Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.Vesicular stomatitis virus (VSV), the prototypic rhabdovirus within the family Rhabdoviridae and the order Mononegavirales, is an enveloped virus with a negative-stranded RNA genome of 11,161 nucleotides. The viral genome encodes five proteins, namely, the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the large polymerase protein (L) (35). The genome is present within the virion core as a ribonucleoprotein (RNP) or nucleocapsid (NC) complex tightly encapsidated by the N protein and associated with the viral RNA-dependent RNA polymerase, a multiprotein complex of the viral L and P proteins. The G protein forms spikes on the viral envelope, binds to cell surface receptors, and plays a role in entry of virus into susceptible cells. The M protein is multifunctional; it plays a role in virus assembly and is responsible for cytopathogenesis observed in virus-infected cells (6).Studies on viral protein transport and virus motility in infected cells have been facilitated by imaging of fluorescent proteins fused to viral structural proteins (for reviews, see references 8 and 23). Recent advances in imaging techniques, coupled with the ability to genetically tag viral structural proteins with fluorescent proteins or to label viral membranes with lipophilic dyes, have allowed studies of the dynamic events of virus entry as well as of virus-cell interactions (17, 27-29, 31, 34, 40, 43, 51, 53). For enveloped viruses, the hallmark event of infection is the fusion of the viral envelope and the release of the NC (or RNP) into the cytoplasm. To examine the infection process by fluorescence microscopy and to distinguish between the enveloped virion and the uncoated NC, it is essential to differentially label the viral envelope and the NC core. Dually fluorescent viruses in which the viral core component, such as the NC or the RNP, is labeled with one fluorescent color and the envelope component is labeled with another color are thus powerful reagents for studies of virus entry and NC uncoating during early stages of infection as well as for studies of virus assembly during late stages of infection. Recently, dually fluorescent rabies virus (27) and human immunodeficiency virus (HIV) (9, 30) have been generated successfully. Using the dually fluorescent rabies virus, it was demonstrated that complete virus particles are transported in a retrograde manner (27).Successful recovery of a recombinant VSV encoding the P protein fused in frame with enhanced green fluorescent protein (PeGFP) allowed us to track the intracellular transport of viral NCs by live-cell imaging (14). This study demonstrated that microtubules were involved in viral NC transport toward the cell periphery (14), presumably to the plasma membrane for virus assembly. Whether the M protein interacts with the viral NCs before transport to the plasma membrane or at the plasma membrane prior to virus assembly remains a fundamental question in VSV assembly. Previous studies have shown that the M protein and the NCs do interact in vitro and in vivo (11, 12, 26), although more recent studies suggest that such interactions may occur only at the plasma membrane (18, 54). To examine the events of virus entry, uncoating, and also assembly, we wanted to generate a dually fluorescent VSV encoding PeGFP and monomeric red fluorescent protein fused in frame with the M protein at its carboxy terminus (MmRFP). Although repeated attempts to recover VSV with the fluorescently tagged M protein in place of wild-type (wt) M were unsuccessful, viruses encoding the fluorescently tagged M protein could be recovered when it was inserted as an extra cistron at the G-L gene junction. Further use of this virus for studies of virus entry, uncoating, and egress was limited because the MmRFP fusion protein was not incorporated into the virions.Recently, a new method of genetic tagging of proteins for fluorescence imaging was developed wherein the protein is tagged with a relatively smaller tetracysteine (tc) motif (CCPGCC). This motif can be recognized specifically by membrane-permeable biarsenical dyes that fluoresce when covalently bound to the cysteine pairs in the tc motif (1, 24, 39). Such a small tag can be fused to the protein of interest with minimal disruption of protein function. This is a powerful approach for real-time visualization of nascent protein synthesis and trafficking, as the existing and newly synthesized pools of proteins can be labeled differentially with the two fluorescent biarsenical dyes, FlAsH (green) and ReAsH (red) (38, 48). Using a tc-tagged M protein (Mtc) encoded in place of the wt M protein in the VSV genome, we rescued recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) and demonstrated that the Mtc was incorporated into infectious virions in amounts similar to that observed for M protein in wt VSV. Moreover, dynamic imaging of newly synthesized M protein by sequential labeling with the two biarsenical dyes revealed that the M protein is transported from the site of synthesis inside the cytoplasm to the plasma membrane in less than 30 min. We have also shown that the M protein reaches the plasma membrane independent of NCs and the microtubules. Additionally, our results show that following adsorption, entry and uncoating of VSV in the infected cells occur with a half-life of approximately 28 min.     

