive of the disease pathology, metabolic involvements, and may be considered as putative markers of PCOS.

     

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a "cosmopolitan" tagging approach to capture the genetic diversity across approximately 2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.     

In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.A large number of inherited or acquired diseases remain promising targets for human gene therapy. One vector that has shown outstanding potential thus far in numerous preclinical and clinical evaluations is based on nonpathogenic adeno-associated virus (AAV). A unique asset among various properties that make AAV especially attractive over its competitors, such as adenoviral or lentiviral vectors, is the availability of a vast number of natural isolates which differ significantly in their properties (24). We and others have shown previously that the function of an AAV vector particle is determined mainly by the capsid protein and that viral Rep proteins and genomic packaging elements are largely interchangeable (24, 27, 85). Paradoxically, the ever-increasing repertoire of naturally occurring and synthetically generated AAV capsid sequences (>300 to date) is currently creating a dilemma for the rational selection of the optimal serotype for a given application. The importance of finding the ideal capsid for efficient and safe gene transfer has been exemplified in many preclinical studies, as well as in a clinical trial using the AAV type 2 (AAV-2) prototype in human liver tissue (36, 47). In one previous study, the treatment of patients with severe hemophilia B with recombinant AAV-2 expressing human factor IX (hFIX) resulted in mildly elevated, yet therapeutic, levels of this blood coagulation factor. However, expression was short lived, and the hFIX decline was accompanied by a transient asymptomatic increase of liver transaminases, due to a T-cell immune response against the AAV-2 capsid (47). Also, preexisting neutralizing anti-AAV-2 antibodies (frequent in humans) in these individuals likely inhibited the linear vector dose response previously observed in animals.We and others have suggested previously that the use of novel AAV serotypes, in particular, nonhuman isolates, will help to overcome some of these problems (19, 24, 63). Important examples are AAV-8 and AAV-9, which can transduce mouse liver far better than AAV-2, albeit the difference in dogs or primates is less clear (17, 52, 54, 75). The potential for the complete transduction of liver tissue and perhaps other tissues makes these two non-AAV-2 serotypes also particularly interesting for therapeutic RNA interference (RNAi) (28). We recently demonstrated the feasibility of efficiently and persistently suppressing hepatitis B virus with RNAi from a double-stranded AAV-8 vector (28). On the other hand, a potential drawback of AAV-8 and AAV-9 is their lack of specific tissue tropism (34, 52). The resulting frequent vector dissemination into all organs, including the brain, even from low peripheral doses in mice or monkeys (52, 54) is a particular concern for RNAi therapies in which control over vector biodistribution and the limitation of off-target effects will be imperative for the success of the approach (28).In order to overcome the constraints of wild-type AAV serotypes, numerous groups have recently begun to develop novel strategies to engineer “designer” AAVs tailored for the therapeutic transduction of clinically relevant organs (reviewed in detail in references 9, 12, 35, 41, 51, and 85). Briefly, the variety of strategies can be grouped into indirect or chemical approaches and direct physical modification strategies. In the indirect approaches, specific molecules (e.g., bispecific antibodies [6] or avidin-coupled ligands [4]) are allowed to react with the viral surface (biotinylated in the case of avidin [4]), as well as a cellular receptor, forming a conjugate ideally able to retarget the capsid to a refractory cell type. Yet, numerous pharmacological problems, such as concerns about in vivo complex stability and difficulties in upscaling complex manufacturing, continue to prevent the broad adaptation of these approaches. Alternative, more powerful strategies rely on the direct physical modification of the AAV capsid protein and gene. Early examples include the generation of mosaic AAV capsids via the mixing of helper plasmids carrying capsid genes from distinct serotypes, such as AAV-1 and AAV-2 (30) and pairwise combinations of AAV-1 through AAV-5 (62). Similar mosaics were generated previously via a marker rescue approach, yielding AAV-2/AAV-3 recombinants with unique properties (8). A related strategy is the rational creation of chimeric virions via domain swapping among multiple parental serotypes, involving either entire capsid loops or parts thereof or individual residues. Notable examples include AAV-1/AAV-2 chimeras with improved tropism in muscle tissue (31), with one of these chimeras presently being studied in a phase I clinical trial for the treatment of Duchenne muscular dystrophy (85). Most recently, our own group described a battery of unique chimeras comprising elements from serotypes 2 and 8, which were exploited to identify capsid subdomains responsible for efficient AAV transduction in murine liver tissue in vivo (64).A special type of chimeric capsids are those containing foreign proteins or peptides inserted into various positions of the virion shell. The methods and strategies used are widely diverse, and again, we refer to comprehensive reviews (12, 35, 41). Noteworthy here are approaches to fuse targeting ligands to the N termini of AAV capsid proteins (ideally, VP2 [45, 83]), or more powerful, to insert short peptides (up to 14 amino acids [21], typically 7) into exposed regions of the assembled virion. This strategy is referred to as viral display, in analogy to phage display, and has already been used extensively to retarget AAV-2 virions to a multitude of refractory or hard-to-infect cell types, such as vascular endothelial, smooth muscle, and pancreatic islet cells (43, 55, 77, 81, 82) and various tumor lines (22, 58, 65, 66). It has particularly benefited from comprehensive mutational analyses by various groups (e.g., references 21, 33, 56, and 83) that have resulted in the identification of prominent locations within the AAV-2 capsid tolerating peptide insertion. Most notable is the heparin binding domain (HBD), consisting of a total of four arginine (R) residues and one lysine residue, with R585 and R588 representing the most crucial components (37, 56). Numerous groups have now consistently shown that the insertion of 7-mer peptides into this region not only is frequently well tolerated and efficiently mediates virus retargeting, but also provides the extra benefit that the endogenous AAV-2 tropism can be abolished, thus enhancing target specificity (e.g., reference 21).In addition to identifying sites for vector engineering, some of the mutational AAV studies directly yielded novel capsid variants with potential benefits for clinical use. A remarkable case was a recent study by Lochrie et al. (42) in which a set of 127 AAV-2 variants with point or insertion mutations were generated and screened for multiple properties. Several capsids were isolated which differed from the wild-type AAV-2 capsid in having better in vitro transduction efficiencies (albeit being equally efficient in vivo) or, clinically most relevant, higher-level resistance to individual or pooled human antisera. Nonetheless, the limitations of the approach also became clear, most notably, the extreme effort required to generate and manually screen a large number of mutants, which in fact prevented the interesting analyses of all possible combinations of beneficial point mutations in further capsids.Indeed, the factors of time and labor are the main reasons why an increasing number of groups have recently begun to develop novel means for AAV vector evolution that no longer rely on the rational modification of the AAV-2 capsid. Instead, the new combinatorial methodologies allow for the far more efficient creation and selection of interesting candidates in a library-based high-throughput format. Thus far, two different strategies have been reported, both principally expanding on previously developed techniques. One is the use of viral display libraries, in which random 7-mer peptides are inserted into the AAV-2 HBD (at amino acid 587 or 588), yielding between 4 × 10⁶ and 1.7 × 10⁸ capsids potentially exposing new ligands on their surfaces (50, 58, 76). Subsequent iterative selection on diverse cell types refractory to the wild type, e.g., coronary artery endothelial cells, cardiomyoblasts, and carcinoma, leukemia, and megakaryocytic cell lines, led to enrichment with peptide mutants with increased target specificities and efficacies (48, 50, 58, 76). The second library type, independently described by two groups in 2006, relies on error-prone PCR amplification of the AAV-2 capsid gene (46, 59). Similar to the methods in earlier mutational studies, this approach resulted in the identification of AAV-2 point mutations (usually up to two per capsid) which yielded mutants that differed from the wild type in having mildly enhanced efficacies in vitro and/or improved transduction efficiencies in the presence of neutralizing anti-AAV-2 antibodies either generated in rabbits or preexisting in individual human sera.Here, for the first time, we introduce the technology of DNA family shuffling into the realm of AAV vector evolution. The basic concept of this technology is the in vitro recombination of related parental genes with >50% homology, which are first fragmented and then reassembled based on partial homology, resulting in libraries of chimeric genes. Iterative amplification under pressure can then yield hybrids not only combining parental assets, but also ideally exhibiting novel and synergistic properties (70, 71). DNA family shuffling has been used extensively in recent years to evolve and improve all types of proteins, from markers and enzymes to vaccines (e.g., references 10, 13-15, and 39). Importantly, a set of reports also suggested its power to enhance viral gene therapy vectors by creating retro- or lentiviruses with improved stability or efficacy compared to that of the parental wild types (57, 61, 69). Here, we describe the novel use of DNA family shuffling for the highly efficient molecular interbreeding of eight multispecies AAVs to create chimeric capsids and, moreover, document its compatibility and synergism with existing AAV vector evolution technology.     

