Isolation of fungi and optimization of process parameters for decolorization of distillery mill effluent

Five different fungi isolated from distillery mill site in which two isolates (DF3 and DF4) had higher capabilities to remove color were identified as Emericella nidulans var. lata and Neurospora intermedia, respectively. Optimization of process parameter for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, %carbon, %nitrogen, duration, pH, temperature, stirring and inoculum size, at two levels applying L-8 orthogonal array were taken for both fungi. Maximum color was removed at pH 3, temperature 30°C, stirring 125 rpm, dextrose (0.05%) and sodium nitrate (0.025%) by both fungi. After optimization, there was two-fold increase in color removal from 38 to 62% (DF3) and 31 to 64% (DF4) indicating significance of Taguchi approach in decolorization of distillery mill effluent. The mechanism for decolorization was determined by enzyme analysis, laccase and glucose oxidase, and indicated significant activity in DF3 as compared to DF4.     

Blind docking of pharmaceutically relevant compounds using RosettaLigand

It is difficult to properly validate algorithms that dock a small molecule ligand into its protein receptor using data from the public domain: the predictions are not blind because the correct binding mode is already known, and public test cases may not be representative of compounds of interest such as drug leads. Here, we use private data from a real drug discovery program to carry out a blind evaluation of the RosettaLigand docking methodology and find that its performance is on average comparable with that of the best commercially available current small molecule docking programs. The strength of RosettaLigand is the use of the Rosetta sampling methodology to simultaneously optimize protein sidechain, protein backbone and ligand degrees of freedom; the extensive benchmark test described here identifies shortcomings in other aspects of the protocol and suggests clear routes to improving the method.     

Cyanobacterial diversity in the rhizosphere of rice and its ecological significance

This investigation was undertaken to characterize the abundance and genera-wise diversity of cyanobacteria in the rice rhizosphere and nitrogen-fixing ability of the isolated strains. The cyanobacterial strains belonging to the genera Nostoc and Anabaena comprised 80% of the rhizosphere isolates, which were also efficient in enhancing the germination and growth of wheat seeds and exhibited significantly high protein accumulation and IAA production. Distinct profiles for the cyanobacterial strains were obtained on amplification with extended Hip 1 primer — HipTG, indicative of the diversity among these strains. Our investigation helped in identifying promising cyanobacterial isolates from the rhizosphere of rice, which can be utilized in developing efficient plant growth promoting cyanobacterial inoculants.     

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

On 15 April and 17 April 2009, novel swineorigin influenza A (H1N1) virus was identifi ed in specimens obtained from two epidemiologically unlinked patients in the United States. The ongoing outbreak of novel H1N1 2009 influenza (swine influenza) has caused more than 3,99,232 laboratory confi rmed cases of pandemic influenza H1N1 and over 4735 deaths globally. This novel 2009 influenza virus designated as H1N1 A/swine/California/04/2009 virus is not zoonotic swine flu and is transmitted from person to person and has higher transmissibility then that of seasonal influenza viruses. In India the novel H1N1 virus infection has been reported from all over the country. A total of 68,919 samples from clinically suspected persons have been tested for influenza A H1N1 across the country and 13,330 (18.9%) of them have been found positive with 427 deaths. At the All India Institute of Medical Sciences, New Delhi India, we tested 1096 clinical samples for the presence of novel H1N1 influenza virus and seasonal influenza viruses. Of these 1096 samples, 194 samples (17.7%) were positive for novel H1N1 influenza virus and 197 samples (18%) were positive for seasonal influenza viruses. During outbreaks of emerging infectious diseases accurate and rapid diagnosis is critical for minimizing further spread through timely implementation of appropriate vaccines and antiviral treatment. Since the symptoms of novel H1N1 influenza infection are not specifi c, laboratory confi rmation of suspected cases is of prime importance.     

Degradation of Trichloroethylene by <Emphasis Type="Italic">Bacillus</Emphasis> sp.: Isolation Strategy,Strain Characteristics,and Cell Immobilization

A novel isolate of a bacterium, capable of degrading trichloroethylene (TCE) and growing on this as the sole carbon source is reported. The test strain was isolated by an enrichment technique with trichloroethylene as the substrate. The isolated strain belongs to the genus Bacillus. The practical utility of cleaning up oil spillage by bioremediation could be extended to this bacterium to degrade the environmental pollutant, which is used in metal degreasing in industries. Cells of the novel bacterium immobilized on calcium alginate were found to have better trichloroethylene degrading activity than the ones which were immobilized on agar-agar or free cells.     

Polymorphism of fecundity genes (BMPR1B, BMP15 and GDF9) in the Indian prolific Black Bengal goat

The Black Bengal is a prolific goat breed in India. Natural mutations in prolific sheep breeds have shown that the transforming growth factor beta (TGF-β) super family ligands such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their type I receptor (bone morphogenetic protein receptor, BMPR1B) are crucial for ovulation and as well as for increasing litter size. Mutations in any of these genes increased prolificacy in sheep. Based on the known mutation information in sheep PCR primers were designed to screen known polymorphism in 88 random Black Bengal goats. Only the BMPR1B gene was polymorphic. Three genotypes of animals were detected in tested animals with mutant (FecB^B) and wild type (FecB⁺) alleles were 0.57 and 0.43, respectively. Non-carrier, heterozygous carrier and homozygous carrier Black Bengal does had 2.7, 3.04 and 3.11 kids, respectively. All known point mutations of BMP15 and GDF9 genes were monomorphic in the animals tested. These results preliminarily showed that the BMPR1B gene might be a major gene that influences prolificacy of Black Bengal goats.     

Sialic acids acquired by Pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated host-cell recognition

The opportunism of Pseudomonas aeruginosa (PA) in immunocompromised hosts prompted us to explore the potential role of sialic acids (Sia) in this phenomenon. Culture of PA in the presence of exogenous Sia resulted in linkage-specific incorporation of Sia which was associated with decreased complement deposition on the bacteria. Sia acquired by PA mediated enhanced binding of bacteria to recombinant-CHO cells expressing human siglec-7 or siglec-9, as well as to human NK-cells and monocytes naturally expressing these siglecs. Therefore, Sia may be acquired by PA in the host and contribute to bacterial pathogenicity and host-cell interactions via reduction of complement deposition and siglec-dependent recognition.     

Novel strategy for selection of monoclonal antibodies against highly conserved antigens: phage library panning against ephrin-B2 displayed on yeast

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as 'EC8') was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeutic applications.     

TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo–N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development.