Global Spectrum of Copy Number Variations Reveals Genome Organizational Plasticity and Proposes New Migration Routes

Global spectrum of CNVs is required to catalog variations to provide a high-resolution on the dynamics of genome-organization and human migration. In this study, we performed genome-wide genotyping using high-resolution arrays and identified 44,109 CNVs from 1,715 genomes across 12 populations. The study unraveled the force of independent evolutionary dynamics on genome-organizational plasticity across populations. We demonstrated the use of CNV tool to study human migration and identified a second major settlement establishing new migration routes in addition to existing ones.     

A Gamma-Knife-Enabled Mouse Model of Cerebral Single-Hemisphere Delayed Radiation Necrosis

Purpose

To develop a Gamma Knife-based mouse model of late time-to-onset, cerebral radiation necrosis (RN) with serial evaluation by magnetic resonance imaging (MRI) and histology.

Methods and Materials

Mice were irradiated with the Leksell Gamma Knife^® (GK) Perfexion^TM (Elekta AB; Stockholm, Sweden) with total single-hemispheric radiation doses (TRD) of 45- to 60-Gy, delivered in one to three fractions. RN was measured using T2-weighted MR images, while confirmation of tissue damage was assessed histologically by hematoxylin & eosin, trichrome, and PTAH staining.

Results

MRI measurements demonstrate that TRD is a more important determinant of both time-to-onset and progression of RN than fractionation. The development of RN is significantly slower in mice irradiated with 45-Gy than 50- or 60-Gy, where RN development is similar. Irradiated mouse brains demonstrate all of the pathologic features observed clinically in patients with confirmed RN. A semi-quantitative (0 to 3) histologic grading system, capturing both the extent and severity of injury, is described and illustrated. Tissue damage, as assessed by a histologic score, correlates well with total necrotic volume measured by MRI (correlation coefficient = 0.948, with p<0.0001), and with post-irradiation time (correlation coefficient = 0.508, with p<0.0001).

Conclusions

Following GK irradiation, mice develop late time-to-onset cerebral RN histology mirroring clinical observations. MR imaging provides reliable quantification of the necrotic volume that correlates well with histologic score. This mouse model of RN will provide a platform for mechanism of action studies, the identification of imaging biomarkers of RN, and the development of clinical studies for improved mitigation and neuroprotection.     

Naturally Occurring Genetic Variants in Human Chromogranin A (CHGA) Associated with Hypertension as well as Hypertensive Renal Disease

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.     

4-deoxy-4-fluoro-xyloside derivatives as inhibitors of glycosaminoglycan biosynthesis

Various 4-deoxy-4-fluoro-xylosides were prepared using click chemistry for evaluating their potential utility as inhibitors of glycosaminoglycan biosynthesis. 2,3-Di-O-benzoyl-4-deoxy-4-fluoro-β-D-xylopyranosylazide, obtained from L-arabinopyranose by six steps, was treated with a wide variety of azide-reactive triple bond-containing hydrophobic agents in the presence of Cu(2+) salt/ascorbic acid, a step known as click chemistry. After click chemistry, benzoylated derivatives were deprotected under Zemplén conditions to obtain 4-deoxy-4-fluoro-xyloside derivatives. A mixture of α:β-isomers of twelve derivatives were then separated on a reverse phase C18 column using HPLC and the resulting twenty four 4-deoxy-4-fluoro-xylosides were evaluated for their ability to inhibit glycosaminoglycan biosynthesis in endothelial cells. We identified two xyloside derivatives that selectively inhibit heparan sulfate and chondroitin sulfate/derman sulfate biosynthesis without affecting cell viability. These novel derivatives can potentially be used to define the biological actions of proteoglycans in model organisms and also as therapeutic agents to combat various human diseases in which glycosaminoglycans participate.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Interaction of Bacillus thuringiensis Vegetative Insecticidal Protein with Ribosomal S2 Protein Triggers Larvicidal Activity in Spodoptera frugiperda

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.Insecticidal proteins produced by strains of Bacillus thuringiensis can broadly be classified into two major categories based on their site of accumulation. Category I consist of proteins that are deposited as crystals in sporangia and are referred to as insecticidal crystalline proteins (ICPs). The second category consists of recently described group of insecticidal proteins, called vegetative insecticidal proteins (8). These proteins are synthesized during the vegetative growth of Bacillus cells and are secreted into the culture medium. Irrespective of the site of accumulation of insecticidal proteins, their ingestion by susceptible insect larvae leads to disruption and lysis of epithelial tissue from the midgut, resulting in larval death (12). The mechanism of lysis of gut epithelial tissue by ICPs has been investigated in detail in several insects (16). Ingestion of ICPs triggers a sequence of biochemical cascade that involves its solubilization and subsequent activation by gut proteases. The activated toxin interacts with specific receptors located at the midgut epithelial tissue. In this sequence of events, the interaction with the receptor is the most significant event since subsequent to interaction, pore formation is initialized, and that leads to lysis of epithelial cells. The identification and characterization of receptors from various insect larvae has led to the identification of following molecules as receptor to ICPs, such as cadherinlike protein (21), glycosyl phosphatidylinositol (GPI)-anchored aminopeptidase N (APN) (1, 9, 11, 17, 19, 20), a GPI-anchored alkaline phosphatase (10, 14), and a 270-kDa glycoconjugate (see references 2, 7, 9, and 16 and references therein for an extensive list of receptors). In addition, certain glycopeptides have been identified as lysis-initiating receptor molecules. Although there is extensive information about the receptor-toxin interaction for ICPs, negligible work has been done toward the identification of receptors to vegetative insecticidal proteins. The ultrastructural changes induced at the midgut epithelial tissue, upon ingestion of ICPs or Vip3As, are common (12). Both ICPs and Vip3As interact at the epithelial layer of midgut, enlarging the affected cells due to osmotic imbalance and eventually causing lysis. In spite of inflicting nearly identical structural damage, the interacting receptor for the Vip3A is not identical (12). In fact, the receptor to Vip3As has not yet been characterized.Our group has been working on the identification, cloning, and evaluation of vegetative insecticidal proteins from strains of B. thuringiensis held in our collection. We have characterized the Vip3A (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"Y17158","term_id":"3135740","term_text":"Y17158"}}Y17158) class of protein and evaluated its toxicity profile (2, 8, 18). Vip3A is active against larvae of Spodoptera litura, among several other lepidopteran pests. In a parallel series of experiments, we identified APN as a receptor to the B. thuringiensis protein Cry1C in S. litura. The heterologously expressed APN did not interact with Vip3A, suggesting that Vip3A toxicity in this insect is not through interaction with APN (1). Our preliminary results on the toxicity of Vip3A revealed that purified insecticidal protein could lyse Sf21 cells, suggesting the presence of receptors in the insect cell line. In the present study, we identified the Vip3A interacting protein in Sf21 cells and the larvae of S. litura. The specificity of the interaction has been examined by a combination of ex vivo and in vitro assays. These assays identified ribosomal S2 protein as the interacting partner of Vip3A. The functional significance of S2-Vip3A protein interaction was examined by monitoring the reduction in Vip3A toxicity in Sf21 cells and larvae of S. litura by the RNA interference-induced knockdown of S2 protein. The results of these experiments are discussed in the context of colocalization of the S2-Vip3A protein interacting complex by confocal microscopy.     

