Antiviral Activity of the Human Cathelicidin,LL-37, and Derived Peptides on Seasonal and Pandemic Influenza A Viruses

Human LL-37, a cationic antimicrobial peptide, was recently shown to have antiviral activity against influenza A virus (IAV) strains in vitro and in vivo. In this study we compared the anti-influenza activity of LL-37 with that of several fragments derived from LL-37. We first tested the peptides against a seasonal H3N2 strain and the mouse adapted H1N1 strain, PR-8. The N-terminal fragment, LL-23, had slight neutralizing activity against these strains. In LL-23V9 serine 9 is substituted by valine creating a continuous hydrophobic surface. LL-23V9 has been shown to have increased anti-bacterial activity compared to LL-23 and we now show slightly increased antiviral activity compared to LL-23 as well. The short central fragments, FK-13 and KR-12, which have anti-bacterial activity did not inhibit IAV. In contrast, a longer 20 amino acid central fragment of LL-37 (GI-20) had neutralizing activity similar to LL-37. None of the peptides inhibited viral hemagglutination or neuraminidase activity. We next tested activity of the peptides against a strain of pandemic H1N1 of 2009 (A/California/04/09/H1N1 or “Cal09”). Unexpectedly, LL-37 had markedly reduced activity against Cal09 using several cell types and assays of antiviral activity. A mutant viral strain containing just the hemagglutinin (HA) of 2009 pandemic H1N1 was inhibited by LL-37, suggested that genes other than the HA are involved in the resistance of pH1N1. In contrast, GI-20 did inhibit Cal09. In conclusion, the central helix of LL-37 incorporated in GI-20 appears to be required for optimal antiviral activity. The finding that GI-20 inhibits Cal09 suggests that it may be possible to engineer derivatives of LL-37 with improved antiviral properties.     

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO₄, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.     

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.     

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.     

Diversity of Glycosyl Hydrolases from Cellulose-Depleting Communities Enriched from Casts of Two Earthworm Species

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales).Microorganisms producing diverse glycosyl hydrolases (GHs) are widespread and typically thrive in environments where plant materials tend to accumulate and deteriorate (42, 73). The habitats of microorganisms with great GH diversity are the ruminant animal rumen, mouse bowel, and rabbit cecum (10, 24, 26, 28, 49, 74). Microorganisms associated with soil invertebrates in general and with soil earthworms in particular carry out metabolic processes that contribute to element cycling and are essential in sustaining processes which their hosts are unable to perform (20, 52, 72, 76). Although some species of earthworms produce cellulases (15, 55), they generally rely on microbes inhabiting their gastrointestinal (GI) tracts to perform cellulose utilization processes (31, 47, 77). Casts are of special interest in this respect. Considering that the overall numbers of cellulolytic microbes in earthworm casts are greater than those in soil (57), earthworm casts seem to play an important role in the decomposition of plant litter, serving as an inoculum for cellulosic substrates (9). It is important to note that microorganisms from preingested substratum (soil or plant litter) are predominant in the gut lumen (20); however, microbial populations in earthworm casts differ from those in soil in terms of diversity and the relative abundance of different taxa (29, 57, 63). It is anticipated that the enzymatic repertoire of such microbial communities must be especially broad toward diverse sugar-based polymeric, oligomeric, and monomeric substrates; however, among approximately 115 families of GHs with thousands of members known to date (12), none of the GHs have been derived from microorganisms of earthworm-associated microbial communities.The aim of the present work was therefore to examine the diversity of GHs in metagenome libraries derived from fresh casts of Aporrectodea caliginosa and Lumbricus terrestris earthworms via functional screening. Other important tasks of this work were to characterize individual enzymes and to gain insight into their structural-functional features. Finally, we performed sequence analysis of large contiguous DNA fragments of fosmids harboring the genes for GHs to associate them with the organism(s) that may produce them, which was complemented by conventional small-subunit (SSU) rRNA clone library sequencing analysis.     

AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.     

Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown on arsenic-contaminated soils of West Bengal

The exposure of paddy fields to arsenic (As) through groundwater irrigation is a serious concern that may not only lead to As accumulation to unacceptable levels but also interfere with mineral nutrients in rice grains. In the present field study, profiling of the mineral nutrients (iron (Fe), phosphorous, zinc, and selenium (Se)) was done in various rice genotypes with respect to As accumulation. A significant genotypic variation was observed in elemental retention on root Fe plaque and their accumulation in various plant parts including grains, specific As uptake (29–167 mg kg^?1 dw), as well as As transfer factor (4–45%). Grains retained the least level of As (0.7–3%) with inorganic As species being the dominant forms, while organic As species, viz., dimethylarsinic acid and monomethylarsonic acid, were non-detectable. In all tested varieties, the level of Se was low (0.05–0.12 mg kg^?1 dw), whereas that of As was high (0.4–1.68 mg kg^?1 dw), considering their safe/recommended daily intake limits, which may not warrant their human consumption. Hence, their utilization may increase the risk of arsenicosis, when grown in As-contaminated areas.