Rapid onsite detection of bacterial spores of biothreat importance by paper-based colorimetric method using erbium-pyrocatechol violet complex.

Dipicolinic acid (DPA) is an important chemical marker for the detection of bacterial spores. In this study, complexes of lanthanide series elements such as erbium, europium, neodymium, and terbium were prepared with pyrocatechol violet and effectively immobilized the pyrocatechol violet (PV)-metal complex on a filter paper using polyvinyl alcohol. These filter paper strips were employed for the onsite detection of bacterial spores. The test filter papers were evaluated quantitatively with different concentrations of DPA and spores of various bacteria. Among the four lanthanide ions, erbium displayed better sensitivity than the other ions. The limit of detection of this test for DPA was 60 µM and 5 × 10(6) spores. The effect of other non-spore-forming bacteria and interfering chemicals on the test strips was also evaluated. The non-spore-forming bacteria did not have considerable effect on the test strip whereas chemicals such as EDTA had significant effects on the test results. The present test is rapid and robust, capable of providing timely results for better judgement to save resources on unnecessary decontamination procedures during false alarms.

Serum levels of IL-8, IFNγ, IL-10, and TGF ß and their gene expression levels in severe and non-severe cases of dengue virus infection.

Increased serum and mRNA levels of cytokines in patients with dengue virus (DV) infection suggest that cytokines are one of the key factors in the pathogenesis of disease caused by this virus. Here, we tested 211 serum and 56 mRNA samples from an equal number of dengue cases to determine the levels of interleukin-8 (IL-8), interferon gamma (IFN-γ), interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß). A total 70 serum and 15 mRNA samples from healthy individual were also tested for cytokines and served as controls. Serum and mRNA levels of IL-8 were highest in the earlier days of dengue infection. IFNγ levels peaked one or two days before defervescence. Levels of IL-10 and TGF-ß were highest later in dengue infection, and TGF-ß levels peaked on the day of defervescence. Mean levels of IFNγ, TGF ß and IL-10 were higher in samples from dengue cases, irrespective of severity, than in healthy controls. In contrast, the level of IL-8 was significantly higher in samples from severe dengue cases and lower in cases of dengue without warning signs than in healthy controls. Children (82.2 % of 101 paediatric cases) commonly had severe dengue illness. Samples that were positive for anti-DV IgG antibody had higher levels of IL-8 and TGF ß. DV-2 infections were associated with severe dengue illness. IL-8 and IFNγ levels were higher in the presence of warning signs of severe dengue. Levels of IL-8, IL-10 and TGF ß were independently associated with disease outcome. These data provide evidence of an association of IL-8, IFNγ, TGF ß and IL-10 levels with the severity of dengue illness. Especially, IL-8 levels can be used as a predictor of severe DV infection.

Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells.

T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.

Plant toxin abrin induced oxidative stress mediated neurodegenerative changes in mice.

Abrin is a potent plant toxin. It is a heterodimeric protein toxin which is obtained from the seeds of Abrus precatorius plant. At cellular level abrin causes protein synthesis inhibition by removing the specific adenine residue (A4324) from the 28s rRNA of the 60S - ribosomal subunit. In the present study we investigated the role of oxidative stress in neurotoxic potential and demyelinating effects of abrin on brain. The mechanism by which abrin induces oxidative damage and toxicity in brain are relatively unknown. Animals were exposed to 0.4 and 1.0 LD50 abrin dose by intraperitoneal route and observed for 1 and 3 day post-toxin exposure. Oxidative stress occurred in brain due to abrin was confirmed in terms of increased reactive oxygen species (ROS), glutathione depletion and increased lipid peroxidation. Significant increase in blood and brain ROS was observed at day 3, 1 LD50. Abrin induced changes in the neurotransmitters (5-hydroxy tryptamine, norepinephrine, dopamine and monoamine oxidase) levels were evaluated by spectroflourometry. Increase in the levels of 5-HT and NE was observed after abrin exposure. MAO activity was found to be decreased in abrin exposed animals compared to control. Significant inhibition in the activity of acetylcholine esterase enzyme in brain and serum was reported for both the doses and time points. Western blot analysis of iNOS expression indicated that abrin treatment resulted in dose and time dependent increase. Furthermore, protein expression of myelin basic protein (MBP) was down regulated in a dose and time dependent manner. Brain histopathology was carried out and cortical brain region showed demyelination after abrin exposure. Results confirmed that abrin poisoning leads to neurodegeneration and neurotoxicity mediated through oxidative stress, AChE inhibition, lipid peroxidation and decrease in MBP levels.

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

T-2 toxin induced skin inflammation and cutaneous injury in mice.

