Assessment of textile industry effluent (untreated and microbially treated) induced genotoxic, haematological, biochemical, histopathological and ultrastructural alterations in fresh water fish Channa punctata.

The unregulated expulsion of untreated textile water into water bodies is a major hazard to aquatic ecosystems. The present investigation was contrived to estimate the impact of textile dye bath effluent (untreated and microbially treated) on fish Channa punctata. Untreated effluent-exposed fish showed extremely altered behaviour (air gulping, erratic and speedy movements, increased opercular activity) and morphology (deposition of dyes on skin and scales, high pigmentation, mucus exudation). Significantly increased micronuclei (1.61-, 1.28-, 1.38-fold) and aberrant cell frequency (1.37-, 1.45-, 1.28-fold) was observed in untreated group as compared to treated group after 15, 30, and 45 days of exposure. Tail length, % tail intensity, tail moment and olive tail moment were also enhanced in all the exposed tissues. However, maximum damage was noticed in gill tissues showing 1.19-, 1.37-, 1.34- and 1.50-fold increased TL, %TI, TM and OTM in untreated group as compared to treated group after 45 days of exposure. On comparing untreated and treated groups, increased blood parameters and significantly reduced white blood cell count (WBC) were noticed in treated group. Significantly enhanced alterations in biochemical parameters were also analysed in untreated group. Reduced alterations in enzymological levels of fishes exposed to treated effluent indicate lesser toxic nature of the degraded metabolites of dye. Histological analysis in fishes exposed to untreated effluent showed several deformities in liver (necrosis, congestion, fusion of cells and melanomacrophage infiltration) and gill tissues (necrosis, bending of lamellae and severe aneurysm). Scanning electron microscopy (SEM) analysis further reaffirmed the pathologies observed in histological analysis. Fewer structural alterations were noticed in treated effluent fishes. The results concluded that untreated effluent inflicted toxicity potential on morphology as well as physiological defects in fish, and the severity increased with increasing duration of exposure, whereas reduction in toxicity in microbially treated groups can be analysed for aquacultural purposes owing to their lesser toxic nature.

Synergistic and additive interactions of Shewanella sp., Pseudomonas sp. and Thauera sp. with chlorantraniliprole and emamectin benzoate for controlling Spodoptera litura (Fabricius).

The imprudent use of insecticides causes the development of resistance in insect pest populations, contamination of the environment, biological imbalance and human intoxication. The use of microbial pathogens combined with insecticides has been proposed as an alternative strategy for insect pest management. This IPM approach may offer effective ways to control pests, in addition to lowering the risk of chemical residues in the environment. Spodoptera litura (Fabricius) is a major pest of many crops like cotton, maize, tobacco, cauliflower, cabbage, and fodder crops globally. Here, we evaluated the combined effects of new chemistry insecticides (chlorantraniliprole and emamectin benzoate) and entomopathogenic bacterial strains, Shewanella sp. (SS4), Thauera sp. (M9) and Pseudomonas sp. (EN4) against S. litura larvae inducing additive and synergistic interactions under laboratory conditions. Both insecticides produced higher larval mortality when applied in combination with bacterial isolates having maximum mortality of 98 and 96% with LC50 of chlorantraniliprole and emamectin benzoate in combination with LC50 of Pseudomonas sp. (EN4) respectively. The lower concentration (LC20) of both insecticides also induced synergism when combined with the above bacterial isolates providing a valuable approach for the management of insect pests. The genotoxic effect of both the insecticides was also evaluated by conducting comet assays. The insecticide treatments induced significant DNA damage in larval hemocytes that further increased in combination treatments. Our results indicated that combined treatments could be a successful approach for managing S. litura while reducing the inappropriate overuse of insecticides.

Larvicidal, growth inhibitory and biochemical effects of soil bacterium, Pseudomonas sp. EN4 against Spodoptera litura (Fab.) (Lepidoptera: Noctuidae).

