Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients

Collagen-Like Proteins in Pathogenic E. coli Strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages

Thermal Sensitivity of the Enzymatic Activity of β‑Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations

A crucial prerequisite for industrial applications of enzymes is the maintenance of specific activity across wide thermal ranges. β-Glucosidase (EC 3.2.1.21) is an essential enzyme for converting cellulose in biomass to glucose. While the reaction mechanisms of β-glucosidases from various thermal ranges (hyperthermophilic, thermophilic, and mesophilic) are similar, the factors underlying their thermal sensitivity remain obscure. The work presented here aims to unravel the molecular mechanisms underlying the thermal sensitivity of the enzymatic activity of the β-glucosidase BglB from the bacterium Paenibacillus polymyxa. Experiments reveal a maximum enzymatic activity at 315 K, with a marked decrease in the activity below and above this temperature. Employing in silico simulations, we identified the crucial role of the active site tunnel residues in the thermal sensitivity. Specific tunnel residues were identified via energetic decomposition and protein–substrate hydrogen bond analyses. The experimentally observed trends in specific activity with temperature coincide with variations in overall binding free energy changes, showcasing a predominantly electrostatic effect that is consistent with enhanced catalytic pocket–substrate hydrogen bonding (HB) at Topt. The entropic advantage owing to the HB substate reorganization was found to facilitate better substrate binding at 315 K. This study elicits molecular-level insights into the associative mechanisms between thermally enabled fluctuations and enzymatic activity. Crucial differences emerge between molecular mechanisms involving the actual substrate (cellobiose) and a commonly employed chemical analogue. We posit that leveraging the role of fluctuations may reveal unexpected insights into enzyme behavior and offer novel paradigms for enzyme engineering

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Not AvailableOccurrence of natural populations of lac insects (Hemiptera: Tachardiidae) were observed on Amherstia nobilis at Thrissur of Kerala; on Albizia saman at Madurai, Salem, Vellore and Thiruvallur of Tamil Nadu and on Ficus religiosa at Erode and Vellore of Tamil Nadu. Dead lac insects were observed on A. saman, at Thenkasi and Theni in Tamil Nadu. Albizia saman, Albizia lebbek, Ficus religiosa, Ficus bengalensis and Ziziphus mauritiana were the most common lac host plants found in surveyed areas, but lac insect populations were observed only at some locations. The survey led to identification of two new species of Kerria. The molecular analysis based on cox1 sequence classified the collected lac insects from Tamil Nadu and Kerala under rangeeni strain. Newly collected lac insect lines from Tamil Nadu and Kerala clustered with K. lacca in phylogenetic tree. Hence, there is a need for a detailed survey in unexplored parts. The study also revealed that Madurai and Thrissur accessions did not perform well in Jharkhand. Agroecological region wise survey and maintenance is suggested for conserving lac associated faunal and floral diversity.Not Availabl

Natural Clay-Modified Piezocatalytic Membrane for Efficient Removal of Coliform Bacteria from Wastewater

In the modern era, water pollution, especially from industries, agricultural farms, and residential areas, is caused by the release of a large scale of heavy metals, organic pollutants, chemicals, etc., into the environment, posing a serious threat to aquatic ecosystems and nature. Moreover, untreated sewage waste discharged directly into nearby water bodies can cause various diseases to mankind due to the high load of fecal coliform bacteria. This work demonstrates the development of a biocompatible, cost-effective, highly robust, efficient, flexible, freestanding, and reusable membrane using naturally formed biocompatible kaolinite clay-doped poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP) for effective piezodynamic destruction of coliform bacteria. In this study, Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) have been used to evaluate the mechanical stimulus-responsive antibacterial efficacy of the nanocomposite membrane. The membrane can effectively eradicate nearly 99% viable E. coli and 97% E. faecalis within a span of 40 min under mechanical stimulation (soft ultrasound ∼15 kHz). To further understand the mechanism, an evaluation of reactive oxygen species and bacterial FESEM was performed. These studies revealed that bacterial cells suffered severe visible cell damage after 40 min of piezocatalysis, elucidating the fact that the synthesized membrane is capable of producing a superior piezodynamic antibacterial effect

Molecular dimensions of <i>r</i>EPclA and its domains.