Exploring hydrogen bond in the excited state leading toward intramolecular proton transfer: detailed analysis of the structure and charge density topology along the reaction path using QTAIM

Excited state intramolecular proton transfer (ESIPT) reaction along the O-H[Symbol: see text][Symbol: see text][Symbol: see text][Symbol: see text]O hydrogen bond of o-hydroxy benzaldehyde (OHBA), methyl salicylate (MS) and salicylic acid (SA) was investigated by ab-initio quantum chemical calculation and theory of atoms and molecules (QTAIM) for the first time. Variation in several geometric as well as QTAIM parameters along the reaction coordinate was monitored in the fully relaxed excited state potential energy curve (PEC) obtained from intrinsic reaction coordinate (IRC) analysis. Although, the excited state barrier height for the forward reaction (?E (0) (#) ) reduces substantially in all the systems, MS and SA do not show any obvious asymmetry for proton transfer. For MS and SA, the crossover of the bond index as well as the lengths of the participating bonds at the saddle point is assigned due to this symmetry in accordance with bond energy - bond order (BEBO) model, which does not hold true in OHBA both in the ground and excited states. Bond ellipticity, covalent and metallic character were examined for different structures along the reaction path within the QTAIM framework. The QTAIM analysis was found to be able to uniquely distinguish between the ground and excited states of the OHBA molecule as well as both determining the effects on the bonding character of adding different substituent groups and differentiating between the ESIPT reactions in the SA and MS molecules.     

Comparative molecular field analysis (CoMFA) of phthalazine derivatives as phosphodiesterase IV inhibitors

A comparative molecular field analysis (CoMFA) of phthalazine class of phosphodiesterase IV (PDE IV) inhibitors has been performed to correlate their chemical structures with their observed biological activity. A statistically valid model with good correlative and predictive power is reported. The leave one out cross-validation study gave cross-validation r(2)(cv) of value 0.507 at six optimum components and conventional r(2) of value 0.98. The predictive ability of the model was tested by predicting the seven molecules belonging to the test set giving predictive correlation coefficient of 0.59. This model is potentially helpful in the design of novel and more potent PDE IV inhibitors.     

Bottom-up effects of species diversity on the functioning and stability of food webs

1. The importance of species diversity for the stability of populations, communities and ecosystem functions is a central question in ecology. 2. Biodiversity experiments have shown that diversity can impact both the average and variability of stocks and rates at these levels of ecological organization in single trophic-level ecosystems. Whether these impacts hold in food webs and across trophic levels is still unclear. 3. We asked whether resource species diversity, community composition and consumer feeding selectivity in planktonic food webs impact the stability of resource or consumer populations, community biomass and ecosystem functions. We also tested the relative importance of resource diversity and community composition. 4. We found that resource diversity negatively affected resource population stability, but had no effect on consumer population stability, regardless of the consumer's feeding selectivity. Resource diversity had positive effects on most ecosystem functions and their stability, including primary production, resource biomass and particulate carbon, nitrogen and phosphorus concentrations. 5. Community composition, however, generally explained more variance in population, community and ecosystem properties than species diversity per se. This result points to the importance of the outcomes of particular species interactions and individual species' effect traits in determining food web properties and stability. 6. Among the stabilizing mechanisms tested, an increase in the average resource community biomass with increasing resource diversity had the greatest positive impact on stability. 7. Our results indicate that resource diversity and composition are generally important for the functioning and stability of whole food webs, but do not have straightforward impacts on consumer populations.     

