Saurabh Mishra

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1.

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30-90 microg/ml) of SQGD for different time (3-12h) intervals at 37°C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30μg/ml) stimulation for 3h at 37°C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3-6h, which was found decrease at 12h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30-90 microg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25mg/kgbwt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH. Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.

Extremophiles: Sustainable Resource of Natural Compounds-Extremolytes

Microorganisms that thrive in extreme adverse environmental conditions are extremophiles. Examples of these conditions are temperature (>45°C; 500 atmosphere), pH (>8.0; 1.0 M NaCl), high concentrations of calcitrant, heavy metals, high levels of radiation exposure etc. The discovery of extremophiles has enabled the biotechnology industry to innovate corresponding bioproducts, extremolytes, for people’s benefit. The production of Taq DNA polymerase has revolutionized biotechnology research in many ways. Many thermostable enzymes including cellulase, lipase, amylase, and proteases have contributed significantly as industrial bioproducts. Extremophilic radioresistant bacteria and fungi can be used strategically for the development of radioprotective drugs to protect against radiation exposure. Further these extremophiles can be used to develop cryoprotectants. Categorically, the piezophilic microorganisms in the deep sea are a prominent source of specific bio-molecules that has ability to stabilize cell membrane blebbing by maintaining the membrane fluidity. Extremophiles are a sustainable resource for biotechnology industry, which needs to be explored. This chapter provides a comprehensive view of the extremophiles and their products with the possible implications in human interest.

A semiquinone glucoside derivative provides protection to male reproductive system of the mice against gamma radiation toxicity.

Present investigation was carried out to evaluate the radioprotective efficacy of a novel Semiquinone glucoside derivative (SQGD), isolated from Bacillus sp. INM‐1, in the male reproductive system of BALB/c mice. Animals were administered 50 mg/kg b.wt. (i.p.) SQGD 2 h before whole body γ‐irradiation (10 Gy). Radiation‐induced cellular toxicity and its modulation by SQGD pretreatment was evaluated in the mice testes by quantitative histological and protein expression analysis. SQGD pretreatment protects irradiated mice from radiation‐induced testicular atrophy and germ cells degeneration, which may lead to emptiness of seminiferous tubules. Significant decrease in P53 and P21(Cip/WAF‐1) expression was observed in the irradiated mice pretreated (2 h) by SQGD at 6 h compared with only irradiated mice. However, contrary to P53, expressions of P21 at latter time, that is, 24–72 h was found to be increased significantly in the irradiated mice pretreated by SQGD. Significant increase in the intact PARP‐1 protein expression were observed in the testes of the mice pretreated by SQGD 2 h before irradiation at 24–72 h compared with the only irradiated mice, whereas significant increase in PARP‐1 cleaved fragment was noticed at 24 h. Similarly, significant increase in NF‐kB and BCL‐2/BAX expressions ratio was noticed in SQGD‐treated mice (± irradiation) compared with irradiated mice, suggested a role of SQGD in the activation of prosurvival signaling in the testicular germinal cells population of the irradiated mice and thus contributed to protection against lethal γ‐irradiation.

Semiquinone glucoside derivative provides protection against γ‐radiation by modulation of immune response in murine model

Present study was undertaken to evaluate radioprotective and immunomodulatory activities of a novel semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM‐1 in C57BL/6 mice. Whole body survival study was performed to evaluate in vivo radioprotective efficacy of SQGD. To observe effect of SQGD on immunostimulation, Circulatory cytokine (i.e., interleukin‐2 (IL‐2), IFN‐γ, IL‐10, granulocyte colony stimulating factor (G‐CSF), granulocyte macrophage colony stimulating factor (GM‐CSF), and macrophage colony stimulating factor (M‐CSF) expression was analyzed in serum of irradiated and SQGD treated mice at different time intervals using ELISA assay. Results of the present investigation indicated that SQGD pre‐treatment (‐2 h) to lethally irradiated mice provide ∼83% whole body survival compared with irradiated mice where no survival was observed at 30th post irradiation day. Significant (p < 0.05) induction in IL‐2 and IFN‐γ expression was observed at all tested time intervals with SQGD pre‐treated irradiated mice as compared with irradiated mice alone. However, sharp increase in IL‐10 expression was observed in irradiated mice which were found to be subsidized in irradiated mice pre‐treated with SQGD. Similarly, significant (p < 0.05%) induction in G‐CSF, M‐CSF and GM‐CSF expression was observed in irradiated mice treated with SQGD as compared with irradiated control mice at tested time intervals. In conclusion, SQGD pre‐treatment to irradiated mice enhanced expression of IL‐12 and IFN‐γ while down‐regulated IL‐10 expression and thus modulates cytoprotective pro‐inflammatory TH1 type immune response in irradiated mice. Further, SQGD pre‐treatment to irradiated mice accelerate G‐CSF, GM‐CSF and M‐CSF expression suggesting improved haematopoiesis and enhanced cellular immune response in immuno‐compromised irradiated mice that may contribute to in vivo radiation protection.

Semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 protects against gamma radiation-induced oxidative stress.

In the present study, radioprotective potential of Semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 was evaluated. γ-Radiation induced protein carbonylation, plasmid DNA damage, enzyme functional impairment, lipid peroxidation, HO radicals generation and their protection by SQGD was assessed. As a result of SQGD treatment, significant inhibition (p<0.05) in protein carbonylation was observed with BSA. SQGD treatment was found to restore supercoiled (~70±3.21%) form of irradiated plasmid DNA against γ-irradiation. SQGD protects enzymes (EcoR1 and BamH1) against radiation-induced dysfunctioning. SQGD significantly inhibited (p<0.05) lipid peroxidation in liposomes, brain and liver homogenate. Higher HO(•) radicals-averting activity of SQGD was observed in the serum and liver homogenate of C57BL/6 mice against H2O2-induced oxidative stress. In conclusion, SQGD demonstrates excellent radical-scavenging activity towards bio-macromolecules in irradiated environment and can be developed as an ideal radioprotector against radiation-induced oxidative stress in future.

A semiquinone glucoside derivative isolated from Bacillus sp. INM-1 provides protection against 5-fluorouracil-induced immunotoxicity.

Abstract 5-Fluorouracil (5-FU) is a widely used anti-cancer agent; however, it induces immunosuppression in patients undergoing a chemotherapy regime. The mode of action by which 5-FU induces immunosuppression is primarily via inhibition of hematopoietic growth factors. In the present study, immunoprotective effects of a semiquinone glucoside derivative (SQGD), a bacterial metabolite isolated from Bacillus sp. INM-1, were evaluated in a model of 5-FU-induced immunotoxicity in C57Bl/6 male mice. The evaluation was done by analyzing G-CSF, GM-CSF, and M-CSF expression in the serum, spleen, and bone marrow cells of the mice at different timepoints after 5-FU treatment. Mice received a single intraperitoneal injection of either 5-FU (75 mg/kg) alone, SQGD (50 mg/kg) alone, or SQGD 2 h prior to the 5-FU treatment. Control mice received saline vehicle only. The results demonstrated that 5-FU treatment significantly inhibited G-CSF, GM-CSF, and M-CSF expression in all three sites at all timepoints from 6–72 h post 5-FU. In SQGD treated mice, up-regulation of factor expression was observed in each compartment, and significantly so most often after 12 h. SQGD treatment prior to 5-FU administration to the mice significantly increased in all sites evaluated – relative to values in both control mice and 5-FU only-treated mice – G-CSF, M-CSF, and GM-CSF expression at almost every timepoint. The present findings suggest that SQGD provides protection against 5-FU-induced immunotoxicity in mice and could protect bone marrow progenitor cells against the effects of cytotoxic drugs used for treatment of cancer. The findings also suggested to us that SQGD is a potential immunomodulator and could protect hematopoiesis against toxic assault caused by anti-cancer drugs in the clinical setting.

Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage

Abstract Purpose: A simple, sensitive and novel method was developed to screen out potential agents able to protect functional activity of DNA ligase against gamma irradiation-induced damage. Repeatability, authenticity and sensitivity of the method was verified by analyzing DNA ligase protecting activities of well-known radioprotectors such as amifostine, trolox, melatonin, semiquinone glucoside derivative (SQGD) and an antioxidant gallic acid in extremely low concentration (1 μg/reaction). Material and methods: Two different sets (Set A and B) of T4 DNA ligase (1 unit/set) were prepared. Set ‘A’ (negative control) was exposed to different doses (3–5 kGy) of gamma radiation in the absence of radioprotective compounds. Set B (test) was exposed to similar doses of gamma radiation in the presence of radioprotective compounds. Following irradiation, DNA ligase was mixed with λ DNA (250 ng) pre-digested with Hind III restriction endonuclease. Ligation reaction was performed in both sets simultaneously at 22°C for 20 min and reaction product was analyzed using agarose gel electrophoresis. Results: Complete DNA ligation was observed in samples where DNA ligase was irradiated in the presence of radioprotectective compounds, i.e., amifostine, trolox, melatonin and a natural radioprotector semiquinone glucoside derivative (SQGD) individually, while, functional impairment in ligation activity of DNA ligase was evident in samples in which DNA ligase was irradiated in the absence of a radioprotective compound. Conclusion: The current method was able to provide significant input to screen out radioprotective compounds able to protect DNA ligase functional activity against gamma radiation-induced functional impairment.

