The 3' untranslated regions of influenza genomic sequences are 5'PPP-independent ligands for RIG-I

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to ~300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.     

In vitro antibacterial potential of Eugenia jambolana seed extracts against multidrug-resistant human bacterial pathogens

The present study was carried out to evaluate the possible in vitro antibacterial potential of extracts of Eugenia jambolana seeds against multidrug-resistant human bacterial pathogens. Agar well diffusion and microbroth dilution assay methods were used for antibacterial susceptibility testing. Kill-kinetics study was done to know the rate and extent of bacterial killing. Phytochemical analysis and TLC-bioautography were performed by colour tests to characterize the putative compounds responsible for this antibacterial activity. Cytotoxic potential was evaluated on human erythrocytes by haemolytic assay method and acute oral toxicity study was done in mice. The plant extracts demonstrated varying degrees of strain specific antibacterial activity against all the test isolates. Further, ethyl acetate fraction obtained from fractionation of most active ethanol extract showed maximum antibacterial effect against all the test isolates. Phytochemical analysis and TLC-bioautography of ethyl acetate fraction revealed that phenolics were the major active phytoconstituents. Ethyl acetate fraction also demonstrated no haemolytic activity on human erythrocytes and no gross behavioural changes as well as toxic symptoms were observed in mice at recommended dosage level. The results provide justification for the use of E. jambolana in folk medicine to treat various infectious diseases and may contribute to the development of novel antimicrobial agents for the treatment of infections caused by these drug-resistant bacterial pathogens.     

Spectrum of MTHFR gene SNPs C677T and A1298C: a study among 23 population groups of India

Elevated homocysteine is a risk factor for many complex disorders. The role of methylenetetrahydrofolate reductase (MTHFR) gene in methylation of homocysteine makes it one of the most important candidate genes for these disorders. Considering the heterogeneity in its distribution in world populations, we screened MTHFR C677T and A1298C single nucleotide polymorphisms in a total of 23 Indian caste, tribal and religious population groups from five geographical regions of India and belonging to four major linguistic groups. The frequencies of MTHFR 677T and 1298C alleles were found to be 10.08 and 20.66%, respectively. MTHFR homozygous genotype 677TT was absent in eight population groups and homozygous 1298CC was absent in two population groups. 677T allele was found to be highest among north Indian populations with Indo-European tongue and 1298C was high among Dravidian-speaking tribes of east India and south India. The less common mutant haplotype 677T-1298C was observed among seven population groups and overall the frequency of this haplotype was 0.008, which is similar to that of African populations. cis configuration of 677T and 1298C was 0.94%. However, we could not find any individual with four mutant alleles which supports the earlier observation that presence of more than two mutant alleles may decrease the viability of foetus and possibly be a selective disadvantage in the population.     

Expression of synthetic human tumor necrosis factor is toxic to Escherichia coli

The overlap forward-primer-walk polymerase chain reaction method was used to synthesize the human tumor necrosis factor α (hTNF) gene in Escherichia coli cells. Growth curves for hTNF and pET23d vector cultures exhibited slower doubling rates than cultures containing the pET23d vector alone. Cell cultures transformed with hTNF reached peak densities (0.4-0.6 OD₆₀₀) 3 to 4 h post-induction, then decreased prior to growth recovery. This inhibition occurred in the BL21DE3 strain of E. coli, whereas no inhibition of growth and no expression of hTNF were observed in the JM109 strain of E. coli containing hTNF. Induced hTNF cultures hyperexpressed the hTNF-histidine fusion protein for the first 3 to 4 h of induction; subsequently, growth retardation was observed. Hyperexpression and continuous growth were observed in the extracellular expression system. Electron microscopy revealed that accumulation of hTNF inclusion bodies was apparent only in the intracellular expression system — no accumulation was observed with regard to the secretory system. The hTNF-pET23d vector was purified from cells expressing the fusion protein and from cells with recovered growth curves. Sequencing of the vector demonstrated the complete hTNF gene and T7 promoter in cells expressing the fusion protein and mutations of the T7 promoter site from recovered cells.     

Analysis of Delta-Notch interaction by molecular modeling and molecular dynamic simulation studies

Notch is a single-pass transmembrane receptor protein. Delta (member of the DSL protein family), a Notch ligand, is also single-pass transmembrane protein that can interact with Notch to form the Delta-Notch complex. It has been demonstrated that of the 36 Epidermal Growth Factor (EGF) repeats of Notch, 11th and 12th are sufficient to mediate interactions with Delta. Crystal structure of mammalian Notch1 extracellular ligand binding domain shows the presence of 11th and 12th EGF-like repeats. Here a portion of the Drosophila Delta protein, known to interact with Notch extracellular domain, has been modeled using homology modeling. The structure of the Delta-Notch complex was subsequently modeled by protein-docking method using GRAMM. Molecular dynamic simulations of the modeled structures were performed. The probable structures for Delta-Notch complex have been proposed based on interaction energy parameter and planarity studies.     

Interaction between HIV awareness, knowledge, safe sex practice and HIV prevalence: evidence from Botswana

This paper makes methodological and empirical contributions to the study of HIV in the context of Botswana, a country with high HIV prevalence. Comparable evidence is presented from India to put the Botswana results in perspective. The results point to the strong role played by affluence and education in increasing HIV knowledge, promoting safe sex and reducing HIV prevalence. The study presents African evidence on the role played by the empowerment of women in promoting safe sex practices such as condom use. The lack of significant association between HIV prevalence and safe sex practice points to the danger of HIV-infected individuals spreading the disease through multiple sex partners and unprotected sex. This danger is underlined by the finding that females with multiple sex partners are at higher risk of being infected with HIV. These results take on special policy significance in the context of Botswana, where the issue of multiple sex partners has not been adequately addressed in the programme to contain the spread of HIV.     

Influenza A virus neuraminidase protein enhances cell survival through interaction with carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) protein

The influenza virus neuraminidase (NA) protein primarily aids in the release of progeny virions from infected cells. Here, we demonstrate a novel role for NA in enhancing host cell survival by activating the Src/Akt signaling axis via an interaction with carcinoembryonic antigen-related cell adhesion molecule 6/cluster of differentiation 66c (C6). NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3β, and Bcl-2, which affects cell survival, proliferation, migration, differentiation, and apoptosis. siRNA-mediated suppression of C6 resulted in a down-regulation of activated Src, FAK, and Akt, increased apoptosis, and reduced expression of viral proteins and viral titers in influenza virus-infected human lung adenocarcinoma epithelial and normal human bronchial epithelial cells. These findings indicate that influenza NA not only aids in the release of progeny virions, but also cell survival during viral replication.     

Noncoding variations in Cyp24a1 gene are associated with Klotho‐mediated aging phenotypes in different strains of mice

In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging‐related phenotypes caused by Klotho down‐regulation. The hypomorphic Klotho [kl/kl] allele that decreases Klotho expression in C3H, BALB/c, 129, and C57BL/6 genetic backgrounds substantially increases 1,25(OH)2D3 levels in the sera of susceptible C3H, BALB/c, and 129, but not C57BL/6 mice. This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super‐enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6‐kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.     

Flexible Nanopipettes for Minimally Invasive Intracellular Electrophysiology In Vivo

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