Ram Kumar Dhaked

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.

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature.

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.

Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice

Shigella dysenteriae is the causative agent of the third commonest bacterial disease for childhood diarrhoea and responsible for millions of deaths per year. It produces potent toxin termed Shiga toxin which is listed in category B biological warfare agent of CDC, USA. Earlier we have reported production of recombinant Shiga toxin B subunit that produced antibodies which neutralized Shiga toxin toxicity in HeLa cells. In the present study, we have evaluated the immunomodulatory potential of rStxB protein in Balb/c mice using Freunds adjuvants. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses as indicated by an increased antibody titre with predominance of IgG2a and IgG2b isotypes along with elevated levels of IgG1 subtype. Cytokine profile of the mice antiserum and splenocyte also indicates Th2 and Th1 type of immune responses where former dominates in early stage of immunization. Histopathology study of kidneys of vaccinated mice showed remarkable differences when compared to the animals infected with Shigella dysenteriae type1. The present study indicates that recombinant StxB is a promising vaccine candidate and can be used for production of therapeutic antibodies to counter Shiga intoxication.

MILITARY POTENTIAL OF BIOLOGICAL NEUROTOXINS

Toxins are produced by thousands of living species for increasing their chance of survival by modifying the physiology of other species. They are used either for defense or predation. They vary in their structural complexities ranging from formic acid used by ants to the multimillion Dalton protein toxins produced by several bacteria. All the most poisonous, fastest-acting toxins are neurotoxins. They specifically affect the nervous system of animals, including humans, by interfering with nerve impulse transmission. There are four major components of the nervous system on which these neurotoxins generally act, namely, ion channels, acetylcholine receptors, synaptic vesicle peptides, and acetylcholine esterase. Six different receptor sites on the sodium channel have been described on which some of the biological neurotoxins act. Different protein and nonprotein neurotoxins, their structure, source, targets, and mode of action are discussed in view of the current developments. These neurotoxins have gained increasing significance as potential candidates for weaponization. They are generally more lethal than microbial pathogens, causing incapacitation or death within minutes or hours. Despite extensive studies on the physiology and structure of these neurotoxins; appropriate detection systems for most of these toxins are lacking. Many of these toxins cause serious illnesses, showing overlapping clinical signs and symptoms. This makes specific and timely diagnosis extremely difficult in the absence of a suitable analytical system. Among several applications of these neurotoxins, their use as therapeutic agents for various disorders are remarkable. Many more neurotoxins remain to be explored.

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.

Surface Plasmon Resonance Sensing of Biological Warfare Agent Botulinum Neurotoxin A

A label free real time method was developed for the detection as well as quantification of botulinum neurotoxin A (BoNT/A) using surface plasmon resonance (SPR). In the present work, antibody against rBoNT/A-HCC fragment and synaptic vesicles (SV) were immobilized on carboxymethyldextran modified gold chip. The immobilization of BoNT/A antibody and interaction of BoNT/A with immobilized antibody were in-situ characterized by SPR and electrochemical impedance spectroscopy. A sample solution containing BoNT/A antigen in the concentration ranging from 0.225 fM to 4.5 fM and 0.045 fM to 5.62 fM was interacted with immobilized antibody and immobilized SV, respectively. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) values were calculated and found to be 0.53 fM and 38.23 mo for immobilized antibody and 0.22 fM and 116.0 mo for immobilized SV, respectively. Moreover, thermodynamic parameters such as change in Gibb’s free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between BoNT/A antigen and BoNT/A antibody as spontaneous, endothermic and entropy driven one. In order to optimize the detection method, temperature and pH variation studies were also performed.

Applications of rat brain synaptic vesicle proteins for sensitive and specific detection of Botulinum Neurotoxins.

We propose here the application of synaptic vesicle proteins isolated from rat brain as a sole substrate for the specific endoproteinase activities of all seven serotypes of Botulinum Neurotoxin (BoNT/A to G). In this study, we used these proteins for evaluating endopeptidase and receptor binding activity for detecting BoNT/A by western blot and surface plasmon resonance with 6.25 pM and 0.22 fM limit of detection, respectively. Substrate and receptor present in the synaptic vesicle proteins are very robust and stable for more than 6 months to use in BoNT detection.

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.