N-acetyl tryptophan glucopyranoside (NATG) as a countermeasure against gamma radiation-induced immunosuppression in murine macrophage J774A.1 cells

Abstract Radiation exposure to immune system induces imbalance in cytokines expression involved in Th1/Th2 homeostasis perturbations. In the present study, N-acetyl tryptophan glucoside (NATG), a bacterial secondary metabolite, was evaluated for its possible radioprotective potential to immune system using J774A.1 murine macrophages. In this study, expression of IFN-γ, TNF-α, IL-10, IL-2, IL-12, IL-13 and IL-17A cytokines was analyzed in irradiated and NATG pretreated cells using ELISA assay. Results of the study indicated that irradiated macrophages (NK-1R+ cells) pretreated with NATG showed higher (p < .05) survival at all observed time-intervals (2 h-48 h) as compared to irradiated (20Gy) cells that were not pretreated with NATG. However, NATG pretreatment to irradiated HEK293T cells (that did not express NK-1Receptor) did not provide significant survival, suggesting NK-1R involvement in NATG-mediated radioprotection. Cytokine expression analysis demonstrated that NATG pre-treated plus irradiated J774A.1 murine macrophages exhibited increased IFN-γ levels (∼90%) with significant decrease in TNF-α at 24h as compared to irradiated cells. Further, significant decrease (∼20%) in IL-10 and IL-2 (∼26%) levels was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. A sharp improvement in IL-17A (∼92%) and IL-12 (∼116%) expression was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. Hence, NATG pre-treatment to irradiated macrophages induced IFN-γ, IL-17A and IL-12 expression, but suppresses TNF-α, IL-10 and IL-2 expressions. Conclusively, NATG pretreatment overcomes radiation-induced Th2 immune response by improving Th1 responsive cytoprotective cytokines IFN-γ, IL-17A and IL-12 in irradiated macrophages possibly by NK-1R antagonistic mechanism, and thus contributes to radioprotection.

Semiquinone fraction isolated from Bacillus sp. INM-1 protects hepatic tissues against γ-radiation induced toxicity

Present study was focused on evaluation of a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM‐1 for its ability against γ radiation induced oxidative stress in irradiated mice. Animals were divided into four group, i.e., (i) untreated control mice; (ii) SQGD treated (50 mg/kg b. wt. i.p.) mice; (iii) irradiated (10 Gy) mice; and (iv) irradiated mice which were pre‐treated (−2 h) with SQGD (50 mg/kg b. wt. i.p.). Following treatment, liver homogenates of the treated mice were subjected to endogenous antioxidant enzymes estimation. Result indicated that SQGD pre‐treatment, significantly (P < 0.05) induced superoxide dismutase (SOD) (19.84 ± 2.18% at 72 h), catalase (CAT) (26.47 ± 3.11% at 12 h), glutathione (33.81 ± 1.99% at 24 h), and glutathione‐S‐transferase (24.40 ± 2.65% at 6 h) activities in the liver of mice as compared with untreated control. Significant (P < 0.05) induction in SOD (50.04 ± 5.59% at 12 h), CAT (62.22 ± 7.50 at 72 h), glutathione (42.92 ± 2.28% at 24 h), and glutathione‐S‐transferase (46.65 ± 3.25 at 12 h) was observed in irradiated mice which were pre‐treated with SQGD compared with only irradiated mice. Further, significant induction in ABTS+ radicals (directly proportional to decrease mM Trolox equivalent) was observed in liver homogenate of H2O2 treated mice which were found to be significantly inhibited in H2O2 treated mice pre‐treated with SQGD. Thus, it can be concluded that SQGD treatment neutralizes oxidative stress caused by irradiation not only by enhancing endogenous antioxidant enzymes but also by improving total antioxidant status of cellular system and thus cumulative effect of the phenomenon may contributes to radioprotection.

Radiation Biomarkers : Applications in Triage Management of Radiation Victims

Human exposure to ionizing radiation disrupts normal metabolic processes in cells and organs by inducing complex biological responses that interfere with gene and protein expression. Conventional dosimetry, monitoring of prodromal symptoms and peripheral lymphocyte counts are of limited value as organ- and tissue-specific biomarkers for personnel exposed to radiation, particularly, weeks or months after exposure. Analysis of metabolites generated in known stress-responsive pathways by molecular profiling helps to predict the physiological status of an individual in response to environmental or genetic perturbations. There is a need for research to rapidly determine an individual’s absorbed dose and its potential health effects after a potential radiological or nuclear event that could expose large portions of a population to ionizing radiation. Studies on biomarker identification after radiation exposure could contribute in biodosimetry, identifying individual dose absorbed, as well as biologic response, and administering immediate and proper medical care. In the recent scenario development of biomarker is major thrust area. In the present review paper articles related to gene biomarker, protein biomarker and metabolic biomarker are reviewed in order to sketch an overview on the recent advances related to developing an biomarker to assess the radiation induced toxicity.