BACKGROUND: Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) also known as tobacco caterpillar, is one of the most serious polyphagous pests that cause economic losses to a variety of commercially important agricultural crops. Over the past few years, many conventional insecticides have been used to control this pest. However, the indiscriminate use of these chemicals has led to development of insecticide resistant populations of S. litura in addition to harmful effects on environment. Due to these ill effects, the emphasis is being laid on alternative eco-friendly control measures. Microbial control is one of the important components of integrated pest management. Thus, in search for novel biocontrol agents, the current work was carried out with the aim to evaluate the insecticidal potential of soil bacteria against S. litura. RESULTS: Among the tested soil bacterial isolates (EN1, EN2, AA5, EN4 and R1), maximum mortality (74%) was exhibited by Pseudomonas sp. (EN4). The larval mortality rate increased in a dose-dependent manner. Bacterial infection also significantly delayed the larval development, reduced adult emergence, and induced morphological deformities in adults of S. litura. Adverse effects were also detected on various nutritional parameters. The infected larvae showed a significant decrease in relative growth and consumption rate as well as efficiency of conversion of ingested and digested food to biomass. Histopathological studies indicated damage to the midgut epithelial layer of larvae due to the consumption of bacteria treated diet. The infected larvae also showed a significantly decreased level of various digestive enzymes. Furthermore, exposure to Pseudomonas sp. also caused DNA damage in the hemocytes of S. litura larvae. CONCLUSION: Adverse effects of Pseudomonas sp. EN4 on various biological parameters of S. litura indicate that this soil bacterial strain may be used as an effective biocontrol agent against insect pests.

Schizophyllum commune induced oxidative stress and immunosuppressive activity in Spodoptera litura.

BACKGROUND: In the last few decades, considerable attention has been paid to fungal endophytes as biocontrol agents, however little is known about their mode of action. This study aimed to investigate the toxic effects of an endophytic fungus Schizophyllum commune by analyzing activities of antioxidant and detoxifying enzymes as well as morphology of haemocytes using Spodoptera litura as a model. RESULTS: Ethyl acetate extract of S. commune was fed to the larvae of S. litura using the artificial diet having 276.54 µg/ml (LC50 of fungus) concentration for different time durations. Exposed groups revealed significant (p ≤ 0.05) increase in the activities of various enzymes viz. Catalase, Ascorbate peroxidase, Superoxide dismutase, Glutathione-S-Transferase. Furthermore, haemocytes showed various deformities like breakage in the cell membrane, cytoplasmic leakage and appearance of strumae in the treated larvae. A drastic reduction in the percentage of normal haemocytes was recorded in the treated groups with respect to control. CONCLUSION: The study provides important information regarding the oxidative stress causing and immunosuppressant potential of S. commune against S. litura and its considerable potential for incorporation in pest management programs.

Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) plays a key role in multiple events during infection including virus entry, cell-to-cell spread, and virus-induced syncytia formation. Here, we provide evidence that an arginine/lysine cluster located at the transmembrane-cytoplasm interface of gD critically contributes to viral spread and cell-cell fusion. Our studies began with the discovery that packaging of gD into virions is almost completely blocked in the absence of tegument protein UL16. We subsequently identified a novel, direct, and regulated interaction between UL16 and gD, but this was not important for syncytia formation. However, a mutational analysis of the membrane-proximal basic residues of gD revealed that they are needed for the gBsyn phenotype, salubrinal-induced fusion of HSV-infected cells, and cell-to-cell spread. Finally, we found that these same gD tail basic residues are not required for cell fusion induced by a gKsyn variant.

Antioxidant and in vivo genoprotective effects of phenolic compounds identified from an endophytic Cladosporium velox and their relationship with its host plant Tinospora cordifolia.

