Sequence elements controlling expression of Barley stripe mosaic virus subgenomic RNAs in vivo.

Barley stripe mosaic virus (BSMV) contains three positive-sense, single-stranded genomic RNAs, designated alpha, beta, and gamma, that encode seven major proteins and one minor translational readthrough protein. Three proteins (alphaa, betaa, and gammaa) are translated directly from the genomic RNAs and the remaining proteins encoded on RNAbeta and RNAgamma are expressed via three subgenomic messenger RNAs (sgRNAs). sgRNAbeta1 directs synthesis of the triple gene block 1 (TGB1) protein. The TGB2 protein, the TGB2' minor translational readthrough protein, and the TGB3 protein are expressed from sgRNAbeta2, which is present in considerably lower abundance than sgRNAbeta1. A third sgRNA, sgRNAgamma, is required for expression of the gammab protein. We have used deletion analyses and site-specific mutations to define the boundaries of promoter regions that are critical for expression of the BSMV sgRNAs in infected protoplasts. The results reveal that the sgRNAbeta1 promoter encompasses positions -29 to -2 relative to its transcription start site and is adjacent to a cis-acting element required for RNAbeta replication that maps from -107 to -74 relative to the sgRNAbeta1 start site. The core sgRNAbeta2 promoter includes residues -32 to -17 relative to the sgRNAbeta2 transcriptional start site, although maximal activity requires an upstream hexanucleotide sequence residing from positions -64 to -59. The sgRNAgamma promoter maps from -21 to +2 relative to its transcription start site and therefore partially overlaps the gammaa gene. The sgRNAbeta1, beta2, and gamma promoters also differ substantially in sequence, but have similarities to the putative homologous promoters of other Hordeiviruses. These differences are postulated to affect competition for the viral polymerase, coordination of the temporal expression and abundance of the TGB proteins, and constitutive expression of the gammab protein

Autocatalytic Processing of the 223-kDa Protein of Blueberry Scorch Carlavirus by a Papain-like Proteinase

AbstractThe first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative hellcase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro , supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 significantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants

The genome sequence and effector complement of the flax rust pathogen Melampsora lini

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.This work was funded by a grant from the CSIRO Transformational Biology Capability Platform to Adnane Nemri. Claire Anderson was supported by an ARC Discovery Grant (DP120104044) awarded to David A. Jones and Peter N. Dodds

A Theory of Organization-EHR Affordance Actualization

While organizations implement information technology (IT) to effect change, current theories of IT-associated organizational change pay insufficient attention to the change goals, the role of IT in organizational change, and the multilevel nature of change processes. We take a fresh look at IT-associated organizational change using grounded theory methods. Our longitudinal study of an electronic health record (EHR) system implementation in a multi-site medical group found user behaviors that did not fit well with existing theories. Instead, we found that they fit better with the concept of affordances from ecological psychology. In developing our affordance-based theory of IT-associated organizational change from our field data, we discovered three gaps in the affordance literature; namely, the lack of theory for (1) the process of actualizing an affordance’s potential, (2) affordances in an organizational context, and (3) bundles of interrelated affordances. This paper extends the theory of affordances to handle these three gaps and, in doing so, develops a mid-range theory for EHR-associated organizational change in a healthcare organization. While the resulting theory is specific to EHR implementations, it offers a template for other mid-range affordance-actualization theories and a more general affordance-actualization lens. Our affordance-actualization lens considers the materiality of the IT artifact, the non-deterministic process by which IT leads to organizational effects, the multilevel nature of IT-associated change processes, and the intentionality of managers and users as agents of change, and thus addresses important criteria for theories of IT effects in organizations. The paper also provides practical guidance for implementing EHR systems and other organizational systems

Spending per Medicare Beneficiary Is Higher in Hospital‐Owned Small‐ and Medium‐Sized Physician Practices

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145215/1/hesr12765.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145215/2/hesr12765-sup-0001-AppendixSA1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145215/3/hesr12765_am.pd