T-2 toxin is one of the most toxic among several trichothecenes involved in both human and animal poisoning cases. We investigated the biochemical and histological alterations behind inflammation and cutaneous injury caused by T-2 toxin. Swiss albino mice were exposed to T-2 toxin topically at doses of 0.5, 1 and 2 LD50 (2.97, 5.94 and 11.88 mg/kg respectively) and observed till 3, 24 and 72 h. Topical application of T-2 toxin resulted in skin oxidative stress in terms of increased reactive oxygen species generation, lipid peroxidation and neutrophil mediated myeloperoxidase activity. The histological alterations include degenerative changes like vacuolation, ballooning of basal keratinocytes and infiltration of inflammatory cells in dermis. The mRNA levels of skin pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß showed significant up regulation. Anti-inflammatory cytokines IL-10 showed significant up regulation at 24h whereas IL-4 showed down regulation for all the doses and time points. Gelatin zymography and immunoblot analysis of matrix metalloproteinases (MMP)-9 and 2 indicated MMP activation and their role in degenerative skin histological changes. Time dependent increase in inducible nitric oxide synthase levels was seen. Immunoblot analysis revealed significant increase in the levels of phosphorylated p38 mitogen activated protein kinase (MAPK). Flow cytometry analysis of propidium iodide stained epidermal cells showed increase in sub-G1 population at all the doses and time points indicating apoptosis. In summary, T-2 toxin induced skin inflammation and cutaneous injury is mediated through oxidative stress, activation of myeloperoxidase, MMP activity, increase in inflammatory cytokines, activation of p38 MAPK and apoptosis of epidermal cells leading to degenerative skin histological changes.

Production, characterization, and application of monoclonal antibodies specific to recombinant (E2) structural protein in antigen-capture ELISA for clinical diagnosis of Chikungunya virus.

The resurgence of Chikungunya (CHIK) virus in the form of an explosive, unprecedented epidemic with high virulence and unusual numbers of fatalities has created an immense public health concern in recent years. In the absence of an effective vaccine and specific antiviral therapy, early accurate diagnosis is essential for the best patient management. The present study describes the production and characterization of high-affinity and selective monoclonal antibodies (Mabs) against recombinant E2 protein (rE2) of the CHIK virus. The reactivity of Mabs for rE2 protein was demonstrated using ELISA. The specificity of the generated Mabs for rE2 was demonstrated by Western blot and indirect immunofluorescence. The application of this CHIK virus E2-specific monoclonal antibody in early clinical diagnosis was demonstrated by various analytical methods, such as immunoblotting, indirect immunofluorescence assay (IFA), and antigen-capture ELISA (AC-ELISA), for the detection as well as the identification of the novel ECSA genotypes of CHIK virus. These findings suggest that the high-affinity E2-specific monoclonal antibodies reported in this study will be useful for early clinical diagnosis and epidemiological studies of CHIK virus in developing countries.

Evaluation of protective efficacy of CC-2 formulation against topical lethal dose of T-2 toxin in mice.

T-2 toxin is the type-A trichothecene and a common contaminant of food and cereals, produced by Fusarium species. T-2 toxin easily penetrates skin due to its lipophilic nature and causes skin irritation and blisters in humans. Physical protection of the skin and airway is the only proven effective method of protection. To date, no chemical antidotes are available to prevent T-2 induced lethality. In the present study, we evaluated the protective efficacy of 20% N,N'-dichloro-bis(2,4,6-trichlorophenyl) urea (CC-2) formulation against lethal topical exposure dose of T-2 toxin in mice. None of the animals exposed to only T-2 toxin at lethal dose of 2 and 4 LD50 (11.8 and 23.76 mg/kg body weight) survived beyond 36 and 16 h, respectively. CC-2 application at 5 and 15 min post-exposure protected mice 100% from lethality at 2 LD50. Survival rate was 100% and 50% at 4LD50 dose if CC-2 was applied dermally within 5 and 15 min post-exposure. Recovery profile of surviving animals after 2LD50 T-2 toxin exposure at 1, 3, 7, and 14 days was assessed in terms of hepatic GSH, lipid peroxidation, serum ALP, ALT and AST. Hepatic lipid peroxidation significantly increased in all groups exposed to T-2 toxin by 3 day but normalized by day 7. A delayed GSH depletion was noted in surviving animals on day 7 but recovered by day 14. ALT and AST levels were elevated in all CC-2 protected mice on day 1 and normalized by day 3. ALP level decreased till day 7 in all protected groups. The biochemical variables recovered to control values by 14th day. GC-MS analysis after in vitro interaction of CC-2 formulation with T-2 toxin had shown that nearly 86% of T-2 toxin is decontaminated in 5 min but 8-10% of T-2 toxin was still present even after 60 min of interaction. Results of our study suggest that CC-2 may be an effective dermal decontaminant against lethal topical exposure of T-2 toxin.

Development of a simple fed-batch process for the high-yield production of recombinant shiga toxin B-chain protein.

