Adoptive Autophagy Activation: a Much-Needed Remedy Against Chemical Induced Neurotoxicity/Developmental Neurotoxicity

The profound significance of autophagy as a cell survival mechanism under conditions of metabolic stress is a well-proven fact. Nearly a decade-long research in this area has led scientists to unearth various roles played by autophagy other than just being an auto cell death mechanism. It is implicated as a vital cell survival pathway for clearance of all the aberrant cellular materials in case of cellular injury, metastasis, disease states, cellular stress, neurodegeneration and so on. In this review, we emphasise the critical role of autophagy in the environmental stressors-induced neurotoxicity and its therapeutic implications for the same. We also attempt to shed some light on the possible protective role of autophagy in developmental neurotoxicity (DNT) which is a rapidly growing health issue of the human population at large and hence a point of rising concern amongst researchers. The intimate association between DNT and neurodegenerative disorders strongly indicates towards adopting autophagy activation as a much-needed remedy for DNT.     

Proteogenomics

The ability to sequence DNA rapidly, inexpensively and in a high-throughput fashion provides a unique opportunity to sequence whole genomes of a large number of species. The cataloging of protein-coding genes from these species, however, remains a non-trivial task with the majority of initial genome annotation dependent on the use of gene prediction algorithms. Recent advances in mass spectrometry-based proteomics now enable generation of accurate and comprehensive protein sequence of tissues and organisms. Proteogenomics allows us to harness the wealth of information available at the proteome level and apply it to the available genomic information of organisms. This includes identifying novel genes and splice isoforms, assigning correct start sites and validating predicted exons and genes. It is also possible to use proteogenomics to identify protein variants that could cause diseases, to identify protein biomarkers and to study genome variation. We anticipate proteogenomics to become a powerful approach that will be routinely employed by 'Genome and Proteome Centers' of the future.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.     

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.     

DNA biosensor for detection of Neisseria gonorrhoeae causing sexually transmitted disease

A sequence-specific electrochemical sexually transmitted disease (STD) sensor based on self-assembled monolayer of thiolated DNA probe specific to target opa gene for detection of Gonorrhoea, a sexually transmitted disease has been fabricated. 6-Mercapto-1-hexanol (MCH) has been used as a blocking agent to facilitate oligos "stand" up at the surface, a configuration favoring subsequent DNA hybridization and to repel non-specific adsorption of undesired DNA. The results of differential pulse voltammetric studies of this STD sensor reveal low detection limit (1.0 × 10(-18)M) and a wide dynamic range (from 1.0 × 10(-6)M to 0.5 × 10(-18)M) arising due to the stable hybridization using methylene blue as an electro-active DNA hybridization indicator. The experimental results with genomic DNA, clinical patient sample of Neisseria gonorrhoeae, culture of non-N. gonorrhoeae Neisseria species (NgNS) and gram negative bacteria indicate that the fabricated sensor is specific to this STD.     

The role of calcium nutrition in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) microplants in relation to minimal growth over prolonged storage <Emphasis Type="Italic">in vitro</Emphasis>

The present study investigated the effect of different levels of Ca_[ext] (0.3, 3.0, 5.0, 7.0, 9.0 and 11.0 mM) on potato over minimal growth in vitro in relation to varying water stress levels and moisture vapour transmission regimes using ⁴⁵Ca as an isotopic tracer. Ca nutrition was substantially limited when the microplants were grown at enhanced water stress level (MS + 40 g l^-1 sucrose + 20 g l^-1 mannitol) under minimal growth. Ca_[ext] in excess of standard level (3.0 mM), however, resulted in a significant increase in Ca content in microplants. The differential Ca uptake in microplants in relation to water stress and moisture vapour transmission has been discussed in terms of transpiration stream and root pressure water flow under minimal growth. The study showed that poor microplant quality at standard Ca_[ext] over prolonged storage under minimal growth was due to limiting Ca nutrition, and this could be improved by using Ca_[ext]-enriched (5.0-7.0 mM) minimal growth medium for conservation of potato microplants. The role of high Ca_[ext] in reducing the phenotypic abnormalities such as vitrification, flaccidity, hyperhydricity, etc. in potato microplants over extended storage under minimal growth has also been discussed.     

Chemotaxis of a Ralstonia sp. SJ98 toward co-metabolizable nitroaromatic compounds

We have earlier reported chemotaxis of a Gram-negative, motile Ralstonia sp. SJ98 towards p-nitrophenol (PNP), 4-nitrocatechol (NC), o-nitrobenzoate (ONB), p-nitrobenzoate (PNB), and 3-methyl-4-nitrophenol (MNP) that also served as sole source of carbon and energy to the strain [S.K. Samanta, B. Bhushan, A. Chauhan, R.K. Jain, Biochem. Biophy. Res. Commun. 269 (2000) 117; B. Bhushan, S.K. Samanta, A. Chauhan, A.K. Chakraborti, R.K. Jain, Biochem. Biophy. Res. Commun. 275 (2000) 129]. In this paper, we report chemotaxis of a Ralstonia sp. SJ98 toward seven different nitroaromatic compounds (NACs) by drop assay, swarm plate assay, and capillary assay. These NACs do not serve as sole carbon and energy source to strain SJ98 but are partially transformed in the presence of an alternate carbon source such as succinate. This is the first report showing chemotaxis of a bacterial strain toward co-metabolizable NACs.     

Functional characterization of EngA(MS), a P-loop GTPase of Mycobacterium smegmatis

Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngA(MS)) is highly conserved in mycobacteria. Homology modeling of EngA(MS) reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngA(MS) purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngA(MS) protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngA(MS) with 50S subunit of ribosome and specifically C-terminal domains of EngA(MS) are required to facilitate this interaction. Moreover, EngA(MS) devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngA(MS)-50S interaction.     

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.