Isolation and characterization of heavy metal tolerant Gram-positive bacteria with bioremedial properties from municipal waste rich soil of Kestopur canal (Kolkata), West Bengal,India

The present study was conducted to determine the culturable bacterial profile from Kestopur canal (Kolkata, India) and analyze their heavy metal tolerance. In addition to daily sewage including solid and soluble wastes, a considerable load of toxic metals are released into this water body from industries, tanneries and agriculture, household as well as health sectors. Screening out microbes from such an environment was done keeping in mind their multifunctional application especially for bioremediation. Heavy metals are major environmental pollutants when present in high concentration in soil and show potential toxic effects on growth and development in plants and animals. Some edible herbs growing in the canal vicinity, and consumed by people, were found to harbour these heavy metals at sub-toxic levels. The bioconcentration factor of these plants being <1 indicates that they probably only absorb but not accumulate heavy metals. All the thirteen Grampositive bacteria isolated from these plants rhizosphere were found to tolerate high concentration of heavy metals like Co, Ni, Pb, Cr, Fe. Phylogenetic analysis of their 16S rDNA genes revealed that they belonged to one main taxonomic group — the Firmicutes. Seven of them were found to be novel with 92–95% sequence homology with known bacterial strains. Further microbiological analyses show that the alkaliphilic Bacillus weihenstephanensis strain IA1 and Exiguobacterium aestuarii strain CE1, with selective antibiotic sensitivity along with high Ni²⁺ and Cr⁶⁺ removal capabilities, respectively, can be prospective candidates for bioremediation.     

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.     

Carbonic anhydrase I and II inhibition with natural products: caffeine and piperine

Novel chemotypes with carbonic anhydrase (CA; EC 4.2.1.1) inhibitory action, in addition to the sulphonamide and sulphamate were discovered, many of which are based on natural products. Caffeine and piperine were extracted and tested for inhibition of the human (h) cytosolic isoforms hCA I and II. The IC(50) values of caffeine against hCA I was of 55?mM, whereas that of piperine of 60?mM. The IC(50) values of caffeine and piperine against hCA II were of 2?mM. Although these are quite weak inhibitors they may constitute leads for developing tighter binding compounds.     

In silico modeling and docking studies of Soap Nut Trypsin Inhibitor

Soap nut trypsin inhibitor (SNTI), an inhibitory protein was isolated and purified from the seeds of Sapindus trifoliatus by ammonium sulphate precipitation followed by ion-exchange and gel filtration chromatographic techniques and was found to be homogenous by PAGE. Using advanced proteomic techniques like MALDI-TOF the inhibitor was sequenced and analyzed using MASCOT software. A refined 3D model of the structure was predicted by in silico technique like homology modeling. The docked interactions between the predicted structure of SNTI and bovine trypsin were studied using ClusPro 2.0. Further docking results indicate that residues within the receptor binding domain include N145, R241, P242, L243, R244, R249, E266 and R275 respectively which play a key role in protein–protein interaction between SNTI and 3MFJ (bovine trypsin). SNTI was also known to exhibit potent anti-fungal activity against dandruff causing fungi. This study provides an insight into the structure of SNTI and also gives an idea about potential sites responsible for inhibitory action that could further be substantiated by experimental investigations.     

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.     

T-cell assays for tuberculosis infection: deriving cut-offs for conversions using reproducibility data

Background

Although interferon-gamma release assays (IGRA) are promising alternatives to the tuberculin skin test, interpretation of repeated testing results is hampered by lack of evidence on optimal cut-offs for conversions and reversions. A logical start is to determine the within-person variability of T-cell responses during serial testing.