Shiga toxins are one of the very potent agents for causing dysentery, diarrhoea and haemolytic uremic syndrome with very low LD50. For better understanding of their biology, detection and neutralization, the components of toxins are needed to be expressed and purified in bulk amounts. However, following traditional expression procedures, this task is very tedious as the yield of the toxin is very low. In this manuscript, we have described the optimization of media for enhanced production of recombinant Shiga toxin B (rStx-B) chain protein in Escherichia coli. This protein is known to have neutralization ability against shiga toxins. Furthermore, fed-batch cultivation process in E. coli was also developed in the optimized medium. Expression was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG). The purification of protein involved Ni-NTA affinity chromatography under native conditions followed by gel filtration chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell weight and purified protein of about 19.41 g/l (dry cell weight, 11.38 g/l) and 30 mg/l of culture, respectively. The purity of the recombinant StxB protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Reactivity of this protein was determined by Western blotting as well as indirect ELISA using specific antibodies. These results establish the application of this protein for diagnosis of shiga toxin infection or for neutralizing the toxicity.

Rapid and Real-time Detection of Human Viral Infections: Current Trends and Future Perspectives.

The development of technologies with rapid and sensitive detection capabilities and increased throughput have become crucial for responding to greater number of threats posed by emerging and re-emerging viruses in the recent past. The conventional identification methods require time-consuming culturing, and/or detection of antibodies, which are not very sensitive and specific. The recent advances in molecular biology techniques in the field of genomics and proteomics greatly facilitate the rapid identification with more accuracy. The real-time assays viz; SYBR green I based real time RT-PCR and RT-LAMP have been developed for rapid detection as well as typing of some of the emerging arboviruses of biomedical importance viz; Dengue, Japanese Encephalitis, Chikungunya, West Nile, SARS and Swine Flu etc. Both these techniques are capable of detection and differentiation as well as quantifying viral load with higher sensitivity, rapidity and specificity. One of the most important advantages of RT-LAMP is its field applicability, without requirement of any sophisticated equipments. The establishment of these real time molecular assays will certainly facilitate the rapid detection of viruses with high degree of precision and accuracy in future.

Evaluation of a commercial Dengue NS1 enzyme-linked immunosorbent assay for early diagnosis of dengue infection.

PURPOSE: Dengue is one of the most serious mosquito-borne viral infections affecting tropical and subtropical countries in the world. Since there is no immunoprophylactic or specific antiviral therapy available, timely and rapid diagnosis plays a vital role in patient management and implementation of control measures. This paper evaluates a commercially available NS1 antigen capture ELISA vis-a-vis SD bioline Dengue NS1 antigen test for early detection of dengue virus. MATERIALS AND METHODS: To evaluate a commercial NS1 antigen detection kit vis-a-vis SD bioline Dengue NS1 antigen test, a total of 91 clinical samples were tested. Virological investigations with regard to dengue virus, viz. NS1 antigen capture ELISA (Panbio, Australia), SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were performed. RESULTS: Out of 91 samples, 24 (26%) were positive by NS1 antigen capture ELISA, 15 (16%) by SD bioline Dengue NS1 antigen test and 11(12%) positive by RT-PCR analysis. The RT-PCR-positive samples were further subjected to virus isolation and resulted in three isolates. The results of the Panbio NS1 antigen capture ELISA, SD bioline Dengue NS1 antigen test, RT-PCR and virus isolation were correlated among themselves. CONCLUSIONS: The present study comprehensively established the utility of NS1 antigen ELISA in early diagnosis of dengue infection.

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via, mitochondrial mediated caspase-dependent mechanism.

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.

Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against Chikungunya virus in vero cells.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented and explosive epidemics in India and the Indian Ocean islands after a gap of 32 years is a major public health concern. Currently, there is no specific therapy available to treat CHIKV infection. In the present study, the in vitro prophylactic and therapeutic effects of chloroquine on CHIKV replication in Vero cells were investigated. Inhibitory effects were observed when chloroquine was administered pre-infection, post-infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. The inhibitory effects were confirmed by performing a plaque reduction neutralization test (PRNT), real-time reverse transcriptase (RT)-PCR analysis of viral RNA levels, and cell viability assays. Chloroquine diminished CHIKV infection in a dose-dependent manner, with an effective concentration range of 5-20 microM. Concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by >/=99.99%. The maximum inhibitory effect of chloroquine was observed within 1-3 hr post-infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. The mechanism of inhibition of virus activity by chloroquine involved impaired endosomal-mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent.

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein.

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l(-1) and 720 mg l(-1) of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.

Bacterially expressed recombinant envelope protein domain III of Japanese encephalitis virus (rJEV-DIII) elicits Th1 type of immune response in BALB/c mice.