ETHNOPHARMACOLOGICAL RELEVANCE: Tinospora cordifolia (Willd. Hook. f. & Thomson; family: Menispermaceae), has a long history of use in various traditional medicinal systems including "Ayurveda". It is reported to possess anticancer, antidiabetic, antimicrobial, antispasmodic, and antiinflammatory activities. T. cordifolia has also been well documented for production of various bioactive metabolites and their antioxidant activity, but the microorganisms associated with it have been least explored for the same properties. AIM OF THE STUDY: Aim of the present study was to evaluate antioxidant and in vivo genoprotective potential of phenolic compounds produced by an endophytic fungus Cladosporium velox TN-9S isolated from T. cordifolia. MATERIALS AND METHODS: The isolate of C. velox TN-9S was cultivated in malt extract medium and extracted with ethyl acetate. Total phenol content was determined by Folin Ciocalteu reagent. The antioxidant activity was measured in terms of DPPH and FRAP assay. The in vivo genoprotective activity was assessed using fish Channa punctatus as model. Identification of phenolic compounds was carried out using RP-HPLC. The fungal extract was evaluated for biosafety using Salmonella typhimurium His- strain and CHO cell lines for mutagenicity and cytotoxicity, respectively. RESULTS: The total phenolic content in the ethyl acetate extract of the fungus was determined to be 730µg gallic acid equivalent/mL. The extract evinced significant antioxidant activity with IC50 value of 22.5µg/mL in DPPH scavenging assay. The phenolic extract showed good in vivo genoprotective activity against the genetic damage induced in fish C. punctatus after treatment with a non-ionic surfactant 4-nonylphenol. RP-HPLC analysis revealed the presence of peaks corresponding to various phenolic compounds in the extract. Mutagenicity and cytotoxicity results revealed the extract to be nonmutagenic and non cytotoxic in nature. CONCLUSION: The results indicate the potential of an endophytic C. velox isolated from T. cordifolia as a producer of phenolic compounds with antioxidant and genoprotective activities which could be exploited in pharmaceutical industry. The ability of endophytes to produce similar compounds as the host, is also revealed in the present study.

Textile industry and occupational cancer.

BACKGROUND AND SUMMARY: Thousands of workers are engaged in textile industry worldwide. Textile industry involves the use of different kinds of dyes which are known to possess carcinogenic properties. Solvents used in these industries are also associated with different health related hazards including cancer. In previous studies on textile and iron industries, the authors have reported genotoxicity among them and observed occurrence of cancer deaths among textile industry workers. Thus, an attempt has been made to compile the studies on the prevalence of different types of cancers among textile industry workers. LITERATURE SEARCH: A wide literature search has been done for compiling the present paper. Papers on cancer occurrence among textile industry workers have been taken from 1976 to 2015. A variety of textile dyes and solvents, many of them being carcinogenic, are being used worldwide in the textile industry. The textile industry workers are therefore, in continuous exposure to these dyes, solvents, fibre dusts and various other toxic chemicals. The present study evaluates the potential of different chemicals and physical factors to be carcinogenic agents among occupationally exposed workers by going through various available reports and researches. Papers were collected using different databases and a number of studies report the association of textile industry and different types of cancer including lung, bladder, colorectal and breast cancer. After going through the available reports, it can be concluded that workers under varied job categories in textile industries are at a higher risk of developing cancer as various chemicals used in the textile industry are toxic and can act as potential health risk in inducing cancer among them. Assessing the cancer risk at different job levels in textile industries may be found useful in assessing the overall risk to the workers and formulating the future cancer preventive strategies.

Assessment of DNA damage as an index of genetic toxicity in welding microenvironments among iron-based industries.

