Myxoma Virus Encodes an α2,3-Sialyltransferase That Enhances Virulence

A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galβ1,3(4)GlcNAc α2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that α2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host’s defenses against infection are discussed.     

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.     

A Simplified Approach for the Molecular Classification of Glioblastomas

Glioblastoma (GBM) is the most common malignant primary brain tumors in adults and exhibit striking aggressiveness. Although GBM constitute a single histological entity, they exhibit considerable variability in biological behavior, resulting in significant differences in terms of prognosis and response to treatment. In an attempt to better understand the biology of GBM, many groups have performed high-scale profiling studies based on gene or protein expression. These studies have revealed the existence of several GBM subtypes. Although there remains to be a clear consensus, two to four major subtypes have been identified. Interestingly, these different subtypes are associated with both differential prognoses and responses to therapy. In the present study, we investigated an alternative immunohistochemistry (IHC)-based approach to achieve a molecular classification for GBM. For this purpose, a cohort of 100 surgical GBM samples was retrospectively evaluated by immunohistochemical analysis of EGFR, PDGFRA and p53. The quantitative analysis of these immunostainings allowed us to identify the following two GBM subtypes: the “Classical-like” (CL) subtype, characterized by EGFR-positive and p53- and PDGFRA-negative staining and the “Proneural-like” (PNL) subtype, characterized by p53- and/or PDGFRA-positive staining. This classification represents an independent prognostic factor in terms of overall survival compared to age, extent of resection and adjuvant treatment, with a significantly longer survival associated with the PNL subtype. Moreover, these two GBM subtypes exhibited different responses to chemotherapy. The addition of temozolomide to conventional radiotherapy significantly improved the survival of patients belonging to the CL subtype, but it did not affect the survival of patients belonging to the PNL subtype. We have thus shown that it is possible to differentiate between different clinically relevant subtypes of GBM by using IHC-based profiling, a method that is advantageous in its ease of daily implementation and in large-scale clinical application.     

一种简易SNP检测方法的建立

目的：建立一种快速、简单的SNP（Single Nucleotide Polymorphisms）检测方法。方法：设计带生物素标记的扩增引物对检测用具有单碱基差异的野生型和突变型靶序列分别进行扩增,然后通过紫外交联的方式将相应检测靶序列的探针固定在硝酸纤维素膜上,借助Taq酶完成膜上单引物延伸,从而对探针捕获的靶序列进行延伸固定在膜上,最后使用生物素．亲和素酶联显色（ABs-ELISA）反应肉眼观察结果。结果：阳性和阴性对照探针显示正常。野生型探针和突变型探针能够分别特异性结合靶序列,并通过生物素和亲和索显色系统放大为一种肉眼可判断结果的检测形式。结论：建立了一种基于硝酸纤维素膜载体上进行核酸扩增的SNP检测方法。     

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.     

A Hepatitis C Virus NS5A Phosphorylation Site That Regulates RNA Replication

The hepatitis C virus NS5A protein is essential for RNA replication and virion assembly. NS5A is phosphorylated on multiple residues during infections, but these sites remain uncharacterized. Here we identify serine 222 of genotype 2a NS5A as a phosphorylation site that functions as a negative regulator of RNA replication. This site is a component of the hyperphosphorylated form of NS5A, which is in good agreement with previous observations that hyperphosphorylation negatively affects replication.     

Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.     

Glycine-Like Modulation of N-Methyl-d-Aspartate Receptors by a Monoclonal Antibody That Enhances Long-Term Potentiation

We have identified a monoclonal antibody, B6B21, that significantly elevates long-term potentiation when applied to CA1 pyramidal cell apical dendrites in rat hippocampal slices and characterized its binding to N-methyl-D-aspartate-receptor complexes using extensively washed hippocampal membranes. Five micrograms of affinity-purified B6B21 per 100 micrograms of membranes gave a two- to threefold elevation in N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) binding. When [3H]TCP binding was stimulated by the combined addition of maximal concentrations of glutamate, glycine, and magnesium, B6B21 no longer stimulated [3H]TCP binding. Like glycine, B6B21 enhanced the effect of N-methyl-D-aspartate and glutamate in stimulating [3H]TCP binding. Moreover, B6B21 reversed 7-chlorokynurenic acid inhibition of [3H]TCP binding, but it had no effect on the inhibition of [3H]TCP binding by D-(-)-2-amino-5-phosphonovaleric acid. B6B21 increased the rate of association and dissociation of [3H]TCP, but had no effect on equilibrium binding. Glutamate, but not glycine, however, increased B6B21-enhancement of [3H]TCP association and dissociation. B6B21 binding at strychnine-insensitive glycine sites was confirmed by direct measurement of [3H]glycine binding. These results suggest that B6B21 binds directly to N-methyl-D-aspartate receptors and displays properties similar to glycine.     

