Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.     

Characterizing associations and SNP-environment interactions for GWAS-identified prostate cancer risk markers--results from BPC3

Genome-wide association studies (GWAS) have identified multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer risk. However, whether these associations can be consistently replicated, vary with disease aggressiveness (tumor stage and grade) and/or interact with non-genetic potential risk factors or other SNPs is unknown. We therefore genotyped 39 SNPs from regions identified by several prostate cancer GWAS in 10,501 prostate cancer cases and 10,831 controls from the NCI Breast and Prostate Cancer Cohort Consortium (BPC3). We replicated 36 out of 39 SNPs (P-values ranging from 0.01 to 10⁻²⁸). Two SNPs located near KLK3 associated with PSA levels showed differential association with Gleason grade (rs2735839, P = 0.0001 and rs266849, P = 0.0004; case-only test), where the alleles associated with decreasing PSA levels were inversely associated with low-grade (as defined by Gleason grade <8) tumors but positively associated with high-grade tumors. No other SNP showed differential associations according to disease stage or grade. We observed no effect modification by SNP for association with age at diagnosis, family history of prostate cancer, diabetes, BMI, height, smoking or alcohol intake. Moreover, we found no evidence of pair-wise SNP-SNP interactions. While these SNPs represent new independent risk factors for prostate cancer, we saw little evidence for effect modification by other SNPs or by the environmental factors examined.     

No effect of menstrual cycle phase on glucose kinetics and fuel oxidation during moderate-intensity exercise

Resting and exercise fuel metabolism was assessed in three different phases of the menstrual cycle, characterized by different levels of estrogen relative to progesterone: early follicular (EF, low estrogen and progesterone), midfollicular (MF, elevated estrogen, low progesterone), and midluteal (ML, elevated estrogen and progesterone). It was hypothesized that exercise glucose utilization and whole body carbohydrate oxidation would decrease sequentially from the EF to the MF to the ML phase. Normal-weight healthy females, experiencing a regular menstrual cycle, were recruited. Subjects were moderately active but not highly trained. Testing occurred after 3 days of diet control and after an overnight fast (12-13 h). Resting (2 h) and exercise (50% maximal O(2) uptake, 90 min) measurements of whole body substrate oxidation, tracer-determined glucose flux, and substrate and hormone concentrations were made. No significant difference was observed in whole body fuel oxidation during exercise in the three phases (nonprotein respiratory exchange ratio: EF 0.84 +/- 0.01, MF 0.85 +/- 0.01, ML 0.85 +/- 0.01) or in rates of glucose appearance or disappearance. There were, however, significantly higher glucose (P < 0.05) and insulin (P < 0.001) concentrations during the first 45 min of exercise in the ML phase vs. EF and MF phases. In conclusion, whole body substrate oxidation and glucose utilization did not vary significantly across the menstrual cycle in moderately active women, either at rest or during 90 min of moderate-intensity exercise. During the ML phase, however, this similar pattern of substrate utilization was associated with greater glucose and insulin concentrations. Both estrogen and progesterone are elevated during the ML phase of the menstrual cycle, suggesting that one or both of these sex steroids may play a role in this response.     

Sequencing of the Dutch Elm Disease Fungus Genome Using the Roche/454 GS-FLX Titanium System in a Comparison of Multiple Genomics Core Facilities

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.     

Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum

Interactions with bacteria play a major role in immune responses, ecology, and evolution of all animals, but they have been neglected until recently in the case of C. elegans. We report a genetic investigation of the interaction of C. elegans with the nematode-specific pathogen Microbacterium nematophilum, which colonizes the rectum and causes distinctive tail swelling in its host. A total of 121 mutants with altered response to infection were isolated from selections or screens for a bacterially unswollen (Bus) phenotype, using both chemical and transposon mutagenesis. Some of these correspond to known genes, affecting either bacterial adhesion or colonization (srf-2, srf-3, srf-5) or host swelling response (sur-2, egl-5). Most mutants define 15 new genes (bus-1-bus-6, bus-8, bus-10, bus-12-bus-18). The majority of these mutants exhibit little or no rectal infection when challenged with the pathogen and are probably altered in surface properties such that the bacteria can no longer infect worms. A number have corresponding alterations in lectin staining and cuticle fragility. Most of the uninfectable mutants grow better than wild type in the presence of the pathogen, but the sur-2 mutant is hypersensitive, indicating that the tail-swelling response is associated with a specific defense mechanism against this pathogen.     

Steps in reductive activation of the disulfide-generating enzyme Ero1p

Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity.     

Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem

Hydrothermal venting and the formation of carbonate chimneys in the Lost City hydrothermal field (LCHF) are driven predominantly by serpentinization reactions and cooling of mantle rocks, resulting in a highly reducing, high-pH environment with abundant dissolved hydrogen and methane. Phylogenetic and terminal restriction fragment length polymorphism analyses of 16S rRNA genes in fluids and carbonate material from this site indicate the presence of organisms similar to sulfur-oxidizing, sulfate-reducing, and methane-oxidizing Bacteria as well as methanogenic and anaerobic methane-oxidizing Archaea. The presence of these metabolic groups indicates that microbial cycling of sulfur and methane may be the dominant biogeochemical processes active within this ultramafic rock-hosted environment. 16S rRNA gene sequences grouping within the Methylobacter and Thiomicrospira clades were recovered from a chemically diverse suite of carbonate chimney and fluid samples. In contrast, 16S rRNA genes corresponding to the Lost City Methanosarcinales phylotype were found exclusively in high-temperature chimneys, while a phylotype of anaerobic methanotrophic Archaea (ANME-1) was restricted to lower-temperature, less vigorously venting sites. A hyperthermophilic habitat beneath the LCHF may be reflected by 16S rRNA gene sequences belonging to Thermococcales and uncultured Crenarchaeota identified in vent fluids. The finding of a diverse microbial ecosystem supported by the interaction of high-temperature, high-pH fluids resulting from serpentinization reactions in the subsurface provides insight into the biogeochemistry of what may be a pervasive process in ultramafic subseafloor environments.