Welding is used extensively in different industries. Welders are always at a risk of exposure to a number of gases and metal-containing fumes in their respective microenvironments in which they work. Welding fumes consist of a wide range of complex metal oxide particles which can deposit in different parts of their bodies causing serious health problems. In the present study, 35 welders (age: 33.80 ± 1.04 years) from two iron-based industries have been assessed for DNA damage in peripheral blood lymphocytes using single-cell gel electrophoresis. An equal number of subjects (N = 35; age: 30.40 ± 1.51 years) matched to exposed subjects with respect to sex, age, socioeconomic status, smoking, and alcoholic habits were taken as controls. The results revealed that the damaged cell frequency (DCF) and mean comet tail length (CTL) in welders were significantly higher as compared to the controls (DCF: 69.74 ± 1.68 vs. 31.14 ± 1.67 and CTL: 29.21 ± 1.48 vs. 1.47 ± 0.08; p < 0.05). The effect of confounding factors such as age, duration of exposure, smoking, and drinking habits was also studied. Blood lead levels also showed a positive correlation with duration of exposure and CTL, and the overall results indicated an increased genetic damage as an index of genotoxicity in workers occupationally engaged in welding microenvironments.

Elucidation of the block to herpes simplex virus egress in the absence of tegument protein UL16 reveals a novel interaction with VP22.

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a "bridging" function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production.

Evaluation of oxidative stress and genotoxicity in battery manufacturing workers occupationally exposed to lead.

Battery manufacturing workers are occupationally exposed to lead (Pb), which is a highly toxic heavy metal. The aim of this study was to investigate the blood lead levels (BLL) of 30 battery manufacturing workers and find the correlation between BLL, micronucleated cell (MNC) frequency, binucleated cell (BNC) frequency in buccal mucosal cells and malondialdehyde concentrations in serum. 30 subjects of the BMW group, exposed to lead, and 30 control subjects, matched with the exposed subjects with respect to age, socio-economic status, sex, diet, smoking and drinking habits, were monitored for this study. BLL was found to have highly significant difference between both the groups (P < 0.001). The serum MDA levels were observed at significantly higher levels (6.76 ± 3.26) for the exposed group as compared to the control group (2.10 ± 1.02; P < 0.001). Buccal micronucleus test showed that both MNC and BNC frequencies were higher among the workers, in comparison to the control subjects. A positive correlation has been found between BLL and all the parameters. Our results indicate an increased health associated risk for workers occupationally exposed to lead.

Function of glycoprotein E of herpes simplex virus requires coordinated assembly of three tegument proteins on its cytoplasmic tail.

Glycoprotein E (gE) of HSV plays a key role in cell-to-cell spread and virus-induced cell fusion. Here, we report that this function of gE requires the cooperation of tegument proteins UL11, UL16, and UL21. We found that the four proteins come together with very high efficiency to form a complex in transfected cells and in a manner that is regulated and coordinated. In particular, the inefficient interaction of UL16 with each membrane protein (UL11 and gE) observed in pairwise transfections became efficient when other binding partners were present. The significance of these interactions was revealed in studies of viral mutants, which showed that each of these tegument proteins is critical for processing, transport, and biological activity of gE. These findings provide insights into the mechanisms of how gE executes its function and also have implications in understanding HSV assembly and budding.

Regulated interaction of tegument proteins UL16 and UL11 from herpes simplex virus.

It is well known that proteins in the tegument (located between the viral capsid and envelope proteins) play critical roles in the assembly and budding of herpesviruses. Tegument proteins UL16 and UL11 of herpes simplex virus (HSV) are conserved among all the Herpesviridae. Although these proteins directly interact in vitro, UL16 was found to colocalize poorly with UL11 in cotransfected cells. To explain this discrepancy, we hypothesized that UL16 is initially made in an inactive form and is artificially transformed to the binding-competent state when cells are disrupted. Consistent with a regulated interaction, UL16 was able to fully colocalize with UL11 when a large C-terminal segment of UL16 was removed, creating mutant UL16(1-155). Moreover, membrane flotation assays revealed a massive movement of this mutant to the top of sucrose gradients in the presence of UL11, whereas both the full-length UL16 and the C-terminal fragment (residues 156 to 373) remained at the bottom. Further evidence for the presence of a C-terminal regulatory domain was provided by single-amino-acid substitutions at conserved cysteines (C269S, C271S, and C357S), which enabled the efficient interaction of full-length UL16 with UL11. Lastly, the binding site for UL11 was further mapped to residues 81 to 155, and to our surprise, the 5 Cys residues within UL16(1-155) are not required, even though the modification of free cysteines in UL16 with N-ethylmaleimide does in fact prevent binding. Collectively, these results reveal a regulatory function within the C-terminal region of UL16 that controls an N-terminal UL11-binding activity.

