Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 _{(55AAAAAAA61)} and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.     

Transcriptomic Identification of ADH1B as a Novel Candidate Gene for Obesity and Insulin Resistance in Human Adipose Tissue in Mexican Americans from the Veterans Administration Genetic Epidemiology Study (VAGES)

Type 2 diabetes (T2D) is a complex metabolic disease that is more prevalent in ethnic groups such as Mexican Americans, and is strongly associated with the risk factors obesity and insulin resistance. The goal of this study was to perform whole genome gene expression profiling in adipose tissue to detect common patterns of gene regulation associated with obesity and insulin resistance. We used phenotypic and genotypic data from 308 Mexican American participants from the Veterans Administration Genetic Epidemiology Study (VAGES). Basal fasting RNA was extracted from adipose tissue biopsies from a subset of 75 unrelated individuals, and gene expression data generated on the Illumina BeadArray platform. The number of gene probes with significant expression above baseline was approximately 31,000. We performed multiple regression analysis of all probes with 15 metabolic traits. Adipose tissue had 3,012 genes significantly associated with the traits of interest (false discovery rate, FDR ≤ 0.05). The significance of gene expression changes was used to select 52 genes with significant (FDR ≤ 10^-4) gene expression changes across multiple traits. Gene sets/Pathways analysis identified one gene, alcohol dehydrogenase 1B (ADH1B) that was significantly enriched (P < 10^-60) as a prime candidate for involvement in multiple relevant metabolic pathways. Illumina BeadChip derived ADH1B expression data was consistent with quantitative real time PCR data. We observed significant inverse correlations with waist circumference (2.8 x 10^-9), BMI (5.4 x 10^-6), and fasting plasma insulin (P < 0.001). These findings are consistent with a central role for ADH1B in obesity and insulin resistance and provide evidence for a novel genetic regulatory mechanism for human metabolic diseases related to these traits.     

Micro-Economic Impact of Congenital Heart Surgery: Results of a Prospective Study from a Limited-Resource Setting

Introduction

The microeconomic impact of surgery for congenital heart disease is unexplored, particularly in resource limited environments. We sought to understand the direct and indirect costs related to congenital heart surgery and its impact on Indian households from a family perspective.

Methods

Baseline and first follow-up data of 644 consecutive children admitted for surgery for congenital heart disease (March 2013 – July 2014) in a tertiary referral hospital in Central Kerala, South India was collected prospectivelyfrom parents through questionnaires using a semi-structured interview schedule.

Results

The median age was 8.2 months (IQR: 3.0– 36.0 months). Most families belonged to upper middle (43.0%) and lower middle (35.7%) socioeconomic class. Only 3.9% of families had some form of health insurance. The median expense for the admission and surgery was INR 201898 (IQR: 163287–266139) [I$ 11989 (IQR: 9696–15804)], which was 0.93 (IQR: 0.52–1.49) times the annual family income of affected patients. Median loss of man-days was 35 (IQR: 24–50) and job-days was 15 (IQR: 11–24). Surgical risk category and hospital stay duration significantly predicted higher costs. One in two families reported overwhelming to high financial stress during admission period for surgery. Approximately half of the families borrowed money during the follow up period after surgery.

Conclusion

Surgery for congenital heart disease results in significant financial burden for majority of families studied. Efforts should be directed at further reductions in treatment costs without compromising the quality of care together with generating financial support for affected families.     

fastSTRUCTURE: Variational Inference of Population Structure in Large SNP Data Sets

Tools for estimating population structure from genetic data are now used in a wide variety of applications in population genetics. However, inferring population structure in large modern data sets imposes severe computational challenges. Here, we develop efficient algorithms for approximate inference of the model underlying the STRUCTURE program using a variational Bayesian framework. Variational methods pose the problem of computing relevant posterior distributions as an optimization problem, allowing us to build on recent advances in optimization theory to develop fast inference tools. In addition, we propose useful heuristic scores to identify the number of populations represented in a data set and a new hierarchical prior to detect weak population structure in the data. We test the variational algorithms on simulated data and illustrate using genotype data from the CEPH–Human Genome Diversity Panel. The variational algorithms are almost two orders of magnitude faster than STRUCTURE and achieve accuracies comparable to those of ADMIXTURE. Furthermore, our results show that the heuristic scores for choosing model complexity provide a reasonable range of values for the number of populations represented in the data, with minimal bias toward detecting structure when it is very weak. Our algorithm, fastSTRUCTURE, is freely available online at http://pritchardlab.stanford.edu/structure.html.     

Relatedness of Indian Flax Genotypes (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.): An Inter-Simple Sequence Repeat (ISSR) Primer Assay

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03–0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard’s similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.     

A Novel NAD<Superscript>+</Superscript>-dependent aldehyde dehydrogenase encoded by the <Emphasis Type="Italic">puuC</Emphasis> gene of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate

3-Hydroxypropionic acid (3-HP), a versatile and valuable platform chemical, has diverse industrial applications; but its biological production from glycerol is often limited by the capability of the enzyme aldehyde dehydrogenase (ALDH) to convert an intermediary compound, 3-hydroxypropionaldehyde (3-HPA), to 3-HP. In this study, we report a new ALDH, PuuC, from Klebsiella pneumoniae DSM 2026, that efficiently converts 3-HPA to 3-HP. The identified gene puuC was cloned, expressed in Escherichia coli, purified, and characterized for its properties. The recombinant enzyme with a molecular weight of 53.8 kDa exhibited broad substrate specificity for various aliphatic aldehydes, especially C₂–C₅ aldehydes. NAD⁺ was the preferred coenzyme for the oxidation of most aliphatic and aromatic aldehydes tested. The optimum pH and temperature for PuuC activity were pH 8.0 and 45°C. The K _m values for 3-HPA and NAD⁺ were 0.48 and 0.09 mM, respectively. The activity of PuuC was enhanced in the presence of reducing agents such as 2-mercaptoethanol or dithiothreitol, while several metal ions, particularly Hg²⁺, Ag⁺, and Cu²⁺ inhibited its activity. The predicted structure of PuuC indicated the presence of K191 and E194 in close proximity to the glycine motif, suggesting that PuuC belongs to class 2 ALDHs.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

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.