The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV’s largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial

Differences in environmental stress response between yeasts is consistent with species-specific lifestyles

Defining how organisms respond to environmental change has always been an important step toward understating their adaptive capacity and physiology. Variation in transcription during stress has been widely described in model species, especially in the yeastSaccharomyces cerevisiae, which helped to shape general rules regarding how cells cope with environmental constraints as well as decipher the functions of many genes. Now, comparison of the environmental stress response (ESR) across species is essential to obtain a better insight into the common and species-specific features of stress defense. In this context, we explored the transcriptional landscape of the yeastLachancea kluyveri(formerlySaccharomyces kluyveri) in response to diverse stresses, using RNA-seq. We investigated variation in gene expression and observed a link between genetic plasticity and environmental sensitivity. We identified the ESR genes in this species and compared them to those already found inS. cerevisiae We observed common features between the two species as well as divergence in the regulatory networks involved. Interestingly, some changes were related to differences in species lifestyle. Thus, we were able to decipher how adaptation to stress has evolved among different yeast species. Finally, by analyzing patterns of coexpression, we were able to propose potential biological functions for 42% of genes and furthermore annotate 301 genes for which no function could be assigned by homology. This large dataset allowed for the characterization of the evolution of gene regulation and provides an efficient tool to assess gene function

Sources of Nonnative Centrarchids in the Upper Colorado River Revealed by Stable Isotope and Microchemical Analyses of Otoliths

Nonnative fishes represent a significant impediment to the recovery of imperiled fishes, including those endemic to the Colorado River in the southwestern United States. Efforts to control nonindigenous fish abundance in the upper Colorado River basin have been unsuccessful owing in part to lack of knowledge regarding nonnative fish recruitment sources. We determined the source habitat (floodplain pond versus riverine habitats) for nonnative centrarchid fishes (largemouth bass Micropterus salmoides, green sunfish Lepomis cyanellus, bluegill L. macrochirus, and black crappie Pomoxis nigromaculatus) in the upper Colorado River using stable hydrogen isotopic composition (δD) and strontium:calcium (Sr:Ca) ratios in fish otoliths as natural markers of environmental history. Stable hydrogen isotope analysis revealed that 59% of centrarchids exhibited the otolith core signatures expected for riverine-origin fish, while 22% had emigrated from floodplain ponds and 19% were of uncertain origin. Strontium:calcium ratio data were consistent with the δD assays and indicated that relatively few fish immigrated to the river from high-salinity habitats. Black crappie was the only species that originated primarily from floodplain ponds. Efforts to control the abundance of most of the fishes included in this study should be concentrated in riverine habitats given the hydrologic conditions (below-average river discharge) present during our study. However, the proportion of pond-origin fish increased with fish age, which, coupled with historical river discharge data, suggested that floodplain pond contributions to riverine nonnative fish populations fluctuate with the interannual variations in flow regime and river–pond connectivity. Our results are the first to demonstrate the utility of δD as a natural marker of fish environmental history that will probably provide valuable insights into the management of fish in other environments

Habitat, Fish Species, and Fish Assemblage Associations of the Topeka Shiner in West-Central Iowa

Our goal was to identify habitat, fish species, and fish assemblages associated with the occurrence of Topeka Shiners Notropis topeka in stream and off-channel habitat (OCH) of west-central Iowa. Fish assemblages and habitat characteristics were estimated in 67 stream and 27OCHsites during 2010–2011. Topeka Shiners were sampled in 52% of OCH sites, but in only 9% of stream sites, which supports the hypothesis that OCH is an important component of their life history. Fish assemblages containing Topeka Shiners were different from those that did not contain Topeka Shiners in OCH sites, but this was not evident in stream sites. Results from logistic regression models suggested that Topeka Shiner presence was associated with increased submerged vegetation and abundance of Fathead Minnow Pimephales promelas. Contrary to the findings of other studies, the abundance of large piscivorous fishes was not associated with the occurrence of Topeka Shiners. Our results provide new information about the biology and life history of the Topeka Shiner that will guide habitat restoration and other recovery efforts

Growth Rate Responses of Missouri and Lower Yellowstone River Fishes to a Latitudinal Gradient

