Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Heterotypic Humoral and Cellular Immune Responses following Norwalk Virus Infection

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-γ) was measured. As seen with blockade responses, IFN-γ secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.Noroviruses are the second-most important cause of severe viral gastroenteritis in young children and cause approximately 20% of endemic familial diarrheal disease and traveler''s diarrhea in all ages (reviewed in references 45 and 70). Noroviruses are genetically grouped into five different genogroups (GI to GV). GI and GII genogroups are responsible for the majority of human infections and are subdivided into more than 25 different genotypes (for example, GI.1 is genogroup I genotype 1). Most norovirus outbreaks are caused by the GII.4 genotype (65). Although genogroup I strains are associated with fewer reported outbreaks, they are frequently identified in environmental samples and in children (7, 21, 33, 58, 74, 82). The severity of norovirus disease is usually moderate although infection can be especially virulent, even fatal, in the elderly (14, 24, 31, 38, 46, 67). An effective vaccine would be particularly advantageous to vulnerable older populations, food handlers, child and health care providers, and military personnel. One major obstacle to norovirus vaccine development is the lack of understanding of the extensive antigenic relationships between heterogenic norovirus family members and of how this antigenic heterogeneity affects host protective immunity. Norovirus heterogeneity can be examined through sequence, structural, ligand binding, and host immune studies.Structurally, noroviruses are ∼38-nm icosahedral viruses with an ∼7.5 kb single-stranded, positive-sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the replicase polyprotein, while ORFs 2 and 3 encode the major and minor capsid proteins, respectively. The ORF2 major capsid protein sequence can vary by up to 60% between genogroups and by ∼20 to 30% between the genotypes (91). Expression of the major capsid protein (ORF2) in baculovirus and Venezuelan equine encephalitis (VEE) results in formation of virus-like particles (VLPs) composed of 180 copies of the monomeric protein (72). The monomer is structurally divided into the shell domain (S) that forms the structural core of the particle and the protruding domain (P) that protrudes away from the core. The P domain is further subdivided into the P1 subdomain (residues 226 to 278 and 406 to 520) and the P2 subdomain (residues 279 to 405) (72). P2 represents the most exposed surface of the viral particle and determines interaction with both potential neutralizing antibody recognition sites and putative cellular receptors, the histo-blood group antigens (HBGAs) (13, 16, 54, 57).The P domain has been shown to independently form dimers and P particles comprised of 12 monomers (85). Dimers and P particles share structural and HBGA binding similarities with the VLP generated with the same monomers (9, 85, 87). Three norovirus-HBGA binding profiles have been identified: (i) those that bind A/B and/or H epitopes, (ii) those that bind Lewis and/or H epitopes, and (iii) those that do not bind any available HBGA (86). Elegant structural analyses of Norwalk virus VLPs in complex with synthetic HBGAs identified a highly conserved binding site within the G1 noroviruses and predicted that structural constraints within the GI strains would restrict HBGA binding patterns to either a terminal Gal-Fuc or GalNAc (18, 88).Norwalk virus (NV; GI.1-1968) is the prototypic GI strain and typically infects individuals who encode a functional FUT2 α-1,2-fucosyltransferase enzyme resulting in expression of HBGAs on mucosal surfaces (secretor-positive phenotype) (53). Individuals who do not encode a functional FUT2 enzyme have a secretor-negative phenotype, do not express ABH HBGAs on mucosal surfaces, and are resistant to NV infection. Outbreak investigations have confirmed the association between HBGA expression and norovirus infection for some GI and GII strains (37, 39, 43, 49, 89). It remains likely that enzymes other than FUT2 may function as norovirus susceptibility factors because secretor-negative individuals have low-level norovirus-reactive antibodies (49, 52, 53) and can become infected after challenge with a GII.2 strain (52); in addition, some norovirus strains bind to FUT2-independent HBGAs in vitro (35, 54, 79).Early challenge studies (reviewed in reference 50) suggested that short-term protective immunity may occur following NV challenge (96). Demonstration of long-term protective immunity has been more complex. One early rechallenge study found that 50% of NV-challenged volunteers experienced repeat infections after ∼3 years while the other 50% remained well initially and after repeated challenge (69). Whether these volunteers remained disease free because of acquired immunity or genetic resistance could not be ascertained (69). However, contemporary norovirus challenge studies suggest that an early mucosal IgA response is associated with protection from NV infection (53). Further, strong gamma interferon (IFN-γ) secretion from CD4⁺ T cells (52) was identified in some uninfected GII.2-1976-challenged volunteers.In the absence of additional rechallenge studies, the most compelling evidence for a long-term protective immune response comes from the growing number of reports from around the world indicating that periods of “high norovirus activity” correlated with the emergence of new GII.4 strains (1, 10, 42, 66, 75, 90). Subsequently, the years following the high activity were characterized by decreased numbers of outbreaks, indicating that herd immunity may be an important regulator of GII.4 noroviruses (54, 80, 81). Clearly, the molecular basis for differential protective immunity/susceptibility following repeat norovirus infection is complex and a major challenge for the field.In this report, we compare the VLP phenotypes of the prototypical norovirus strain NV to an extant GI.1 strain isolated 33 years after NV and to a panel of VLPs representing strains GI.2, GI.3, and GI.4. In the results, we evaluate sequence conservation, carbohydrate (CHO) binding patterns, and antigenic relatedness at the antibody and T-cell levels. In contrast to earlier predictions (19), these data suggest that the GI noroviruses can bind many different HBGAs and that individuals infected with norovirus usually mount robust B- and T-cell responses against homologous strains. Surprisingly, some individuals appear to preferentially mount immune responses against heterologous GI strains.     