Interaction and interdependent packaging of tegument protein UL11 and glycoprotein e of herpes simplex virus.

The UL11 tegument protein of herpes simplex virus plays a critical role in the secondary envelopment; however, the mechanistic details remain elusive. Here, we report a new function of UL11 in the budding process in which it directs efficient acquisition of glycoprotein E (gE) via a direct interaction. In vitro binding assays showed that the interaction required only the first 28, membrane-proximal residues of the cytoplasmic tail of gE, and the C-terminal 26 residues of UL11. A second, weaker binding site was also found in the N-terminal half of UL11. The significance of the gE-UL11 interaction was subsequently investigated with viral deletion mutants. In the absence of the gE tail, virion packaging of UL11, but not other tegument proteins such as VP22 and VP16, was reduced by at least 80%. Reciprocally, wild-type gE packaging was also drastically reduced by about 87% in the absence of UL11, and this defect could be rescued in trans by expressing U(L)11 at the U(L)35 locus. Surprisingly, a mutant that lacks the C-terminal gE-binding site of UL11 packaged nearly normal amounts of gE despite its strong interaction with the gE tail in vitro, indicating that the interaction with the UL11 N terminus may be important. Mutagenesis studies of the UL11 N terminus revealed that the association of UL11 with membrane was not required for this function. In contrast, the UL11 acidic cluster motif was found to be critical for gE packaging and was not replaceable with foreign acidic clusters. Together, these results highlight an important role of UL11 in the acquisition of glycoprotein-enriched lipid bilayers, and the findings may also have important implications for the role of UL11 in gE-mediated cell-to-cell spread.

Serine/threonine kinase (pk-1) is a component of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) very late gene transcription complex and it phosphorylates a 102 kDa polypeptide of the complex.

The baculovirus gene, protein kinase-I (pk-1) encodes a serine/threonine kinase that is essential for very late gene expression. Late and very late genes of the baculoviruses are transcribed by an alpha-amanitin resistant RNA polymerase. The very late gene promoter transcription initiation complex was isolated from nuclei of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells by DNA affinity purification and found to contain 4 major polypeptides of sizes approximately 102, 38, 32, and 18 kDa. The 32 kDa polypeptide was immunoreactive to AcMNPV anti-pk-1 antibody and phosphorylated the 102 kDa polypeptide, earlier reported as late gene expression factor LEF-8. Electrophoretic mobility shift assays with anti-pk-1 antibody indicated the binding of promoter DNA with recombinant AcMNPV-pk-1 and transcription initiation complex proteins. All these results suggested AcMNPV-pk-1 to be a component of the viral very late gene transcription initiation complex.

Inhibition of Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin gene expression by DNAzyme knockout of its serine/threonine kinase (pk1) gene.

DNAzyme is known to selectively cleave RNA at predetermined site. Transfection of Autographa californica nucleopolyhedrovirus (AcNPV) infected Sf9 cells with serine/threonine kinase (pk1) mRNA specific DNAzymes, DZ1 and DZ2 to cleave the viral coded (pk1) mRNA in between 87th and 88th, and 250th and 251st nucleotide, respectively inhibited the pk1 mRNA and its protein expressions. Interestingly, polh mRNA and protein expressions were also inhibited by these DNAzymes despite their inability to cleave polh mRNA. The polyhedrin promoter driven green fluorescent protein (GFP) mRNA and protein expressions were also inhibited by these pk1 specific DZs. Surprisingly the extents of inhibition of polyhedrin and GFP at different concentrations of both DZs were higher than that of pk1 mRNA and protein expressions. These results suggested that pk1 regulates polyhedrin promoter driven transcription of AcNPV, and the effect of one gene expression on that of other can be studied by DNAzyme knockdown.