Microbial Diversity in Ngari,Hentak and Tungtap,Fermented Fish Products of North-East India

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari, hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g, respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus, aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained more than 10² c.f.u./g of Bacillus cereus, 10³ c.f.u./g of Staphylococcus aureus and enterobacteriaceae population, respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their ‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria, Bacillus and yeasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells¹.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.^{(58M, flv)}     

Synthesis and evaluation of alpha-hydroxymethylated conjugated nitroalkenes for their anticancer activity: inhibition of cell proliferation by targeting microtubules

The Morita-Baylis-Hillman (MBH) type reaction of a variety of aromatic and heteroaromatic conjugated nitroalkenes with formaldehyde in the presence of stoichiometric amounts of imidazole and catalytic amounts (10 mol %) of anthranilic acid at room temperature provided the corresponding hydroxymethylated derivatives in moderate to good yield. The parent nitroalkenes and their MBH adducts were subsequently screened for their anticancer activity. Some of the MBH adducts were found to inhibit cervical cancer (HeLa) cell proliferation at low micromolar concentrations with half-maximal inhibitory concentrations in the range of 1-2 microM. The antiproliferative activity of 3-((E)-2-nitrovinyl)furan and three potent MBH adducts, namely, hydroxymethylated derivatives of 3-((E)-2-nitrovinyl)thiophene, 1-methoxy-4-((E)-2-nitrovinyl)benzene, and 1,2-dimethoxy-4-((E)-2-nitrovinyl)benzene was correlated well with their antimicrotubule activity. At their effective concentration range, the tested compounds perturbed the organization of mitotic spindle microtubules and chromosomes. In the presence of hydroxymethylated nitroalkenes, abnormal bipolar or multipolar mitotic spindles were apparent. Interphase microtubules were found to be significantly depolymerized at relatively higher concentrations of the tested compounds. These compounds inhibited tubulin assembly into microtubules in vitro by binding to tubulin at a site distinct from the vinblastine and colchicine binding sites. The compounds reduced the intrinsic tryptophan fluorescence of tubulin and the fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid (ANS) complex indicating that they induced conformational changes in the tubulin. The results suggest that hydroxymethylated nitroalkenes exert their antiproliferative activity at least in part by depolymerizing cellular microtubules through tubulin binding and indicate that hydroxymethylated nitroalkenes are promising lead compounds for cancer therapy.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

CHIKVPRO - a protein sequence annotation database for chikungunya virus

In the recent past, there has been a resurgence of interest in Chikungunya virus (CHIKV) attributed to massive outbreaks of Chikungunya fever in the South-East Asia Region. This has reflected in substantial increase in submission of CHIKV genome sequences to NCBI (National Center for Biotechnology Information) database. Hereby we submit a database "CHIKVPRO" containing structural and functional annotation of Chikungunya virus proteins (25 strains) submitted in the NCBI repository. The CHIKV genome encodes for 9 proteins:4 non-structural and 5 structural. The CHIKVPRO database aims to provide the virology community with a single accession authoritative resource for CHIKV proteome- with reference to physiochemical and molecular properties, proteolytic cleavage sites, hydrophobicity, transmembrane prediction, and classification into functional families using SVMProt and other Expasy tools. AVAILABILITY: The database is freely available at http://www.chikvpro.info/     

Cultural Adaptation of a Survey to Assess Medical Providers’ Knowledge of and Attitudes towards HIV/AIDS in Albania

Though the HIV/AIDS epidemic in Southeastern Europe is one of low reported prevalence, numerous studies have described the pervasiveness of medical providers’ lack of knowledge of HIV/AIDS in the Balkans. This study sought to culturally adapt an instrument to assess medical providers’ knowledge of and attitudes towards HIV/AIDS in Albania. Cultural adaptation was completed through development of a survey from previously validated instruments, translation of the survey into Albanian, blinded back translation, expert committee review of the draft instrument, focus group pre-testing with community- and University Hospital Center of Tirana-based physicians and nurses, and test-retest reliability testing. Blinded back translation of the instrument supported the initial translation with slight changes to the idiomatic and conceptual equivalences. Focus group pre-testing generally supported the instrument, yet some experiential and idiomatic changes were implemented. Based on unweighted kappa and/or prevalence adjusted bias adjusted kappa (PABAK), 20 of the 43 questions were deemed statistically significant at kappa and/or PABAK ≥0.5, while 12 others did not cross zero on the 95% confidence interval for kappa, indicating their probable significance. Subsequently, an instrument to assess medical providers’ knowledge of and attitudes toward HIV/AIDS for an Albanian population was developed which can be expanded within Albania and potentially to other countries within the Balkans, which have an Albanian-speaking population.     

Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-ε-GG) antibodies. Here, we describe a number of improvements to the K-ε-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ∼20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input.The commercialization of antibodies that recognize lysine residues modified with a di-glycine remnant (K-ε-GG)¹ has significantly transformed the detection of endogenous protein ubiquitination sites by mass spectrometry (1–5). Prior to the development of these highly specific reagents, proteomics experiments were limited to identification of up to only several hundred ubiquitination sites, which severely limited the scope of global ubiquitination studies (6). Recent proteomic studies employing anti-K-ε-GG antibodies have enhanced our understanding of ubiquitin biology through the identification of thousands of ubiquitination sites and the analysis of the change in relative abundance of these sites after chemical or biological perturbation (1–3, 5, 7). Use of stable isotope labeling by amino acids in cell culture (SILAC) for quantification has enabled researchers to better understand the extent of ubiquitin regulation upon proteasome inhibition and precisely identify those protein classes, such as newly synthesized proteins or chromatin-related proteins, that see overt changes in their ubiquitination levels upon drug treatment (2, 3, 5). Emanuel et al. (1) have combined genetic and proteomics assays implementing the anti-K-ε-GG antibody to identify hundreds of known and putative Cullin-RING ligase substrates, which has clearly demonstrated the extensive role of Cullin-RING ligase ubiquitination on cellular protein regulation.Despite the successes recently achieved with the use of the anti-K-ε-GG antibody, increased sample input (up to ∼35 mg) and/or the completion of numerous experimental replicates have been necessary to achieve large numbers of K-ε-GG sites (>5,000) in a single SILAC-based experiment (1–3, 5). For example, it has been recently shown that detection of more than 20,000 unique ubiquitination sites is possible from the analysis of five different murine tissues (8). However, as the authors indicate, only a few thousands sites are detected in any single analysis of an individual tissue sample (8). It is recognized that there is need for further improvements in global ubiquitin technology to increase the depth-of-coverage attainable in quantitative proteomic experiments using moderate amounts of protein input (9). Through systematic study and optimization of key pre-analytical variables in the preparation and use of the anti-K-ε-GG antibody as well as the proteomic workflow, we have now achieved, for the first time, routine quantification of ∼20,000 nonredundant K-ε-GG sites in a single SILAC triple encoded experiment starting with 5 mg of protein per SILAC channel. This represents a 10-fold improvement over our previously published method (3).