Chronobiological factors for compassion satisfaction and fatigue among ambulatory oncology caregivers

Primary caregivers for victims of chronic illness and or trauma experience both positive and negative emotional consequences. These are broadly classified as compassion satisfaction (CS) and compassion fatigue (CF). Because one of the components of CF, burnout, varies with chronotype and sleep quality, we assessed the influence of chronobiological features on the broader constructs of CS and CF. Responses from primary ambulatory care oncology staff working dayshifts were assessed for potential relationships of chronotype and sleep quality with CS and CF using the professional quality of life scale. These were analyzed further in a multivariate model that included personality and job satisfaction as cofactors. We found that sleep quality was a key contributor to CS development and CF reduction. Morningness was positively linked to CS, but the univariate association was masked in the multivariate model. Job satisfaction (contingent rewards, nature of work and operating procedures) heavily influenced CS and CF development. Agreeableness and openness showed positive correlations with CS and negative with burnout, while emotional stability was linked to reduced CF. While job satisfaction and personality predictably played roles in the development of CS and CF, sleep quality and chronotype contributed significantly to benefits and negative consequences of providing care.     

Pathogenomics of fungal plant parasites: what have we learnt about pathogenesis?

Members of the kingdom fungi comprise numerous plant pathogens, including the causal agents of many agriculturally relevant plant diseases such as rust, powdery mildew, rice blast and cereal head blight. Data from recent sequencing projects provide deep insight into the genomes of a range of fungi that infect different organs of monocotyledonous or dicotyledonous hosts and that have diverse pathogenic lifestyles. These studies have revealed that, similar to sequenced phytopathogenic oomycetes, these plant parasites possess very plastic and dynamic genomes, which typically encode several hundred candidate secreted effector proteins that can be highly divergent even among related species. A new insight is the presence of lineage-specific genes on mobile and partly dispensable chromosomes that are transferred intraspecifically and possibly interspecifically, thereby constituting pathogenicity and host range determinants. Convergent lifestyle-specific adaptations have shaped the parasite genomes to maximize pathogenic success according to the different infection strategies employed.     

Multiplicity-adjusted inferences in risk assessment: benchmark analysis with quantal response data

A primary objective in quantitative risk or safety assessment is characterization of the severity and likelihood of an adverse effect caused by a chemical toxin or pharmaceutical agent. In many cases data are not available at low doses or low exposures to the agent, and inferences at those doses must be based on the high-dose data. A modern method for making low-dose inferences is known as benchmark analysis, where attention centers on the dose at which a fixed benchmark level of risk is achieved. Both upper confidence limits on the risk and lower confidence limits on the "benchmark dose" are of interest. In practice, a number of possible benchmark risks may be under study; if so, corrections must be applied to adjust the limits for multiplicity. In this short note, we discuss approaches for doing so with quantal response data.     

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO₃⁻, K⁺, and H₂PO₄⁻ into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.     

Efficient Pre-mRNA Cleavage Prevents Replication-Stress-Associated Genome Instability

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Proteome analysis of early somatic embryogenesis in Picea glauca

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.     

Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.