The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Vertical electric field induced bacterial growth inactivation on amorphous carbon electrodes

The objective of the present work is to understand the vertical electric field stimulation of the bacterial cells, when grown on amorphous carbon substrates in vitro. In particular, the antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli are studied using MTTassay, live/dead assay and inner membrane permeabilization assays. In our experiments, the carbon substrate acts as one electrode and the counter electrode is positioned outside the culture medium, thus suppressing the current, electrokinetic motions and chemical reactions. Guided by similar experiments conducted in our group on neuroblastoma cells, the present experimental results further establish the interdependence of field strength and exposure duration towards bacterial growth inactivation in vitro. Importantly, significant reduction in bacterial viability was recorded at the 2.5 V/cm electric field stimulation conditions, which does not reduce the neural cell viability to any significant extent on an identical substrate. Following electrical stimulation, the bacterial growth is significantly inhibited for S. aureus bacterial strain in an exposure time dependent manner. In summary, our experiments establish the effectiveness of the vertical electric field towards bacterial growth inactivation on amorphous carbon substrates, which is a cell type dependent phenomenon (Gram-positive vs. Gram-negative). (C) 2014 Elsevier Ltd. All rights reserved.

Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum

Three-dimensional positioning of the nuclear genome plays an important role in the epigenetic regulation of genes. Although nucleographic domain compartmentalization in the regulation of epigenetic state and gene expression is well established in higher organisms, it remains poorly understood in the pathogenic parasite Plasmodium falciparum. In the present study, we report that two histone tail modifications, H3K9Ac and H3K14Ac, are differentially distributed in the parasite nucleus. We find colocalization of active gene promoters such as Tu1 (tubulin-1 expressed in the asexual stages) with H3K9Ac marks at the nuclear periphery. By contrast, asexual stage inactive gene promoters such as Pfg27 (gametocyte marker) and Pfs28 (ookinete marker) occupy H3K9Ac devoid zones at the nuclear periphery. The histone H3K9 is predominantly acetylated by the PCAF/GCN5 class of lysine acetyltransferases, which is well characterized in the parasite. Interestingly, embelin, a specific inhibitor of PCAF/GCN5 family histone acetyltransferase, selectively decreases total H3K9Ac acetylation levels (but not H3K14Ac levels) around the var gene promoters, leading to the downregulation of var gene expression, suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in the parasites. Finally, we found that embelin inhibited parasitic growth at the low micromolar range, raising the possibility of using histone acetyltransferases as a target for antimalarial therapy.

Anthrapyrazolone analogues intercept inflammatory JNK signals to moderate endotoxin induced septic shock

Severe sepsis or septic shock is one of the rising causes for mortality worldwide representing nearly 10% of intensive care unit admissions. Susceptibility to sepsis is identified to be mediated by innate pattern recognition receptors and responsive signaling pathways of the host. The c-Jun N-terminal Kinase (JNK)-mediated signaling events play critical role in bacterial infection triggered multi-organ failure, cardiac dysfunction and mortality. In the context of kinase specificities, an extensive library of anthrapyrazolone analogues has been investigated for the selective inhibition of c-JNK and thereby to gain control over the inflammation associated risks. In our comprehensive biochemical characterization, it is observed that alkyl and halogen substitution on the periphery of anthrapyrazolone increases the binding potency of the inhibitors specifically towards JNK. Further, it is demonstrated that hydrophobic and hydrophilic interactions generated by these small molecules effectively block endotoxin-induced inflammatory genes expression in in vitro and septic shock in vivo, in a mouse model, with remarkable efficacies. Altogether, the obtained results rationalize the significance of the diversity oriented synthesis of small molecules for selective inhibition of JNK and their potential in the treatment of severe sepsis.

Paralogous cAMP Receptor Proteins in Mycobacterium smegmatis Show Biochemical and Functional Divergence

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the ?msmeg_0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

Cytotoxicity of TiO2 nanoparticles towards freshwater sediment microorganisms at low exposure concentrations

There is a persistent need to assess the effects of TiO2 nanoparticles on the aquatic ecosystem owing to their increasing usage in consumer products and risk of environmental release. The current study is focused on TiO2 nanoparticle-induced acute toxicity at sub-ppm level (<= 1 ppm) on the three different freshwater sediment bacterial isolates and their consortium under two different irradiation (visible light and dark) conditions. The consortium of the bacterial isolates was found to be less affected by the exposure to the nanoparticles compared to the individual cells. The oxidative stress contributed considerably towards the cytotoxicity under both light and dark conditions. A statistically significant increase in membrane permeability was noted under the dark conditions as compared to the light conditions. The optical and fluorescence microscopic images showed aggregation and chain formation of the bacterial cells, when exposed to the nanoparticles. The electron microscopic (SEM, TEM) observations suggested considerable damage of cells and bio-uptake of nanoparticles. The exopolysaccrides (EPS) production and biofilm formation were noted to increase in the presence of the nanoparticles, and expression of the key genes involved in biofilm formation was studied by RT-PCR. (C) 2014 Elsevier Inc. All rights reserved.

Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence

Background: Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. Results: We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. Conclusions: A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would provide insight into conditions which are encountered by this intracellular enteric pathogen during the course of infection. This would further contribute in identifying novel targets for antimicrobial agents. (C) 2014 Elsevier Ltd. All rights reserved.

Active Viscoelastic Matter: From Bacterial Drag Reduction to Turbulent Solids

A paradigm for internally driven matter is the active nematic liquid crystal, whereby the equations of a conventional nematic are supplemented by a minimal active stress that violates time-reversal symmetry. In practice, active fluids may have not only liquid-crystalline but also viscoelastic polymer degrees of freedom. Here we explore the resulting interplay by coupling an active nematic to a minimal model of polymer rheology. We find that adding a polymer can greatly increase the complexity of spontaneous flow, but can also have calming effects, thereby increasing the net throughput of spontaneous flow along a pipe (a ``drag-reduction'' effect). Remarkably, active turbulence can also arise after switching on activity in a sufficiently soft elastomeric solid.

Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Lobe-specific Expression of Phosphodiesterase 5 in Rat Prostate

OBJECTIVE To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. METHODS The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, alpha-smooth muscle actin, and rat endothelial cell antigen. RESULTS Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. CONCLUSION PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate. (C) 2015 Elsevier Inc.

Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats

Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing. (C) 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH.

Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India

The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p <= 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51 % (n=793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46 % (n=273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85 % (n=107), 94.49 % (n=103), and 90.22 % (n=157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of blaTEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

Enhanced sunlight photocatalytic activity of Ag3PO4 decorated novel combustion synthesis derived TiO2 nanobelts for dye and bacterial degradation

This study demonstrates the synthesis of TiO2 nanobelts using solution combustion derived TiO2 with enhanced photocatalytic activity for dye degradation and bacterial inactivation. Hydrothermal treatment of combustion synthesized TiO2 resulted in unique partially etched TiO2 nanobelts and Ag3PO4 was decorated using the co-precipitation method. The catalyst particles were characterized using X-ray diffraction analysis, BET surface area analysis, diffuse reflectance and electron microscopy. The photocatalytic properties of the composites of Ag3PO4 with pristine combustion synthesized TiO2 and commercial TiO2 under sunlight were compared. Therefore the studies conducted proved that the novel Ag3PO4/unique combustion synthesis derived TiO2 nanobelt composites exhibited extended light absorption, better charge transfer mechanism and higher generation of hydroxyl and hole radicals. These properties resulted in enhanced photodegradation of dyes and bacteria when compared to the commercial TiO2 nanocomposite. These findings have important implications in designing new photocatalysts for water purification.