Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na⁺/H⁺ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2.     

Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant's response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other's production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.     

De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17?855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.     

Phylogeography of the spring and fall waves of the H1N1/09 pandemic influenza virus in the United States

Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the world's largest international travel centers.     

Receptor-ligand requirements for increased NK cell polyfunctional potential in slow progressors infected with HIV-1 coexpressing KIR3DL1*h/*y and HLA-B*57

Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.     

Mesostructure of fibrillar bovine serum albumin gels

The mesostructure of bovine serum albumin (BSA) at low pH was investigated. Rheological measurements were performed to determine the critical percolation concentration (c_p). A decreasing c_p with increasing ionic strength was found. Fibrils with a contour length of about 100–300 nm were found using transmission electron microscopy. The measured conversion of monomers into fibrils was independent of ionic strength (0.20–0.30 M). Dilution of BSA samples showed that the aggregation process is reversible and that there exists a critical concentration for the self-assembly of BSA. We explain the decreasing c_p with increasing ionic strength in terms of an adjusted random contact model.     

Are Niemann‐Pick type C proteins key players in cnidarian–dinoflagellate endosymbioses?

The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host–symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol‐trafficking proteins Niemann‐Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene in contrast to the two genes (NPC1 and NPC1L1) present in vertebrate genomes. While only one NPC2 gene is present in many metazoans, this gene has been duplicated in cnidarians, and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2‐d) was upregulated in symbiotic relative to aposymbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont‐containing tissue) than in the epidermis. We performed immunolabelling experiments on tentacle cross sections and demonstrated that the AvNPC2‐d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2‐d gene expression was strongly downregulated during stress. These data suggest that AvNPC2‐d is involved in both the stability and dysfunction of cnidarian–dinoflagellate symbioses.     

The Conserved nhaAR Operon Is Drastically Divergent between B2 and Non-B2 Escherichia coli and Is Involved in Extra-Intestinal Virulence

The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains.     

Modelling Interactions between Forest Pest Invasions and Human Decisions Regarding Firewood Transport Restrictions

The invasion of nonnative, wood-boring insects such as the Asian longhorned beetle (A. glabripennis) and the emerald ash borer (A. planipennis) is a serious ecological and economic threat to Canadian deciduous and mixed-wood forests. Humans act as a major vector for the spread of these pests via firewood transport, although existing models do not explicitly capture human decision-making regarding firewood transport. In this paper we present a two-patch coupled human-environment system model that includes social influence and long-distance firewood transport and examines potential strategies for mitigating pest spread. We found that increasing concern regarding infestations (f) significantly reduced infestation. Additionally it resulted in multiple thresholds at which the intensity of infestation in a patch was decreased. It was also found that a decrease in the cost of firewood purchased in the area where it is supposed to be burned (C _l) resulted in an increased proportion of local-firewood strategists, and a 67% decrease in C _l from $6.75 to $4.50 was sufficient to eliminate crosspatch infestation. These effects are synergistic: increasing concern through awareness and education campaigns acts together with reduced firewood costs, thereby reducing the required threshold of both awareness and economic incentives. Our results indicate that the best management strategy includes a combination of public education paired with firewood subsidization.