<p>(<b>A</b>) Magnified view of a representative <i>r</i>EPclA molecule from a rotary shadowing electron micrograph. (<b>B</b>) The same molecule with the background masked out showing the different molecular dimensions analyzed below. (<b>C</b>) Average dimensions obtained from multiple measures on electron micrographs: N···C and rEPclA micrographs; <i>D</i>_C, <i>L</i>_Col and <i>T</i>_Col are averages of 76, 35 and 74 measures, respectively, on three Col–PfC micrographs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037872#pone-0037872-g010" target="_blank">Figure 10A</a>); <i>D</i>_N, <i>L</i>_Coil and <i>T</i>_Coil are averages of 200 measures on one PfN–PCoil micrograph (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037872#pone-0037872-g010" target="_blank">Figure 10B</a>). (D) Histograms showing the distribution of N···C and rEPclA molecules.</p

Rotary shadowing electron microscopy of <i>r</i>EPclB.

<p>(<b>A</b>) Internal structure of a small aggregate of <i>r</i>EPclB molecules. The micrograph suggests multiple flexible molecules, reminiscent of those observed for <i>r</i>EPclA, with dark globular structures (presumably globular domains of PfN, PfC and Pf2), and poorly defined linear structures (presumably stalks containing the PCoil and Col domains). The <i>r</i>EPclB molecules seem to aggregate heavily through one of the globular domains. (<b>B</b>) Possible examples of individual <i>r</i>EPclB molecules observed, isolated from the large aggregates, in some electron micrographs. (<b>C</b>) Interpretation of the observed in terms of three globular domains (PfN, Pf2 and PfC) connected by two stalk regions. Approximate molecular dimensions are shown for comparison purposes with <i>r</i>EPclA.</p

Collagen-like proteins from the genomes of <i>E. coli</i> O157:H7 EDL933 and Sakai strains, and their corresponding prophage locations.

*<p>Defective protein sequence, with frame-loss between domains. The original gene sequence may correspond to two open reading frames.</p

Collagen-like proteins from prophages embedded in the genomes of <i>E. coli</i> O157:H7 and other EHEC strains, referred here as EPclA to EPclD (EHEC Prophage collagen-like A to D).

<p>(<b>A</b>) Domain architectures. The collagen triple helical domains are labelled “Col”, and domains predicted to adopt an α-helical coiled-coil conformation (see text) are labelled “PCoil” (for phage coiled-coils). Key to other domain labels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037872#pone-0037872-t001" target="_blank">Table 1</a>): PfN, phage fibre N-terminal domain; PfC, phage fibre C-terminal domain; PfC2, phage fibre C-terminal domain, variant 2; Pf2, phage fibre repeat 2. (<b>B</b>) Sequence of a representative collagen-like protein with EPclA architecture (<u>ECs2717</u>), from the genome of <i>E. coli</i> O157:H7 Sakai. (<b>C</b>) Sequence of a representative collagen-like protein with EPclB architecture (<u>Z1483</u>), from the genome of <i>E. coli</i> O157:H7 EDL933. Amino acid sequences corresponding to the different predicted domains are colour-coded as in (<b>A</b>).</p

Position-specific amino acid preferences in collagen triple-helical domains of EPclPs, human collagens, and collagen-like proteins from different groups of organisms.

<p>The X and Y letters refer to the consensus sequence pattern (Gly-X-Y)<i>_n</i> characteristic of collagen triple-helical domains. The numbers indicate percentage occupation of the X or Y position by a given amino acid type.</p>*<p>Excluding EPclPs from bacteriophages.</p>†<p>Include molecules such as C1q, mannose binding proteins, collectins, macrophage scavenger receptors, or acetyl cholinesterase, which contain in their sequence a collagen domain but are not formally classified as collagen types.</p