Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes

Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.     

BiNChE: A web tool and library for chemical enrichment analysis based on the ChEBI ontology

Background

Ontology-based enrichment analysis aids in the interpretation and understanding of large-scale biological data. Ontologies are hierarchies of biologically relevant groupings. Using ontology annotations, which link ontology classes to biological entities, enrichment analysis methods assess whether there is a significant over or under representation of entities for ontology classes. While many tools exist that run enrichment analysis for protein sets annotated with the Gene Ontology, there are only a few that can be used for small molecules enrichment analysis.

Results

We describe BiNChE, an enrichment analysis tool for small molecules based on the ChEBI Ontology. BiNChE displays an interactive graph that can be exported as a high-resolution image or in network formats. The tool provides plain, weighted and fragment analysis based on either the ChEBI Role Ontology or the ChEBI Structural Ontology.

Conclusions

BiNChE aids in the exploration of large sets of small molecules produced within Metabolomics or other Systems Biology research contexts. The open-source tool provides easy and highly interactive web access to enrichment analysis with the ChEBI ontology tool and is additionally available as a standalone library.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0486-3) contains supplementary material, which is available to authorized users.     

Ant versus bird exclusion effects on the arthropod assemblage of an organic citrus grove

1. Predation‐exclusion experiments have highlighted that top‐down control is pervasive in terrestrial communities, but most of these experiments are simplistic in that they only excluded a single group of predators and the effect of removal was evaluated on a few species from the community. The main goal of our study was to experimentally establish the relative effects of ants and birds on the same arthropod assemblage of canopy trees. 2. We conducted 1‐year long manipulative experiments in an organic citrus grove intended to quantify the independent effects of bird and ant predators on the abundance of arthropods. Birds were excluded with plastic nets whereas ants were excluded with sticky barriers on the trunks. The sticky barrier also excluded other ground dwelling insects, like the European earwig Forficula auricularia L. 3. Both the exclusion of ants and birds affected the arthropod community of the citrus canopies, but the exclusion of ants was far more important than the exclusion of birds. Indeed, almost all groups of arthropods had higher abundance in ant‐excluded than in control trees, whereas only dermapterans were more abundant in bird‐excluded than in control trees. A more detailed analysis conducted on spiders also showed that the effect of ant exclusion was limited to a few families rather than being widespread over the entire diverse spectrum of spiders. 4. Our results suggest that the relative importance of vertebrate and invertebrate predators in regulating arthropod populations largely depends on the nature of the predator–prey system.     

Stable Equilibria at Two Loci in Populations with Large Selfing Rates

The equilibrium structure of two-locus, two-allele models with very large selfing rates is found using perturbation techniques. For free recombination, r = 1/2, the following results hold. If the heterozygotes do not have at least an approximate 30% advantage in fitness relative to homozygotes, a stable equilibrium with all alleles present is possible only if all of the homozygote fitnesses differ at most by approximately the outcrossing rate, t, and all stable polymorphic equilibria have disequilibrium values, D, that are at most on the order of the outcrossing rate. Once the heterozygote fitnesses are above the threshold, there are stable equilibria possible with D near its maximum possible value. The results show that the observed disequilibria in highly selfed plant populations are not likely to result from selection leading to an equilibrium.     

Adaptive amplification and point mutation are independent mechanisms: evidence for various stress-inducible mutation mechanisms

“Adaptive mutation” denotes a collection of processes in which cells respond to growth-limiting environments by producing compensatory mutants that grow well, apparently violating fundamental principles of evolution. In a well-studied model, starvation of stationary-phase lac⁻ Escherichia coli cells on lactose medium induces Lac⁺ revertants at higher frequencies than predicted by usual mutation models. These revertants carry either a compensatory frameshift mutation or a greater than 20-fold amplification of the leaky lac allele. A crucial distinction between alternative hypotheses for the mechanisms of adaptive mutation hinges on whether these amplification and frameshift mutation events are distinct, or whether amplification is a molecular intermediate, producing an intermediate cell type, in colonies on a pathway to frameshift mutation. The latter model allows the evolutionarily conservative idea of increased mutations (per cell) without increased mutation rate (by virtue of extra gene copies per cell), whereas the former requires an increase in mutation rate, potentially accelerating evolution. To resolve these models, we probed early events leading to rare adaptive mutations and report several results that show that amplification is not the precursor to frameshift mutation but rather is an independent adaptive outcome. (i) Using new high-resolution selection methods and stringent analysis of all cells in very young (micro)colonies (500–10,000 cells), we find that most mutant colonies contain no detectable lac-amplified cells, in contrast with previous reports. (ii) Analysis of nascent colonies, as young as the two-cell stage, revealed mutant Lac⁺ cells with no lac-amplified cells present. (iii) Stringent colony-fate experiments show that microcolonies of lac-amplified cells grow to form visible colonies of lac-amplified, not mutant, cells. (iv) Mutant cells do not overgrow lac-amplified cells in microcolonies fast enough to mask the lac-amplified cells. (v) lac-amplified cells are not SOS-induced, as was proposed to explain elevated mutation in a sequential model. (vi) Amplification, and not frameshift mutation, requires DNA polymerase I, demonstrating that mutation is separable from amplification, and also illuminating the amplification mechanism. We conclude that amplification and mutation are independent outcomes of adaptive genetic change. We suggest that the availability of alternative pathways for genetic/evolutionary adaptation and clonal expansion under stress may be exploited during processes ranging from the evolution of drug resistance to cancer progression.     

Influence of leptin on neurotransmitter overflow from the rat brain in vitro.

The 16-kDa polypeptide hormone, leptin along with the neurotransmitters noradrenaline and serotonin (5-HT) have important physiological roles in the regulation of a number of neuroendocrine actions particularly feeding. Leptin receptor mRNA and immunoreactivity has been reported in various brain regions, while recent studies suggest that leptin is released from the human brain. This study investigated the interactions between leptinergic and neurotransmitter systems of the rat brain in vitro. Techniques were established to simultaneously monitor the release of endogenous noradrenaline and its metabolite 3,4 dihydroxyphenylglycol (DHPG), and 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) from the rat brain. The neuromodulatory action of leptin (0.2 and 3 nM) on the overflow of noradrenaline and DHPG from the medulla and hypothalamus was examined. The effect of leptin on 5-HT and 5-HIAA overflow from the hypothalamus was also investigated. Administration of 0.2 and 3 nM leptin significantly increased medullary noradrenaline overflow to 172% and 174% of basal levels, respectively. Leptin had no significant effect on hypothalamic noradrenaline overflow, while leptin perfusion induced a significant increase in 5-HIAA overflow from the hypothalamus. This study lends support to the notion of a complex interaction of the leptinergic and brain neurotransmitters involved in the control of feeding and energy metabolism.     

质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium)细胞壁激发子诱导人参细胞产生激发反应(英文)

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性。这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导。Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproleins)的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30 min 后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人