A Population Density Approach That Facilitates Large-Scale Modeling of Neural Networks: Analysis and an Application to Orientation Tuning

We explore a computationally efficient method of simulating realistic networks of neurons introduced by Knight, Manin, and Sirovich (1996) in which integrate-and-fire neurons are grouped into large populations of similar neurons. For each population, we form a probability density that represents the distribution of neurons over all possible states. The populations are coupled via stochastic synapses in which the conductance of a neuron is modulated according to the firing rates of its presynaptic populations. The evolution equation for each of these probability densities is a partial differential-integral equation, which we solve numerically. Results obtained for several example networks are tested against conventional computations for groups of individual neurons.We apply this approach to modeling orientation tuning in the visual cortex. Our population density model is based on the recurrent feedback model of a hypercolumn in cat visual cortex of Somers et al. (1995). We simulate the response to oriented flashed bars. As in the Somers model, a weak orientation bias provided by feed-forward lateral geniculate input is transformed by intracortical circuitry into sharper orientation tuning that is independent of stimulus contrast.The population density approach appears to be a viable method for simulating large neural networks. Its computational efficiency overcomes some of the restrictions imposed by computation time in individual neuron simulations, allowing one to build more complex networks and to explore parameter space more easily. The method produces smooth rate functions with one pass of the stimulus and does not require signal averaging. At the same time, this model captures the dynamics of single-neuron activity that are missed in simple firing-rate models.     

A Mutant Influenza Virus That Uses an N1 Neuraminidase as the Receptor-Binding Protein

In the vast majority of influenza A viruses characterized to date, hemagglutinin (HA) is the receptor-binding and fusion protein, whereas neuraminidase (NA) is a receptor-cleaving protein that facilitates viral release but is expendable for entry. However, the NAs of some recent human H3N2 isolates have acquired receptor-binding activity via the mutation D151G, although these isolates also appear to retain the ability to bind receptors via HA. We report here the laboratory generation of a mutation (G147R) that enables an N1 NA to completely co-opt the receptor-binding function normally performed by HA. Viruses with this mutant NA grow to high titers even in the presence of extensive mutations to conserved residues in HA''s receptor-binding pocket. When the receptor-binding NA is paired with this binding-deficient HA, viral infectivity and red blood cell agglutination are blocked by NA inhibitors. Furthermore, virus-like particles expressing only the receptor-binding NA agglutinate red blood cells in an NA-dependent manner. Although the G147R NA receptor-binding mutant virus that we characterize is a laboratory creation, this same mutation is found in several natural clusters of H1N1 and H5N1 viruses. Our results demonstrate that, at least in tissue culture, influenza virus receptor-binding activity can be entirely shifted from HA to NA.     

A High Throughput Genotyping Approach Reveals Distinctive Autosomal Genetic Signatures for European and Near Eastern Wild Boar

The lack of a Near Eastern genetic signature in modern European porcine breeds indicates that, although domestic pigs from the Fertile Crescent entered Europe during the Neolithic, they were completely replaced by their European counterparts in a short window of time. Whilst the absence of such genetic signature has been convincingly demonstrated at the mitochondrial level, variation at the autosomal genomes of European and Near Eastern Sus scrofa has not been compared yet. Herewith, we have explored the genetic relationships among 43 wild boar from Europe (N = 21), Near East (N = 19) and Korea (N = 3), and 40 Iberian (N = 16), Canarian (N = 4) and Mangalitza (N = 20) pigs by using a high throughput SNP genotyping platform. After data filtering, 37,167 autosomal SNPs were used to perform population genetics analyses. A multidimensional scaling plot based on genome-wide identity-by-state pairwise distances inferred with PLINK showed that Near Eastern and European wild boar populations are genetically differentiated. Maximum likelihood trees built with TreeMix supported this conclusion i.e. an early population split between Near Eastern and European Sus scrofa was observed. Moreover, analysis of the data with Structure evidenced that the sampled Iberian, Canarian and Mangalitza pigs did not carry any autosomal signature compatible with a Near Eastern ancestry, a finding that agrees well with previous mitochondrial studies.