Why should a grazer browse? Livestock impact on winter resource use by bharal Pseudois nayaur

Many mammalian herbivores show a temporal diet variation between graminoid-dominated and browse-dominated diets. We determined the causes of such a diet shift and its implications for conservation of a medium-sized ungulate—the bharal Pseudois nayaur. Past studies show that the bharal diet is dominated by graminoids (>80%) during summer, but the contribution of graminoids declines to about 50% in winter. We tested the predictions generated by two alternative hypotheses explaining the decline: low graminoid availability during winter causes bharal to include browse in their diet; bharal include browse, with relatively higher nutritional quality, in their diet to compensate for the poor quality of graminoids during winter. We measured winter graminoid availability in areas with no livestock grazing, areas with relatively moderate livestock grazing, and those with intense livestock grazing pressures. The chemical composition of plants contributing to the bharal diet was analysed. The bharal diet was quantified through signs of feeding on vegetation at feeding locations. Population structures of bharal populations were recorded using a total count method. Graminoid availability was highest in areas without livestock grazing, followed by areas with moderate and intense livestock grazing. The bharal diet was dominated by graminoids (73%) in areas with highest graminoid availability. Graminoid contribution to the bharal diet declined monotonically (50, 36%) with a decline in graminoid availability. Bharal young to female ratio was 3 times higher in areas with high graminoid availability than areas with low graminoid availability. The composition of the bharal winter diet was governed predominantly by the availability of graminoids in the rangelands. Our results suggest that bharal include more browse in their diet during winter due to competition from livestock for graminoids. Since livestock grazing reduces graminoid availability, creation of livestock-free areas is necessary for the conservation of grazing species such as the bharal and its predators including the endangered snow leopard in the Trans-Himalaya.     

Androgen receptor status predicts response to chemotherapy, not risk of breast cancer in Indian women

Voltage gated potassium channels have been extensively studied in relation to cancer. In this review, we will focus on the role of two potassium channels, Ether à-go-go (Eag), Human ether à-go-go related gene (HERG), in cancer and their potential therapeutic utility in the treatment of cancer. Eag and HERG are expressed in cancers of various organs and have been implicated in cell cycle progression and proliferation of cancer cells. Inhibition of these channels has been shown to reduce proliferation both in vitro and vivo studies identifying potassium channel modulators as putative inhibitors of tumour progression. Eag channels in view of their restricted expression in normal tissue may emerge as novel tumour biomarkers.     

Reproductive efficiency of Thoroughbred mares under Indian subtropical conditions: A retrospective survey over 7 years

Service records of 253 mares (1181 mare-years) spanning over 7 consecutive years, from nine organized Thoroughbred stud farms, situated in the subtropical northwestern India were retrospectively analyzed to assess their reproductive performance. The overall per cycle pregnancy rate at Day 16 and overall foaling rates were 50.30% and 68.95%, respectively, and were significantly higher in mares aged 3–7 years than ≥18 years old mares. The late embryonic losses (9.86%) that occurred between Days 16 and 39 post-ovulation contributed more than 50% of the overall detected pregnancy losses (19.11%). The overall percent detected pregnancy losses were lower in mares at ages 3–7 years compared to those at ages ≥18 years (14.78% vs. 46.43%, respectively; P < 0.0001). Chronic barren and habitual aborter mares tended to affect reproductive efficiency of mares. Fifty percent of the mares that experienced ≥2 consecutive abortions or barren years, again stayed aborted or barren in the next seasons, respectively. No effect of numbers of matings per oestrus was observed on overall fertility. Neither the induction of oestrus nor ovulation by exogenous hormonal treatment had any effect on most of the analyzed reproductive parameters. Regarding breeding month or years, the reproductive efficiency did not differ significantly. The incidence of multiple pregnancies was 5.40% and percent late embryonic loses were higher (P = 0.0016) in twin (21.98%) than singleton (8.64%) pregnancies. In conclusion, comparatively lower fertility rates were recorded in Thoroughbred mares bred under Indian subtropical climatic conditions than those reported from temperate regions that might be due to difference in breeding management rather than prevailing environment.