A clinico-epidemiological Study of acute Self-poisoning by different Types of herbicidal Substances used in agricultural Fields: A Study from Patients admitted in a Tertiary Care Hospital in West Bengal.

INTRODUCTION: Herbicides are the chemical compounds used to control the growth of unwanted plants or to eliminate them. The common poisonous herbicides available in India are paraquat, glyphosate, pretilachlor, etc. Ingestion of herbicides with suicidal intention is common in rural India and West Bengal but very scanty literature is available. METHODOLOGY: We conducted a unicentric, hospital-based, noninterventional, cross-sectional study comprising 50 consecutive patients to estimate the morbidity, case fatality, and clinical features of acute suicidal poisoning of different herbicides used in agricultural fields. Data were collected from history, clinical features, and laboratory findings. Proper statistical method was used for data analysis. RESULTS: Most of the study population were from 26 to 40 years age group (48%), followed by 13-25 years age group (34%). Paraquat was ingested by 64%, followed by pretilachlor (20%) and glyphosate (16%). Common symptoms were vomiting (60%), abdominal pain (40%), throat discomfort (26%), oral ulcer (24%), decreased urination (50%), and respiratory distress (30%). In laboratory investigation, 64% patients had deranged liver enzymes, 58% patients had acute kidney injury, and 30% patients had alveolar damage. A total of 62% patients were discharged after recovery and 38% patients died. Case fatality rate of paraquat was 56.2%, glyphosate was 12.5%, and pretilachlor was nil (0%). CONCLUSION: Herbicides like paraquat and glyphosate are associated with high morbidity and case fatality. Paraquat has the highest case fatality rate. Pre-emergent herbicide pretilachlor is relatively safe.

Secretome analysis identified extracellular superoxide dismutase and catalase of Macrophomina phaseolina.

Macrophomina phaseolina, a necrotrophic fungal pathogen is known to cause charcoal rot disease in food crops, pulse crops, oil crops and cotton and fibre crops. Necrotrophic fungi survive on dead plant tissue. It is well known that reactive oxygen species (ROS) are produced by the host plant during plant-pathogen interaction. However, it is still unclear how M. phaseolina can overcome the ROS-induced cellular damage. To mimic the invasion of M. phaseolina inside the plant cell wall, we developed solid substrate fermentation where M. phaseolina spore suspension was inoculated on a wheat bran bed and incubated for vegetative growth. To analyse the secretome of M. phaseolina after different day interval, its secretory material was collected and concentrated. Both superoxide dismutase (SOD) and catalase were detected in the secretome by zymogram. The presence of SOD and catalase was further confirmed by liquid chromatography based mass spectrometry. The physicochemical properties of M. phaseolina catalase in terms of stability towards pH, temperature, metal ions and chaotropic agent and inhibitors indicated its fitness at different environmental conditions. Apart from the production of catalase in SSF, the studies on this particular microorganism may also have significance in necrotrophic fungal pathogen and their susceptible host plant interaction.

Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice.

Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cells in vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma.

Nitric oxide production by necrotrophic pathogen Macrophomina phaseolina and the host plant in charcoal rot disease of jute: complexity of the interplay between necrotroph-host plant interactions.

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.

Novel xylanases from Simplicillium obclavatum MTCC 9604: comparative analysis of production, purification and characterization of enzyme from submerged and solid state fermentation.

The production of extracellular xylanase by a newly isolated fungus Simplicillium obclavatum MTCC 9604 was studied in solid-state and submerged fermentation. Multiple xylanases and endoglucanases were produced by the strain during growth on wheat bran in solid state fermentation (SSF). A single xylanase isoform was found to be produced by the same fungus under submerged fermentation (SF) using wheat bran as sole carbon source. Enzyme activity, stability and the protein yield were much higher in SSF than SF. The two dimensional zymogram of the crude enzyme indicated the presence of six isoforms with different pI values starting from pH 3-10. The optimum temperature and pH for the partially purified xylanase activity were 50°C and pH 5.0 respectively; xylanase enzymes exhibited remarkable stability over a broad pH range and the temperature range of 30-60°C which has great potential to be used in biofuels, animal feed and food industry applications.

An Ecofriendly synthesis of silver nano-bioconjugates by Penicillium citrinum (MTCC9999) and its antimicrobial effect.

This report provides for the first time a novel environment friendly extracellular synthesis of stable silver nano-bioconjugates (SNBCs) at room temperature at pH 5.0 using Penicillium citrinum MTCC 9999 biomass. The UV-Visible spectral scan of dispersed SNBCs solution showed absorption in the region 340-450 nm due to surface plasma resonance (SPR). Typical Transmission Electron Microscopic (TEM) images showed that although two populations were present but most of them were in 20-30 nm range. Average zeta potential of SNBCs was -21 mV suggesting some biomolecules capped the nanoparticles imparting a net negative charge over it. FTIR analysis also showed that biomolecules were involved in stabilization. SNBCs showed strong antibacterial activity against both Gram positive (Bacillus subtilis) and Gram negative bacteria (Escherichia coli). SNBCs also showed strong antifungal activity as assessed against Schizosaccharomyces pombe. In the case of E. coli the minimum inhibitory concentrations (MIC) of SNBCs was 4 µg/ml while in B. subtilis it was 8 µg/ml. In the case of E. coli the minimum bactericidal concentrations (MBC) of SNBCs was 8 µg/ml while in B. subtilis it was 32 µg/ml. The SNBCs exerted its antibacterial and antifungal activity through generation of reactive oxygen species (ROS) inside the cell.

Detection of S-nitrosothiol and nitrosylated proteins in Arachis hypogaea functional nodule: response of the nitrogen fixing symbiont.

To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment.

Biochemical characterization of compatible plant-viral interaction: a case study with a begomovirus-kenaf host-pathosystem.

Yellow vein mosaic disease of mesta, a compatible plant virus interaction poses a serious threat to mesta cultivation in India. Plants respond to invasion by pathogens with multi component defense responses particularly in incompatible interaction. With the aim of understanding, a biochemical approach was attempted to study the cellular redox status in early stages of yellow vein mosaic virus infection associated with different age's plant of Hibiscus cannabinus. Comparative analysis of GSH and GSSG content in infected and control plant of different ages indicated that infected plants are under oxidative or nitrosative stress condition. A significant change was observed in Glutathione Reductase, Catalase and Ascorbate Peroxidase level in early stage of infection. We also showed microscopic evidence of nitrosylated thiols in infected leaves, stems and roots of H. cannabinus. Furthermore, we identified few defense related proteins in infected plant using MALDI TOF mass spectrometric analysis.

Production of nitric oxide in host-virus interaction: a case study with a compatible Begomovirus-Kenaf host-pathosystem.

Nitric oxide (NO) plays a key role in plant diseases resistance. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated NO and reactive nitrogen species production during early infection stage not only in infected leaf but also in root and shoot. Production of NO was further confirmed by oxyhemoglobin assay. Furthermore, we used Phenyl alanine ammonia lyase as marker of pathogenesis related enzyme. In addition evidence for protein tyrosine nitration during the early stage of viral infection clearly showed the involvement of nitrosative stress.

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae.

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Deltayhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Deltayhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.

In vivo protein tyrosine nitration in S. cerevisiae: identification of tyrosine-nitrated proteins in mitochondria.

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Deltayhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Deltayhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Deltayhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Deltayhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.