An introduction to the short-term toxicology of respirable industrial fibres

Fibrous materials, exemplified by asbestos, that release respirable fibres are in common use and there is considerable knowledge regarding the toxicology of these common fibres. Newer materials or those that are under development, such as synthetic organic fibres and carbon nanotubes may have a different toxicology paradigms. The existing paradigm for silicate fibres suggests that respirable fibre types vary in their ability to cause lung disease and that this can be understood on the basis of the length of the fibres and their biopersistence in the lungs. Because fibres are regulated on a fibre number basis and the hazard is understood on the basis of the number of long fibres, in fibre testing the dose should always be expressed as fibre number, not mass and the length and diameter distribution need to be known. Short-term biological tests are likely to produce false positives in the case of long non-biopersistent fibres, because whilst they may have effects in vitro, they do not persist long enough in the lungs for sufficient dose to build up and produce effects in vivo. The biopersistence of fibres is therefore a key factor that needs to be known in order to interpret short-term tests that may claim to predict fibre pathogenicity.     

Economic evaluation of weight loss interventions in overweight and obese women

Objective: To conduct a clinical and economic evaluation of outpatient weight loss strategies in overweight and obese adult U.S. women. Research Methods and Procedures: This study was a lifetime cost‐use analysis from a societal perspective, using a first‐order Monte Carlo simulation. Strategies included routine primary care and varying combinations of diet, exercise, behavior modification, and/or pharmacotherapy. Primary data were collected to assess program costs and obesity‐related quality of life. Other data were obtained from clinical trials, population‐based surveys, and other published literature. This was a simulated cohort of healthy 35‐year‐old overweight and obese women in the United States. Results: For overweight and obese women, a three‐component intervention of diet, exercise, and behavior modification cost $12,600 per quality‐adjusted life year gained compared with routine care. All other strategies were either less effective and more costly or less effective and less cost‐effective compared with the next best alternative. Results were most influenced by obesity‐related effects on quality of life and the probabilities of weight loss maintenance. Discussion: A multidisciplinary weight loss program consisting of diet, exercise, and behavior modification provides good value for money, but more research is required to confirm the impacts of such programs on quality of life and the likelihood of long‐term weight loss maintenance.     

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.     

Genetic Identity of Populus tremuloides Litter Influences Decomposition and Nutrient Release in a Mixed Forest Stand

Recent research has shown that genetic variation can directly impact community and ecosystem level processes. Populus tremuloides (trembling aspen) is an extremely widespread and genetically diverse tree species important to many North American forest ecosystems. Using leaf litter from five genotypes grown in a common garden under two nutrient treatments, we tracked litter decomposition in a natural aspen stand for 1 year. Here we show that aspen leaf litter decomposes and releases carbon, nitrogen, and sulfur in relation to its genetic identity. In a secondary experiment, we show that the genetic diversity of aspen litter mixtures can influence decomposition, however weakly so. Overall, nutrient treatments influenced leaf litter decomposition the most, followed by genetic identity, and then by genetic diversity (if at all in some cases). In this widespread, genetically diverse, and dominant species, genetic variation within a single species is important to ecosystem functioning. The relatively weak effect of genetic diversity on the processes measured here does not preclude its importance to ecosystem functioning, but does suggest that genetic identity and composition are more important than genetic diversity per se.     

The Ctf18 RFC-like complex positions yeast telomeres but does not specify their replication time

Chromosome ends in Saccharomyces cerevisiae are positioned in clusters at the nuclear rim. We report that Ctf18, Ctf8, and Dcc1, the subunits of a Replication Factor C (RFC)-like complex, are essential for the perinuclear positioning of telomeres. In both yeast and mammalian cells, peripheral nuclear positioning of chromatin during G1 phase correlates with late DNA replication. We find that the mislocalized telomeres of ctf18 cells still replicate late, showing that late DNA replication does not require peripheral positioning during G1. The Ku and Sir complexes have been shown to act through separate pathways to position telomeres, but in the absence of Ctf18 neither pathway can act fully to maintain telomere position. Surprisingly CTF18 is not required for Ku or Sir4-mediated peripheral tethering of a nontelomeric chromosome locus. Our results suggest that the Ctf18 RFC-like complex modifies telomeric chromatin to make it competent for normal localization to the nuclear periphery.     

