Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entmoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoeLtric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP+-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADHi clone had an open reading frame that was 360 amino acids long and encoded a protein of -39 kDa (calculated size). EhADHi showed a 62% amino acid identity with the tetrameric NADP+-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADHi showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADHi contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADHi lacked a 23-amino acid stretch present in other alcohol dehydrogenases that indudes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-Stransferase fusion protein showed the expected NADP+-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADHi fusion protein were inhibited by pyrazole and 4-methylpyrazole.Entamoeba histolytica is a protozoan parasite that infects -500 million individuals in the developing world, resulting in -40 million cases of dysentery with a consequent annual mortality rate of ==40,000 (1, 2). Entamoebae reproduce under anaerobic conditions in the colonic lumen and cause dysentery when they invade the bowel wall. Because poor countries cannot afford to make the changes in sanitary conditions that might eliminate fecal-oral spread of amoebic infection, amebiasis is primarily controlled by drug treatment of persons sick with the parasite (1-3).Entamoebae are obligate fermenters because they lack mitochondria and the enzymes involved in respiration (4, 5). Amoebae use facilitated diffusion for the uptake of glucose, which is then metabolized to pyruvate via the Embden-

The ability to trigger an innate immune response against opportunistic pathogens associated with HIV-1 infection is an important aspect of AIDS pathogenesis. Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens, but in HIV-1 patients coinfected with opportunistic infections, the regulation of TLR expression has not been studied. In this context, we have evaluated the expression of TLR2 and TLR4 in monocytes, plasmacytoid dendritic cells, and myeloid dendritic cells of HIV-1 patients with or without opportunistic infections. Forty-nine HIV-1-infected individuals were classified according to viral load, highly active antiretroviral therapy (HAART), and the presence or absence of opportunistic infections, and 21 healthy subjects served as controls. Increased expression of TLR2 and TLR4 was observed in myeloid dendritic cells of HIV-1 patients coinfected with opportunistic infections (without HAART), while TLR4 increased in plasmacytoid dendritic cells, compared to both HIV-1 without opportunistic infections and healthy subjects. Moreover, TLR2 expression was higher in patients with opportunistic infections without HAART and up-regulation of TLR expression in HIV-1 patients coinfected with opportunistic infections was more pronounced in dendritic cells derived from individuals coinfected with Mycobacterium tuberculosis. The results indicate that TLR expression in innate immune cells is up-regulated in patients with a high HIV-1 load and coinfected with opportunistic pathogens. We suggest that modulation of TLRs expression represents a mechanism that promotes HIV-1 replication and AIDS pathogenesis in patients coinfected with opportunistic pathogens.

Study DesignProspective observational study.PurposeTo determine the incidence of postoperative urinary retention (POUR) in patients undergoing elective posterior lumbar spine surgery and identify the risk factors associated with the development of POUR.Overview of LiteraturePOUR following surgery can lead to detrusor dysfunction, urinary tract infections, prolonged hospital stay, and a higher treatment cost; however, the risk factors for POUR in spine surgery remain unclear.MethodsA prospective, consecutive analysis was conducted on patients undergoing elective posterior lumbar surgery in the form of lumbar discectomy, lumbar decompression, and single-level lumbar fusions during a 6-month period. Patients with spine trauma, preoperative neurological deficit, previous urinary disturbance/symptoms, multiple-level fusion, and preoperative catheterization were excluded from the study. Potential patient- and surgery-dependent risk factors for the development of POUR were assessed. Univariate analysis and a multiple logistical regression analysis were performed.ResultsA total of 687 patients underwent posterior lumbar spine surgery during the study period; among these, 370 patients were included in the final analysis. Sixty-one patients developed POUR, with an incidence of 16.48%. Significant risk factors for POUR were older age, higher body mass index (BMI), surgery duration, intraoperative fluid administration, lumbar fusion versus discectomy/ decompression, and higher postoperative pain scores (p<0.05 for all). Sex, diabetes, and the type of inhalational agent used during anesthesia were not significantly associated with POUR. Multiple logistical regression analysis, including age, BMI, surgery duration, intraoperative fluid administration, fusion surgery, and postoperative pain scores demonstrated a predictive value of 92% for the study population and 97% for the POUR group.ConclusionsPOUR was associated with older age, higher BMI, longer surgery duration, a larger volume of intraoperative fluid administration, and higher postoperative pain scores. The contribution of postoperative pain scores in the multiple regression analysis was a significant predictor of POUR.