Sirtuin 1 mediates protection against delayed cerebral ischemia in subarachnoid hemorrhage in response to hypoxic postconditioning

Background Many therapies designed to prevent delayed cerebral ischemia (DCI) and improve neurological outcome in aneurysmal subarachnoid hemorrhage (SAH) have failed, likely because of targeting only one element of what has proven to be a multifactorial disease. We previously demonstrated that initiating hypoxic conditioning before SAH (hypoxic preconditioning) provides powerful protection against DCI. Here, we expanded upon these findings to determine whether hypoxic conditioning delivered at clinically relevant time points after SAH (hypoxic postconditioning) provides similarly robust DCI protection. Methods and Results In this study, we found that hypoxic postconditioning (8%

SIRT1 mediates hypoxic preconditioning induced attenuation of neurovascular dysfunction following subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Vasospasm and delayed cerebral ischemia (DCI) contribute significantly to the morbidity/mortality associated with aneurysmal subarachnoid hemorrhage (SAH). While considerable research effort has focused on preventing or reversing vasospasm, SAH-induced brain injury occurs in response to a multitude of concomitantly acting pathophysiologic mechanisms. In this regard, the pleiotropic epigenetic responses to conditioning-based therapeutics may provide an ideal SAH therapeutic strategy. We previously documented the ability of hypoxic preconditioning (PC) to attenuate vasospasm and neurological deficits after SAH, in a manner that depends on the activity of endothelial nitric oxide synthase. The present study was undertaken to elucidate whether the NAD-dependent protein deacetylase sirtuin isoform SIRT1 is an upstream mediator of hypoxic PC-induced protection, and to assess the efficacy of the SIRT1-activating polyphenol Resveratrol as a pharmacologic preconditioning therapy. METHODS: Wild-type C57BL/6J mice were utilized in the study and subjected to normoxia or hypoxic PC. Surgical procedures included induction of SAH via endovascular perforation or sham surgery. Multiple endpoints were assessed including cerebral vasospasm, neurobehavioral deficits, SIRT1 expression via quantitative real-time PCR for mRNA, and western blot for protein quantification. Pharmacological agents utilized in the study include EX-527 (SIRT1 inhibitor), and Resveratrol (SIRT1 activator). RESULTS: Hypoxic PC leads to rapid and sustained increase in cerebral SIRT1 mRNA and protein expression. SIRT1 inhibition blocks the protective effects of hypoxic PC on vasospasm and neurological deficits. Resveratrol pretreatment dose-dependently abrogates vasospasm and attenuates neurological deficits following SAH – beneficial effects that were similarly blocked by pharmacologic inhibition of SIRT1. CONCLUSION: SIRT1 mediates hypoxic preconditioning-induced protection against neurovascular dysfunction after SAH. Resveratrol mimics this neurovascular protection, at least in part, via SIRT1. Activation of SIRT1 is a promising, novel, pleiotropic therapeutic strategy to combat DCI after SAH

Verifying Different-modality Properties for Concepts Produces Switching Costs

According to perceptual symbol systems (Barsalou, 1999), sensory-motor simulations underlie the representation of concepts. It follows that sensory-motor phenomena should arise in conceptual processing. Previous studies have shown that switching from one modality to another during perceptual processing incurs a processing cost. If perceptual simulation underlies conceptual processing, then verifying the properties of concepts should exhibit a switching cost as well. For example, verifying a property in the auditory modality (e.g., BLENDER-loud) should be slower after verifying a property in a different modality (e.g., CRANBERRIES-tart) than in the same modality (e.g., LEAVES-rustling). Only words were presented to subjects, and there were no instructions to use imagery. Nevertheless switching modalities incurred a cost, analogous to switching modalities in perception. A second experiment showed that this effect was not due to associative priming between properties in the same modality. These results support the hypothesis that perceptual simulation underlies conceptual processing