Reduction in Acute Ecotoxicity of Paper Mill Effluent by Sequential Application of Xylanase and Laccase

In order to reduce the ecotoxicity of paper mill, four different enzymatic pretreatment strategies were investigated in comparison to conventional chemical based processes. In strategy I, xylanase-aided pretreatment of pulp was carried out, and in strategy II, xylanase and laccase-mediator systems were used sequentially. Moreover, to compare the efficiency of Bacillus stearothermophilus xylanase and Ceriporiopsis subvermispora laccase in the reduction of ecotoxicity and pollution, parallel strategies (III and IV) were implemented using commercial enzymes. Conventional C_DE_OPD₁D₂ (C_D, Cl₂ with ClO₂; E_OP, H₂O₂ extraction; D₁ and D_2, ClO₂) and X/XLC_DE_OPD₁D₂ (X, xylanase; L, laccase) sequences were employed with non-enzymatic and enzymatic strategies, respectively. Acute toxicity was determined by the extent of inhibition of bioluminescence of Vibrio fischeri with different dilutions of the effluent. Two-fold increase was observed in EC₅₀ values for strategy I compared to the control process. On the other hand, sequential application of commercial enzymes resulted in higher acute toxicity compared to lab enzymes. In comparison to the control process, strategy II was the most efficient and successfully reduced 60.1 and 25.8% of biological oxygen demand (BOD) and color of effluents, respectively. We report for the first time the comparative analysis of the ecotoxicity of industrial effluents.     

High Yield Production and Refolding of the Double-Knot Toxin,an Activator of TRPV1 Channels

A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1∶6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.     

p53 is involved in inducing testicular apoptosis in mice by the altered redox status following tertiary butyl hydroperoxide treatment

In testis, seminiferous epithelium is one of the most productive self-renewing systems in which apoptosis is an important phenomenon. Alteration in the cellular redox status has several detrimental effects on the cells, one of which is increased rate of apoptotic signals disturbing the natural balance. Since apoptotic responses to various therapeutic agents and toxicants follow diverse molecular mechanisms, therefore, the present study was designed to explore apoptosis in testes under the effect of oxidative stress. Tertiary butyl hydroperoxide (tBHP) was used to induce oxidative stress in mice. It was found that ROS production in testes by tBHP resulted in increased apoptosis. The apoptosis was evident from TUNEL staining in Zenker-fixed paraffin-embedded testicular sections of tBHP treated mice testis and DNA fragmentation analysis. Increased mRNA and protein expression of p53 in testis were observed by using RT-PCR and ELISA techniques, respectively. This indicates that p53 expression is linked to ROS generation in mice testes. The functional status of p53 was also assessed by upregulation of cyclin dependent kinase inhibitor, p21. Thus tBHP induced oxidative stress subject testicular cells to apoptosis which seems to involve p53.     

Molecular biology and pathogenesis of hepatitis E virus

The hepatitis E virus (HEV) is a small RNA virus and the etiological agent for hepatitis E, a form of acute viral hepatitis. The virus has a feco-oral transmission cycle and is transmitted through environmental contamination, mainly through drinking water. Recent studies on the isolation of HEV-like viruses from animal species also suggest zoonotic transfer of the virus. The absence of small animal models of infection and efficient cell culture systems has precluded virological studies on the replication cycle and pathogenesis of HEV. A vaccine against HEV has undergone successful clinical testing and diagnostic tests are available. This review describes HEV epidemiology, clinical presentation, pathogenesis, molecular virology and the host response to HEV infection. The focus is on published literature in the past decade. Equal contribution     

Assessment of microbial diversity in effluent treatment plants by culture dependent and culture independent approaches

Microbial community structure of two distinct effluent treatment plants (ETPs) of pesticide and pharmaceutical industries was assessed and defined by (i) culture dependent and culture independent approaches on the basis of 16S rRNA gene sequencing, (ii) diversity index analysis - operational taxonomic units (OTUs). A total of 38 and 44 bacterial OTUs having 85-99% similarity with the closest match in the database were detected among pharmaceutical and pesticide sludge samples, respectively. Fifty percent of the OTUs were related to uncultured bacteria. These OTUs had a Shannon diversity index value of 2.09-2.33 for culturables and in the range of 3.25-3.38 for unculturables. The high species evenness values of 0.86 and 0.95 indicated the vastness of microbial diversity retrieved by these approaches. The dominant cultured bacteria indicative of microbial diversity in functional ETPs were Alcaligenes, Bacillus and Pseudomonas. Brevundimonas, Citrobacter, Pandoraea and Stenotrophomonas were specific to pesticide ETP and Agrobacterium, Brevibacterium, Micrococcus, Microbacterium, Paracoccus and Rhodococcus were specific to pharmaceutical ETP. These microbes can thus be maintained and exploited for efficient functioning and maintenance of ETPs.     