Japanese encephalitis is a major cause of encephalitis in Asia. Cases occur largely in rural areas of the South and East Asian region resulting in significant morbidity and mortality. Multiple vaccines exist to control Japanese encephalitis, but all suffer from problems. Envelope protein domain III of Japanese encephalitis virus is involved in binding to host receptors and it contains specific epitopes that elicit virus-neutralizing antibodies. Earlier, the protective efficacy of domain III has been evaluated in mice by some researchers, but these studies are lacking in explanation of humoral and cellular immune responses. We have earlier reported cloning, expression, purification and in vitro refolding of Japanese encephalitis virus envelope protein domain III (rJEV-DIII). Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80. In the present study, we have evaluated the immunomodulatory potential of refolded rJEV-DIII protein in BALB/c mice with Freunds complete/incomplete adjuvants. Mice were tested for humoral immune response by ELISA. Cell-mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The rJEV-DIII generated high IgG antibody and its isotypes (IgG2a and IgG3) and induced significant expression of INF-gamma and IL-2 cytokines. The rJEV-DIII induced significant lymphoproliferation of splenocytes. In conclusion rJEV-DIII induced Th1 type of immune response which plays an important role in protection for intracellular pathogens.

Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells.

T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.

Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine.

Abrin is a plant glycoprotein toxin, classified as ribosome inactivating protein (RIP) due to its property of damaging ribosomes in an irreversible manner. Many RIPs have direct DNA damaging activity. The objective of the present study was to evaluate the oxidative stress and DNA damaging potential of abrin in U937 human myeloleukemic cells. Cells were treated with abrin at IC50 of 8 ng/ml for 24h. Abrin induced a time dependent increase in reactive oxygen species and levels of antioxidant enzymes. There was significant depletion of reduced glutathione levels. DNA damage was assessed by comet assay in terms of percent head DNA, tail DNA, tail length and Olive tail moment. DNA damage was more pronounced at 4 and 8h at IC50 concentration. Abrin at 4, 8, 16 and 32 ng/ml concentration induced significant DNA damage at 4h. There was time dependent increase in levels of 8-OHdG in abrin treated cells indicating the oxidative stress mediated DNA damage. N-Acetylcysteine pretreatment at 10nM for 1h, considerably reversed the abrin induced DNA damage at 16 and 32 ng/ml. Our results clearly show oxidative DNA damage potential of abrin at low concentration.

RNA interference mediated inhibition of Chikungunya virus replication in mammalian cells.

Chikungunya has emerged as one of the most important arboviral infection of public health significance. Recently several parts of Indian Ocean islands and India witnessed explosive, unprecedented epidemic. So far, there is no effective antiviral or licensed vaccine available against Chikungunya infection. RNA interference mediated inhibition of viral replication has emerged as a promising antiviral strategy. In this study, we examined the effectiveness of small interfering RNAs (siRNAs) against the inhibition of Chikungunya virus replication in Vero cells. Two siRNAs against the conserved regions of nsP3 and E1 genes of Chikungunya virus were designed. The siRNA activity was assessed by detecting both the infectious virus and its genome. The results indicated a reduction of virus titer up to 99.6% in siRNA transfected cells compared to control. The viral inhibition was most significant at 24h (99%), followed by 48 h (65%) post infection. These results were also supported by the quantitative RT-PCR assay revealing similar reduction in Chikungunya viral genomic RNA. The siRNAs used had no effect on the expression of house keeping gene indicating non-interference in cellular mechanism. The specific and marked reduction in viral replication against rapidly replicating Chikungunya virus achieved in this study offers a potential new therapeutic approach. This is the first report demonstrating the effectiveness of siRNA against in vitro replication of Chikungunya virus.

Comparison of a dipstick enzyme-linked immunosorbent assay with commercial assays for detection of Japanese encephalitis virus-specific IgM antibodies.

BACKGROUND: Japanese encephalitis (JE) is a major public health concern in Asia including India. OBJECTIVES: To evaluate an in-house developed dipstick enzyme-linked immunosorbent assay (ELISA) test vis-à-vis two commercial kits for detection of JE virus-specific IgM antibodies. SETTING AND DESIGN: Comparative study carried out in Research and Development centre. MATERIALS AND METHODS: A total of 136 specimens comprising 84 serum and 52 CSF samples were tested by in-house dipstick ELISA, Pan-Bio IgM capture ELISA (Pan-Bio, Australia) and JEV CheX IgM capture ELISA (XCyton, India). RESULTS: The overall agreement among all three tests was found to be 92% with both serum and cerebrospinal fluid (CSF) samples. The sensitivity of the dipstick ELISA was found to be 91% with serum and 89% with CSF samples respectively. The specificity of the dipstick ELISA with reference to both commercial assays was found to be 100% in serum and CSF samples in this study. CONCLUSIONS: The in-house dipstick ELISA with its comparable sensitivity and specificity can be used as a promising test in field conditions since it is simple, rapid and requires no specialized equipment.