Glutamate-induced assembly of bacterial cell division protein FtsZ

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; approximately 90% of the protein was sedimented as polymer in the presence of 1 m glutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 m glutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.     

Mixed culture bioconversion of 16-dehydropregnenolone acetate to androsta-1,4-diene-3,17-dione: optimization of parameters

Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonas diminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16-DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35-40 degrees C was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL(-1) 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9alpha-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend.     

Deteriorative changes of maize,groundnut and soybean seeds by fungi in storage

This paper deals with investigations on fungal infection, moisture content, germinability and deterioration of three seeds, viz., maize (starchy), groundnut (oily) and soybean (proteinaceous) in storage at the locality of Santiniketan, West Bengal, India, under natural condition for 1 year. The airspora of storage environment was trapped using culture plate method. Different species of Aspergillus (A. candidus, A. flavus, A. niger, A. terreus, and A. ruber) were dominant followed by Rhizopus, Penicillium, Curvularia, Fusarium, Alternaria, etc. Seed moisture was maximum in the rainy season followed by a gradual decrease during longer storage. A gradual decrease in field fungi with simultaneous increase in storage fungi accompanied by a reduction in germinability occurred in all seeds as storage proceeded. A gradual loss of carbohydrate (both soluble and insoluble) content in all the seeds were recorded. A loss of protein content was recorded followed by a small increase. Oil content decreased in prolonged storage with simultaneous increase in fatty acid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plants used by andaman aborigines in gathering rock-bee honey

Economic Botany - The giant rock bee,Apis dorsata, of Asia is a migratory and ferocious wild bee, which has not yet been tamed. It is the chief source of honey and beeswax in the Andaman region...     

Interaction between cell division proteins FtsE and FtsZ

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.     

X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43?kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14?Å resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 β-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6?Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with T_m of 76?°C. ¹³C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.