Padma Singh

Biomethanation under psychrophilic conditions

The biomethanation of organic matter represents a long-standing, well-established technology. Although at mesophilic and thermophilic temperatures the process is well understood, current knowledge on psychrophilic biomethanation is somewhat scarce. Methanogenesis is particularly sensitive to temperature, which not only affects the activity and structure of the microbial community, but also results in a change in the degradation pathway of organic matter. There is evidence of psychrophilic methanogenesis in natural environments, and a number of methanogenic archaea have been isolated with optimum growth temperatures of 15-25 °C. At psychrophilic temperatures, large amounts of heat are needed to operate reactors, thus resulting in a marginal or negative overall energy yield. Biomethanation at ambient temperature can alleviate this requirement, but for stable biogas production, a microbial consortium adapted to low temperatures or a psychrophilic consortium is required. Single-step or two-step high rate anaerobic reactors [expanded granular sludge bed (EGSB) and up flow anaerobic sludge bed (UASB)] have been used for the treatment of low strength wastewater. Simplified versions of these reactors, such as anaerobic sequencing batch reactors (ASBR) and anaerobic migrating blanket reactor (AMBR) have also been developed with the aim of reducing volume and cost. This technology has been further simplified and extended for the disposal of night soil in high altitude, low temperature areas of the Himalayas, where the hilly terrain, non-availability of conventional energy, harsh climate and space constraints limit the application of complicated reactors. Biomethanation at psychrophilic temperatures and the contribution made to night-soil degradation in the Himalayas are reviewed in this article.

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC50 values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).

Prevention of aggregation and autocatalysis for sustaining biological activity of recombinant BoNT/A-LC upon long-term storage.

Protein aggregation during expression, purification, storage, or transfer into requisite assay buffers hampers the use of proteins for in vitro studies. The formation of these aggregates represents a major obstacle in the study of biological activity and also restricts the spectrum of protein products being available for the biomedical applications. The catalytic light chain of botulinum neurotoxin type A undergoes autocatalysis and aggregation after purification upon long-term storage and freeze-thawing. In present study the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin were optimized from a synthetic gene. Several co-solvents were screened in order to prevent autocatalysis and aggregation of rBoNT/A-LC. The effect of the co-solvents is studied on endopeptidase activity during long term storage of the recombinant protein. The purified rBoNT/A-LC was also evaluated for its immunogenicity.

Antibodies Against Recombinant Shiga Toxin Subunit B Neutralize Shiga Toxin Toxicity in HeLa Cells

Shigella dysenteriae type 1 and Escherichia coli O157:H7 produce Shiga toxin 1 (Stx) and Shiga toxin1 (Stx1), respectively and these two toxins are almost identical. E. coli O157:H7 is the major cause of diarrhea-associated hemolytic uremic syndrome. Stx and Stx1 are AB5 type of toxin with a molecular weight of 70 kDa, comprising an enzymaticaly-active A subunit (32 kDa) and five receptor-binding B subunits (7.7 kDa). In this study DNA fragment (289 bp, Gene Bank Accn No. EF685161) coding for B chain of Stx was amplified from S. dysenteriae type1 and cloned. Shiga toxin-binding subunit was expressed and purified in native conditions by affinity and gel permeation chromatography with the yield of 5.1 mg/L in shake flask culture. For the purpose of immunization, the polypeptide was polymerized with glutaraldehyde. Hyper immune serum produced in mice reacted with the purified polypeptide and intact Shiga toxin. The anti-StxB antiserum effectively neutralized the cytotoxicity of Shiga toxin towards HeLa cells.

Characterization of LC-HCC Fusion Protein of Botulinum Neurotoxin Type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.

Retraction: Small-molecule quinolinol inhibitor identified provides protection against BoNT/A in mice.

It has been brought to the attention of the PLOS ONE editors that substantial parts of the text in this article were appropriated from text in the following publications: Identification and biochemical characterization of small-molecule inhibitors of Clostridium botulinum neurotoxin serotype A. Roxas-Duncan V, Enyedy I, Montgomery VA, Eccard VS, Carrington MA, Lai H, Gul N, Yang DC, Smith LA. Antimicrob Agents Chemother. 2009 Aug;53(8):3478-86 Eubanks LM, Hixon MS, Jin W, Hong S, Clancy CM, et al. (2007) An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists. Proc Natl Acad Sci USA104: 2602–2607. PLOS ONE therefore retracts this article due to the identified case of plagiarism. PLOS ONE apologizes to the authors of the publications above and to the readers.