Vermiconversion of industrial sludge for recycling the nutrients

The aim of the present study was to investigate the transformation of sugar mill sludge (PM) amended with biogas plant slurry (BPS) into vermicompost employing an epigeic earthworm Eisenia fetida. To achieve the objectives experiments were conducted for 13 weeks under controlled environmental conditions. In all the waste mixtures, a decrease in pH, TOC, TK and C:N ratio, but increase in TKN and TP was recorded. Maximum worm biomass and growth rate was attained in 20% PM containing waste mixture. It was inferred from the study that addition of 30-50% of PM with BPS had no adverse effect on the fertilizer value of the vermicompost as well as growth of E. fetida. The results indicated that vermicomposting can be an alternate technology for the management and nutrient recovery from press mud if mixed with bulking agent in appropriate quantities.     

Intracisternal administration of cholecystokinin-8 counteracts the central cardiovascular effects of adrenaline and NPY. A study based on the coexistence of cholecystokinin, phenylethanolamine N-methyltransferase and neuropeptide Y immunoreactivity in neurons of the nucleus tractus solitarius

(1) In the present study the occlusion method was employed to evaluate the overall coexistence of neuropeptide Y and phenylethanolamine-N-methyl transferase, neuropeptide Y and tyrosine hydroxylase as well as cholecystokinin and phenylethanolamine-N-methyl transferase immunoreactivity in nerve cell bodies of the dorsal subnuclei of the nucleus tractus solitarius of the male rat. A high degree of coexistence was established for neuropeptide Y/phenylethanolamine-N-methyl transferase, cholecystokinin/phenylethanolamine-N-methyl transferase and for tyrosine hydroxylase/neuropeptide Y immunoreactivity. (2) Sulfated [¹²I]cholecystokinin-8 was used as radioligand to study the densities of cholecystokinin-8 binding sites in the dorsal medulla oblongata by means of quantitative receptor autoradiography. High densities of binding sites were observed in parts of the nucleus tractus solitarius and in the area postrema. Labeling was also observed in the dorsal motor nucleus of the vagus. (3) In the physiological studies adrenaline (0.15–1.0 nmol), neuropeptide Y (0.075–0.75 nmol) and sulfated cholecystokinin-8 (0.3–3.0 nmol) were administered alone or in combination with neuropeptide Y or adrenaline intracisternally into -chloralose anaesthetized male rats. Especially the hypotensive and bradycardic responses of adrenaline were counteracted in the adrenaline/cholecystokinin co-treated animals, whereas the cardiovascular effects of neuropeptide Y when co-administered with cholecystokinin-8 (0.3 nmol) appeared to be more resistant to the antagonistic effect of cholecystokinin 8. In addition, cholecystokinin-8 further enhanced the neuropeptide Y-induced bradynpnea and increase in the tidal volume.

The present results indicate the existence of neuropeptide Y, adrenaline and cholecystokinin-8 immunoreactivity in the same neurons of the dorsal subnuclei of the nucleus tractus solitarius. Furthermore, binding sites for cholecystokinin-8 seem to at least partly co-distribute with -2 adrenergic and neuropeptide Y binding sites in the nucleus tractus solitarius. In the functional analysis, an antagonistic interaction between cholecystokinin-8 and adrenaline as well as between cholecystokinin and neuropeptide Y is demonstrated opening up the possibility that cholecystokinin peptides act as intrinsic modulators in the putative cholecystokinin/neuropeptide Y/adrenaline synapses in the nucleus tractus solitarius.     

Synergistic antibacterial and anti-biofilm activity of nisin like bacteriocin with curcumin and cinnamaldehyde against ESBL and MBL producing clinical strains

Abstract

Bacteriocins are small peptides that can inhibit the growth of a diverse range of microbes. There is a need to identify bacteriocins that are effective against biofilms of resistant clinical strains. The present study focussed on the efficacy of purified nisin like bacteriocin-GAM217 against extended spectrum β-lactamase (ESBL) and metallo-beta-lactamase (MBL) producing clinical strains. Bacteriocin-GAM217 when combined with curcumin and cinnamaldehyde, synergistically enhanced antibacterial activity against planktonic and biofilm cultures of Staphylococcus epidermidis and Escherichia coli. Bacteriocin-GAM217 and phytochemical combinations inhibited biofilm formation by >80%, and disrupted the biofilm for selected ESBL and MBL producing clinical strains. The anti-adhesion assay showed that these combinatorial compounds significantly lowered the attachment of bacteria to Vero cells and that they elicited membrane permeability and rapid killing as viewed by confocal microscopy. This study demonstrates that bacteriocin-GAM217 in combination with phytochemicals can be a potential anti-biofilm agent and thus has potential for biomedical applications.     

Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication

Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5′- and 3′-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5′-NCR of the positive-sense genomic RNA - 5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA - 3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.     

Laboratory rearing of the predatory coccinellidCleobora mellyi [Col.: Coccinellidae] for biological control ofParopsis charybdis [Col.: Chrysomelidae] in New Zealand

Cleobora mellyi Mulstant has been introduced into New Zealand in an attempt to controlParopsis charybdis Stål. Artificial diets and a practical method for rearing the predatory Australian ladybird,C. mellyi, are described.