Molecular epidemiology of ceftiofur-resistant Escherichia coli isolates from dairy calves

Healthy calves (n = 96, 1 to 9 weeks old) from a dairy herd in central Pennsylvania were examined each month over a five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur-resistant Escherichia coli isolates (n = 122) were characterized by antimicrobial resistance (disk diffusion and MIC), serotype, pulsed-field gel electrophoresis subtypes, beta-lactamase genes, and virulence genes. Antibiotic disk diffusion assays showed that the isolates were resistant to ampicillin (100%), ceftiofur (100%), chloramphenicol (94%), florfenicol (93%), gentamicin (89%), spectinomycin (72%), tetracycline (98%), ticarcillin (99%), and ticarcillin-clavulanic acid (99%). All isolates were multidrug resistant and displayed elevated MICs. The E. coli isolates belonged to 42 serotypes, of which O8:H25 was the predominant serotype (49.2%). Pulsed-field gel electrophoresis classified the E. coli isolates into 27 profiles. Cluster analysis showed that 77 isolates (63.1%) belonged to one unique group. The prevalence of pathogenic E. coli was low (8%). A total of 117 ceftiofur-resistant E. coli isolates (96%) possessed the bla(CMY2) gene. Based on phenotypic and genotypic characterization, the ceftiofur-resistant E. coli isolates belonged to 59 clonal types. There was no significant relationship between calf age and clonal type. The findings of this study revealed that healthy dairy calves were rapidly colonized by antibiotic-resistant strains of E. coli shortly after birth. The high prevalence of multidrug-resistant nonpathogenic E. coli in calves could be a significant source of resistance genes to other bacteria that share the same environment.     

An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.     

Towards male sterility in Pinus radiata--a stilbene synthase approach to genetically engineer nuclear male sterility

A male cone-specific promoter from Pinus radiata D. Don (radiata pine) was used to express a stilbene synthase gene (STS) in anthers of transgenic Nicotiana tabacum plants, resulting in complete male sterility in 70% of transformed plants. Three plants were 98%-99.9% male sterile, as evidenced by pollen germination. To identify the stage at which transgenic pollen first developed abnormally, tobacco anthers from six different developmental stages were assayed microscopically. Following the release of pollen grains from tetrads, transgenic pollen displayed an increasingly flake-like structure, which gradually rounded up during the maturation process. We further investigated whether STS expression may have resulted in an impaired flavonol or sporopollenin formation. A specific flavonol aglycone stain was used to demonstrate that significant amounts of these substances were produced only in late stages of normal pollen development, therefore excluding a diminished flavonol aglycone production as a reason for pollen ablation. A detailed analysis of the exine layer by transmission electron microscopy revealed minor structural changes in the exine layer of ablated pollen, and pyrolysis-gas chromatography-mass spectroscopy indicated that the biochemistry of sporopollenin production was unaffected. The promoter-STS construct may be useful for the ablation of pollen formation in coniferous gymnosperms and male sterility may potentially be viewed as a prerequisite for the commercial use of transgenic conifers.     

Drosophila Roc1a encodes a RING-H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase

Substrate specificity of SCF E3 ubiquitin ligases is thought to be determined by the F box protein subunit. Another component of SCF complexes is provided by members of the Roc1/Rbx1/Hrt1 gene family, which encode RING-H2 proteins. Drosophila contains three members of this gene family. We show that Roc1a mutant cells fail to proliferate. Further, while the F box protein Slimb is required for Cubitus interruptus (Ci) and Armadillo/beta-catenin (Arm) proteolysis, Roc1a mutant cells hyperaccumulate Ci but not Arm. This suggests that Slimb and Roc1a function in the same SCF complex to target Ci but that a different RING-H2 protein acts with Slimb to target Arm. Consequently, the identity of the Roc subunit may contribute to the selection of substrates by metazoan SCF complexes.