Purpose: The aim of the study is to identify risk factors, clinical characteristics, causative fungi, and treatment outcome of dematiaceous fungal keratitis in North India. Methods: Consecutive cases of culture-proven dematiaceous fungal keratitis between January 2012 and June 2017 were retrieved from the medical record department. Risk factors, clinical signs, and outcome were registered. Results: Eighty-three patients were included. Identified dematiaceous fungal organism were Curvularia sp. ( n = 55/83; 66.3%), Alternaria sp. ( n = 12/83; 14.5%), Ulocladium sp. ( n = 5/83; 6%), Bipolaris sp. ( n = 5/83; 6.1%), Scedosporium sp. ( n = 3/83; 3.6%), Acremonium sp. ( n = 2/83; 2.4%), and Epicoccum sp. ( n = 1/83; 1.2%). Male preponderance was reported. The most common predisposing factor was corneal trauma (67.4%). In cases associated with corneal trauma due to vegetative matter, sugarcane was the most common cause. In all, 89% of the patients were more than 30 years of age. The median infiltrate size was 8 mm 2 . The median time of antifungal therapy was 4.2 weeks (interquartile range [IQR]: 1-25 weeks). Complications were seen in 14 ( n = 14/65; 21.5%) patients. Complete resolution of dematiaceous fungal keratitis was present in 27 ( n = 27/65; 41.5%) eyes. Conclusion: Curvularia sp. and Alternaria sp. were the predominant pathogenic genera causing dematiaceous fungal keratitis. Among the causative fungi, infections due to Scedosporium sp. were associated with the worst outcomes. Ulocladium sp. and Epicoccum sp. were also identified. Both the species are not reported previously as a causal organism of dematiaceous fungal keratitis from North India.

BackgroundRNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application.ResultsThe genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects.ConclusionThe database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-775) contains supplementary material, which is available to authorized users.

Endosulfan has emerged as a major environmental menace worldwide due to  extensive usage and environmental persistence, seeking its remedial by a cheaper and efficient means. Therefore, natural resource (soil) was explored to search a potential candidate for biodegradation of endosulfan. A soil bacterium was enriched and isolated by applying a strong nutritional selection pressure, using a non-sulfur medium supplemented with endosulfan as sole source sulfur. The microbial strain was found to degrade endosulfan as well as its equally toxic metabolite endosulfan sulfate to non-toxic metabolites (endodiol and endosulfan lactone) very efficiently (up to 94.2 %) within 7 days, estimated qualitatively by thin layer chromatography and quantitatively by gas chromatography-electron capture detection methods. The isolate was characterized for its morphological, physiological, biochemical and 16S rRNA sequencing and identified as a new strain of Bacillus subtilis with strain designation AKPJ04, which was deposited with accession number Microbial Type Culture Collection and Gene Bank (MTCC) 8561, at MTCC, Institute of Microbial Technology, Chandigarh, India. The partial 16S rRNA sequence was submitted to Genbank, Maryland, USA, with the accession number EU 258611. The primary investigation for endosulfan degrading gene(s) localization suggested its location on chromosomal DNA.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-013-0176-7) contains supplementary material, which is available to authorized users.

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Protein-protein interaction (PPI) network analysis is a powerful strategy to understand M. tuberculosis (Mtb) system level physiology in the identification of hub proteins. In the present study, the PPI network of 79 Mtb toxin-antitoxin (TA) systems comprising of 167 nodes and 234 edges was investigated. The topological properties of PPI network were examined by 'Network analyzer' a cytoscape plugin app and STRING database. The key enriched biological processes and the molecular functions of Mtb TA systems were analyzed by STRING. Manual curation of the PPI data identified four proteins (i.e. Rv2762c, VapB14, VapB42 and VapC42) to possess the highest number of interacting partners. The top 15% hub proteins were identified in the PPI network by employing two statistical measures, i.e. betweenness and radiality by employing cytohubba. Insights gained from the molecular protein models of VapC9 and VapC10 are also documented.