Polysaccharide-rich fraction of Termitomyces eurhizus accelerate healing of indomethacin induced gastric ulcer in mice

The current study aims to determine the healing activity of water soluble polysaccharide-rich fraction of a wild mushroom, Termitomyces eurhizus (TEps) against the indomethacin induced gastric ulceration in mice model. Gastric tissue histology, myeloperoxidase (MPO) activity, cyclooxygenases (COX) 1 and 2 expression, prostaglandin E₂ (PGE₂) synthesis, and modulation of pro/anti inflammatory cytokines expression were studied for this purpose. Histological study shows that TEps (20 mg/kg) effectively healed the gastric ulceration. Based on biochemical results, the healing capacities of TEps could be attributed to reduction of MPO activity and protection of mucosal mucin content. Enhanced synthesis of PGE₂ by modulation of COX-1 and COX-2 expression and a prominent shift of cytokines expression from pro (TNF-α, IL-1ß) to anti inflammatory (IL-10) side are also held responsible for ulcer healing. The preliminary study highlights the anti-ulcerogenic property of polysaccharide-rich fraction of Termitomyces eurhizus and opens an alternative cure for NSAID induced gastroduodenal diseases.     

Identification of potential targets in Staphylococcus aureus N315 using computer aided protein data analysis

Staphylococcus aureus is a gram positive bacterium, responsible for both community-acquired and hospital-acquired infection, resulting in a mortality rate of 39%. 43.2% resistance to methicilin and emerging resistance to Fluroquinolone and Oxazolidinone, have evoked the necessity of the establishment of alternative and effective therapeutic approach to treat this bacteria. In this computational study, various database and online software are used to determine some specific targets of Staphylococcus aureus N315 other than those used by Penicillin, Quinolone and Oxazolidinone. For this purpose, among 302 essential proteins, 101 nonhomologous proteins were accrued and 64 proteins which are unique in several metabolic pathways of S. aureus were isolated by using metabolic pathway analysis tools. Furthermore, 7 essentially unique enzymes involved in exclusive metabolic pathways were revealed by this research, which can be potential drug target. Along with these important enzymes, 15 non-homologous proteins located on membrane were identified, which can play a vital role as potential therapeutic targets for the future researchers.     

Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe–S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N–S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.     

1H-1H correlations across N–H•••N hydrogen bonds in nucleic acids

In 2HJ(NN)-COSY experiments, which correlate protons with donor/acceptor nitrogens across Nd...HNa bonds, the receptor nitrogen needs to be assigned in order to unambiguously identify the hydrogen bond. For many situations this is a non-trivial task which is further complicated by poor dispersion of (Na,Nd) resonances. To address these problems, we present pulse sequences to obtain direct, internucleotide correlations between protons in uniformly 13C/15N labeled nucleic acids containing Nd...HNa hydrogen bonds. Specifically, the pulse sequence H2(N1N3)H3 correlates H2(A,omega1):H3(U,omega2) protons across Watson-CrickA-U and mismatched G.A base pairs, the sequences H5(N3N1)H1/H6(N3N1)H1 correlate H5(C,omega1)/H6(C,omega1):H1(G,omega2) protons across Watson-Crick G-C base pairs, and the H2(N2N7)H8 sequence correlates NH2(G,A,C;omega1):H8(G,A;omega2) protons across G.G, A.A, sheared G.A and other mismatch pairs. These 1H-1H connectivities circumvent the need for independent assignment of the donor/acceptor nitrogen and related degeneracy issues associated with poorly dispersed nitrogen resonances. The methodology is demonstrated on uniformly 13C/15N labeled samples of (a) an RNA regulatory element involving the HIV-1 TAR RNA fragment, (b) a multi-stranded DNA architecture involving a G.(C-A) triad-containing G-quadruplex and (c) a peptide-RNA complex involving an evolved peptide bound to the HIV-1 Rev response element (RRE) RNA fragment.     

Sensitivity enhanced NMR spectroscopy by quenching scalar coupling mediated relaxation: Application to the direct observation of hydrogen bonds in 13C/15N-labeled proteins

In this paper, we demonstrate that the sensitivity of triple-resonance NMR experiments can be enhanced significantly through quenching scalar coupling mediated relaxation by using composite-pulse decoupling (CPD) or an adiabatic decoupling sequence on aliphatic, in particular alpha-carbons in ¹³C/¹⁵N-labeled proteins. The CPD-HNCO experiment renders 50% sensitivity enhancement over the conventional CT-HNCO experiment performed on a 12 kDa FK506 binding protein, when a total of 266 ms of amide nitrogen–carbonyl carbon defocusing and refocusing periods is employed. This is a typical time period for the direct detection of hydrogen bonds in proteins via trans-hydrogen bond ^3h J _NC couplings. The experimental data fit theoretical analysis well. The significant enhancement in sensitivity makes the experiment more applicable to larger-sized proteins without resorting to perdeuteration.     

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.     

Direct interaction of baculovirus capsid proteins VP39 and EXON0 with kinesin-1 in insect cells determined by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in the nucleus of insect cells to produce nucleocapsids, which are transported from the nucleus to the plasma membrane for budding through GP64-enriched areas to form budded viruses. However, little is known about the anterograde trafficking of baculovirus nucleocapsids in insect cells. Preliminary confocal scanning laser microscopy studies showed that enhanced green fluorescent protein (EGFP)-tagged nucleocapsids and capsid proteins aligned and colocalized with the peripheral microtubules of virus-infected insect cells. A colchicine inhibition assay of virus-infected insect cells showed a significant reduction in budded virus production, providing further evidence for the involvement of microtubules and suggesting a possible role of kinesin in baculovirus anterograde trafficking. We investigated the interaction between AcMNPV nucleocapsids and kinesin-1 with fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) and show for the first time that AcMNPV capsid proteins VP39 and EXON0, but not Orf1629, interact with the tetratricopeptide repeat (TPR) domain of kinesin. The excited-state fluorescence lifetime of EGFP fused to VP39 or EXON0 was quenched from 2.4 ± 1 ns to 2.1 ± 1 ns by monomeric fluorescent protein (mDsRed) fused to TPR (mDsRed-TPR). However, the excited-state fluorescence lifetime of an EGFP fusion of Orf1629 remained unquenched by mDsRed-TPR. These data indicate that kinesin-1 plays an important role in the anterograde trafficking of baculovirus in insect cells.