Notropis atherinoides, freshwater drums Aplodinotus grunniens, river carpsuckers Carpiodes carpio and saugers Stizostedion canadense collected in 1996-1998 from nine river sections of the Missouri and lower Yellowstone rivers at two life-stages (young-of-the-year and age 1+ years) were significantly different among sections. However, they showed no river-wide latitudinal trend except for age 1+ years emerald shiners that did show a weak negative relation between growth and both latitude and length of growing season. The results suggest growth rates of fishes along the Missouri River system are complex and could be of significance in the management and conservation of fish communities in this altered system

Growth, Fecundity, and Diets of Newly Established Silver Carp in the Middle Mississippi River

The silver carp Hypophthalmichthys molitrix has spread throughout the Mississippi River drainage. During 2003, we determined its population status and potential impact in the middle Mississippi River (MMR), the conduit between the lower Mississippi River and the upper Mississippi, Missouri, and Illinois rivers. We quantified growth, age structure, fecundity, and diets of silver carp sampled with trammel nets and AC electrofishing in main-channel areas. Mean length at age in the MMR exceeded that of populations in Asia by as much as 26%. Individuals were typically more than 1 year old and 230 mm total length, suggesting that small, young fish were absent. Individuals in this population matured earlier (age 2) than in the species\u27 native range. Regardless of phytoplankton variation (using chlorophyll a as a surrogate) and zooplankton concentration at MMR sites, phytoplankton was consistently most abundant in diets. Silver carp are finding suitable resources within the MMR, allowing individuals to grow rapidly during early life, persist as adults, and successfully disperse upstream

A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors†

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5′ end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators

Lysine hydroxylation and O-glycosylation in the globular, C-terminal region of mammalian-expressed, recombinant PrP

Conversion of PrPC, the prion protein, to a conformationally altered isoform, PrPSc, is the major pathogenic event in the transmissible spongiform encephalopathies, a family of neurodegenerative diseases including bovine spongiform encephalopathy, Creutzfeldt-Jakob disease and scrapie. Known post-translational modifications to the protein include disulfide bridge formation, addition of a membrane anchor and N-linked glycosylation. We have previously identified the pro-collagen-like hydroxylation of proline 44 in a murine, recombinant prion protein expressed in Chinese hamster ovary cells and herein report the identification of a second pro-collagen-like modification in this protein. In a proportion of the molecules, Lys193, within the C-terminal, folded domain of the protein, is specifically modified to hydroxylysine with subsequent addition of two hexose units, assumed to be the collagen-like disaccharide modifier Gal-Glu. Proof of the existence of these modifications has been obtained by means of tandem mass spectrometry and Edman degradation. Molecular dynamics simulations show that these modifications lead to a pronounced stabilising effect on the β2–α2 loop, a region of PrP crucial for the disease-associated conversion. If present in vivo, these modifications may have important implications in PrP structure, interactions with ligands or may modulate PrP aggregation

Habitat Associations of Fish Species of Greatest Conservation Need at Multiple Spatial Scales in Wadeable Iowa Streams

Fish and habitat data were collected from 84 wadeable stream reaches in the Mississippi River drainage of Iowa to predict the occurrences of seven fish species of greatest conservation need and to identify the relative importance of habitat variables measured at small (e.g., depth, velocity, and substrate) and large (e.g., stream order, elevation, and gradient) scales in terms of their influence on species occurrences. Multiple logistic regression analysis was used to predict fish species occurrences, starting with all possible combinations of variables (5 large-scale variables, 13 small-scale variables, and all 18 variables) but limiting the final models to a maximum of five variables. Akaike’s information criterion was used to rank candidate models, weight model parameters, and calculate model-averaged predictions. On average, the correct classification rate (CCR = 80%) and Cohen’s kappa (κ = 0.59) were greatest for multiple-scale models (i.e., those including both large-scale and small-scale variables), intermediate for small-scale models (CCR = 75%; κ = 0.49), and lowest for large-scale models (CCR = 73%; κ = 0.44). The occurrence of each species was associated with a unique combination of large-scale and small-scale variables. Our results support the necessity of understanding factors that constrain the distribution of fishes across spatial scales to ensure that management decisions and actions occur at the appropriate scale