Murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner

Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.     

Purification of uterine myosin and synthetic filament formation

Rabbit uterine myosin was purified by DEAE-Sephadex column chromatography. The purified myosin was shown to be free from contamination with actin or other impurities by sodium dodecyl sulfate gel electrophoresis. It was also shown to have two light chains with molecular weights of 22,000 and 17,000. The C protein normally found in crude, rabbit skeletal muscle myosin was not found in crude, rabbit uterine myosin.On reducing the ionic strength of a solution of rabbit uterine myosin, the myosin molecules first aggregated to form short, tapered bipolar filaments with bare zones. These were observed in the presence or absence of 10 mm-magnesium. These filaments ranged in length from 0.3 μm to 0.6 μm. On reaching 0.1 m-KCl, and only if 10 mm-magnesium was present, the filaments grew in length by linear overlap of the tapered bipolar filaments and obliteration of the bare zone regions. These filaments ranged in length from 0.7 μm to 1.2 μm.     

Letter: Myosin filaments showing a 430 A axial repeat periodicity

Synthetic myosin filaments with regular projections at intervals of 430 Å along their entire lengths have been observed using the electron microscope. The filaments were formed following dialysis in or rapid dilution to 0.3 m-KC1, 0.01 m-imidazole or phosphate buffer, pH 7.0. It is suggested that these filaments are made up of myosin molecules staggered by 430 Å.     

Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering

Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.     

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization‐defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I‐E CRISPR‐associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild‐type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.     

Designing a sustainable monitoring framework for assessing impacts of climate change at Joshua Tree National Park,USA

Predicting species’ responses to a warming and drying (for North America’s desert southwest region) climate provides focus for monitoring to track shifts in species’ occupancy, and ultimately identifying management options to stem losses to biodiversity. Here we describe a monitoring framework to achieve that objective. A first step is to identify which species to monitor; which species will provide the greatest information for discerning the effects of climate change versus the myriad of other stressors that may impact their distributions and abundance. To select focal species we employed two complimentary approaches. One tool, vulnerability assessments (VAs), use available scientific literature to assess exposure to environmental stressors and adaptive capacity or resilience to climate change. Another approach is habitat suitability modeling (HSM) coupled with simulated temperature shifts. This method statistically combines environmental variables at known species’ locations, such as climate and terrain, to model the complex interaction of factors that constrain a species’ distribution. All other variables held constant, simulated temperature shifts can identify species’ sensitivities to those shifts and identify potential refugia. We used these tools to assess risk of local extinction due to predicted levels of climate change, as well as to identify where to locate monitoring plots to best capture the shifts in species distributions over time. A challenge in developing a monitoring program to document the effects of climate change on biodiversity is program sustainability. One way to support and enhance the sustainability of such a program will be to couple trained biologists with volunteer citizen scientists.     

Toward a molecular equivalent dose: use of the medaka model in comparative risk assessment

Recent changes in the risk assessment landscape underscore the need to be able to compare the results of toxicity and dose-response testing between a growing list of animal models and, quite possibly, an array of in vitro screening assays. How do we compare test results for a given compound between vastly different species? For example, what dose level in the ambient water of a small fish model would be equivalent to 10 ppm of a given compound in the rat's drinking water? Where do we begin? To initially address these questions, and in order to compare dose-response tests in a standard rodent model with a fish model, we used the concept of molecular dose. Assays that quantify types of DNA damage that are directly relevant to carcinogenesis integrate the factors such as chemical exposure, uptake, distribution, metabolism, etc. that tend to vary so widely between different phyletic levels. We performed parallel exposures in F344 rats and Japanese medaka (Oryzias latipes) to the alkylating hepatocarcinogen, dimethylnitrosamine (DMN). In both models, we measured the DNA adducts 8-hydroxyguanine, N(7)-methylguanine and O(6)-methylguanine in the liver; mutation frequency using lambda cII transgenic medaka and lambda cII transgenic (Big Blue(R)) rats; and early morphological changes in the livers of both models using histopathology and immunohistochemistry. Pulse dose levels in fish were 0, 10, 25, 50, or 100 ppm DMN in the ambient water for 14 days. Since rats are reported to be especially sensitive to DMN, they received 0, 0.1, 1, 5, 10, or 25 ppm DMN in the drinking water for the same time period. While liver DNA adduct concentrations were similar in magnitude, mutant frequencies in the DMN-exposed medaka were up to 20 times higher than in the Big Blue rats. Future work with other compounds will generate a more complete picture of comparative dose response between different phyletic levels and will help guide risk assessors using "alternative" models.     

Identification of Saccharomyces cerevisiae spindle pole body remodeling factors

The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.