Methodology/Principal Findings

We performed a pilot study in India, to evaluate the short-term reproducibility of QuantiFERON-TB Gold In Tube assay (QFT) among 14 healthcare workers (HCWs) who underwent 4 serial QFT tests on day 0, 3, 9 and 12. QFT ELISA was repeated twice on the same sets of specimens. We assessed two types of reproducibility: 1) test-retest reproducibility (between-test variability), and 2) within-person reproducibility over time. Test-retest reproducibility: with dichotomous test results, extremely high concordance was noticed between two tests performed on the same sets of specimens: of the 56 samples, the test and re-test results agreed for all but 2 individuals (κ = 0.94). Discordance was noted in subjects who had IFN-γ values around the cut-off point, with both increases and decreases noted. With continuous IFN-γ results, re-test results tended to produce higher estimates of IFN-γ than the original test. Within-person reproducibility: when continuous IFN-γ data were analyzed, the within-person reproducibility was moderate to high. While persons with negative QFT results generally stayed negative, positive results tended to vary over time. Our data showed that increases of more than 16% in the IFN-γ levels are statistically improbable in the short-term.

Conclusions

Conservatively assuming that long-term variability might be at least twice higher than short-term, we hypothesize that a QFT conversion requires two conditions to be met: 1) change from negative to positive result, and 2) at least 30% increase in the baseline IFN-γ response. Larger studies are needed to confirm our preliminary findings, and determine the conversion thresholds for IGRAs.     

New bichalcone analogs as NF-κB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis

A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a limited panel of four human cancer cell lines. All analogs inhibited NO production. Compounds 4 and 11 exhibited optimal activity with IC(50) values of 0.3 and 0.5 μM, respectively, and were at least 38-fold better than the positive control. A mechanism of action study showed that both compounds significantly blocked the nuclear translocation of NF-κB p65 and up-regulation of iNOS at 1.0 μM. Compound 4 and three other analogs (3, 20, and 23) exerted significant in vitro anticancer activity GI(50) values ranging from 0.70 to 13.10 μM. A mode of action study using HT-29 colon cancer cells showed that 23 acts by inducing apoptosis signaling.     

Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup

The Mouse Lymphoma Expert Workgroup of the International Workshop for Genotoxicity Tests (IWGT) met in Basel, Switzerland in August of 2009. The Workgroup (WG) was tasked with discussing the appropriate top concentration for non-pharmaceuticals that would be required for the conduct of the mouse lymphoma assay (MLA) when sufficient cytotoxicity [to between 10 and 20% relative total growth (RTG)] has not been attained. The WG approached this task by (1) enumerating the various regulatory decisions/use for MLA data, (2) discussing the appropriate assays to which MLA data and assay performance should be compared and (3) discussing all the proposals put forth concerning the top concentration for non-pharmaceuticals. In addition, one of the members presented a summary of a re-evaluation of the National Toxicology Program MLA data using the IWGT harmonized guidance that was underway as a separate (non IWGT) activity, being conducted by two members of the Expert WG. The WG was asked to vote on each of the various proposals for top concentration for when cytotoxicity is not concentration limiting. While there was general agreement that the top concentration for non-pharmaceuticals should be re-evaluated and likely lowered from the current recommended levels, there was no agreement on a specific new recommendation.     

Screening and Identification of New Isolate: Thermostable <Emphasis Type="Italic">Escherichia coli</Emphasis> with Novel Thermoalkalotolerant Cellulases

A cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth on CMC-Na agar screening plate at 37°C. It was identified as Escherichia coli using the method of 16S rRNA and intergenic spacer gene analysis combined with morphological, physiological, and biochemical tests. Three major components of the cellulases [carboxymethyl cellulase (CMCase), filter paper cellulase, and β-glucosidase] were produced with maximal activities (0.23, 0.08, and 0.15 U/ml) and maximum specific activities 4.13, 0.56, and 0.50 U/mg protein after 72, 96, and 120 h growth, respectively. Maximum CMCase activity was measured at 50°C and pH 6.0, respectively, and it also retained more than 60% of its maximal activity for at least 20 min at 50–70°C and 10 min at 80°C, respectively, and retained approximately 50% of its maximal activity after incubating at 90°C for 10 min. The enzyme could be applied in bioconversion of lignocellulosic agricultural wastes.