Selective activation of peripheral blood T cell subsets by endotoxin infusion in healthy human subjects corresponds to differential chemokine activation

Although activation of human innate immunity after endotoxin administration is well established, in vivo endotoxin effects on human T cell responses are not well understood. Most naive human T cells do not express receptors for LPS, but can respond to endotoxin-induced mediators such as chemokines. In this study, we characterized the in vivo response of peripheral human T cell subsets to endotoxin infusion by assessing alterations in isolated T cells expressing different phenotypes, intracellular cytokines, and systemic chemokines concentration, which may influence these indirect T cell responses. Endotoxin administration to healthy subjects produced T cell activation as confirmed by a 20% increase in intracellular IL-2, as well as increased CD28 and IL-2R alpha-chain (CD25) expression. Endotoxin induced indirect activation of T cells was highly selective among the T cell subpopulations. Increased IL-2 production (36.0 +/- 3.7 to 53.2 +/- 4.1) vs decreased IFN-gamma production (33.8 +/- 4.2 to 19.1 +/- 3.2) indicated selective Th1 activation. Th2 produced IL-13 was minimally increased. Differentially altered chemokine receptor expression also indicated selective T cell subset activation and migration. CXCR3+ and CCR5+ expressing Th1 cells were decreased (CXCR3 44.6 +/- 3.2 to 33.3 +/- 4.6 and CCR5 24.8 +/- 2.3 to 12 +/- 1.4), whereas plasma levels of their chemokine ligands IFN-gamma-inducible protein 10 and MIP-1alpha were increased (61.4 +/- 13.9 to 1103.7 +/- 274.5 and 22.8 +/- 6.2 to 55.7 +/- 9.5, respectively). In contrast, CCR4+ and CCR3 (Th2) proportions increased or remained unchanged whereas their ligands, eotaxin and the thymus and activation-regulated chemokine TARC, were unchanged. The data indicate selective activation among Th1 subpopulations, as well as differential Th1/Th2 activation, which is consistent with a selective induction of Th1 and Th2 chemokine ligands.     

Three-dimensional quantitative structure (3-D QSAR) activity relationship studies on imidazolyl and N-pyrrolyl heptenoates as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitors by comparative molecular similarity indices analysis (CoMSIA)

A comparative molecular similarity indices analysis (CoMSIA) of a set of 29 imidazolyl and N-pyrrolyl heptenoates have been performed to find out the structural requirements for 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitory activity. The HMG like side chain, a common moiety of statins, was used to align the molecules. The results guide to design new chemical entities with high potency.     

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.     

Characterization of Functional,Safety, and Probiotic Properties of Enterococcus faecalis AG5 Isolated From Wistar Rat,Demonstrating Adherence to HCT 116 Cells and Gastrointestinal Survivability

Gut microbiota remains a prominent source for a diverse range of potential probiotics. In this context, the current study explored the rectal region of experimental Wistar rat for the isolation of potent probiotic. Sixteen lactic acid bacteria (LAB), from rectal swab of Wistar rats, were subjected to evaluation of probiotic properties. Among all, AG5 was found unique with consistent probiotic properties and was further identified as Enterococcus faecalis AG5 (NCBI accession number KT248537) using 16S rDNA sequencing, followed by BLAST analysis. Since the Enterococci strains inhibit various food-borne pathogens efficiently while proving itself as a safe probiotic candidate, the study further evaluated the safety of the strain AG5 using primer specific PCR amplification which revealed the existence of gene encoding gelE, asa1, efaA, ace, vanA, and vanB and negative for cylA, hyl, and esp respectively. SEM analysis confirmed the adherence ability of AG5 to HCT 116 cells. Adherence was found to be non-colonial and scattered manner. Furthermore, the strain demonstrated a significant survivability during simulated gastrointestinal transit. Taken together, the E. faecalis AG5 was found potential probiotic candidate with future implication in both food and health industry.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.