The elemental mechanism of transcriptional pausing.

Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI

Characterizing Molecular Modifiers of Pathogenesis in Spinobulbar Muscular Atrophy.

Spinobulbar muscular atrophy (SBMA), or Kennedy’s disease, is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) tract expansion in the androgen receptor (AR). This mutation initiates misfolding and aggregation of AR, eliciting toxicity in motor neurons, progressive weakness, and muscle atrophy. PolyQ expansion also compromises the transactivation function of AR in response to androgens, resulting in androgen insensitivity. Although considerable progress has been made in characterizing molecular consequences of the polyQ mutation in SBMA, many aspects of pathogenesis, and in particular the cellular processes that modify disease development, remain incompletely understood. Based on previous work suggesting a pathogenic role of autophagy in SBMA, I use cellular and mouse models to delineate the state of autophagy in SBMA. I show that autophagy is induced in SBMA cells and diseased tissues, and that this is due to depressed mTOR activity. These changes correlate with activity of the transcription factors TFEB and ZKSCAN3, which coordinate expression of autophagy-related genes in SBMA mice and human patients. Furthermore, these alterations in autophagy regulators lead to enhanced responsiveness to stimulation by nutrient deprivation and exercise. These results indicate that dysregulated transcriptional programming promotes induction of autophagy in SBMA and provide evidence for targeting autophagy for therapeutic inhibition. Given the previously established role of small ubiquitin-like modifier (SUMO) on AR function, I characterize a novel knock-in mouse model of SBMA to address the influence of SUMO on SBMA pathogenesis. We introduce mutations that prevent SUMOylation of polyQ AR (AR113Q-KRKR) and demonstrate that, despite unaltered androgen insensitivity and neuromuscular pathology, AR113Q-KRKR mice display a striking extension of lifespan and recovery of exercise tolerance. Complementary expression analysis of the non-SUMOylatable polyQ AR reveals substantial expansion of the receptor’s transactivation activity. These findings suggest that abrogating SUMO modification of polyQ AR mediates amelioration of the SBMA phenotype, in part by improving skeletal muscle physiology. Additionally, these studies not only reveal new insights in the comparative roles of polyQ AR toxicity versus loss of function in affected tissues, but they also establish the benefits of enhancing AR function in SBMA for therapeutic design.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111409/1/jaschua_1.pd

LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang).

A number of three LC–MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100–1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35 °C and 100 °C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35 °C and 100 °C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100 °C

Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor-mediated disease.

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA

Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells

Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin–mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI

Translating policy into practice for community-based management of rheumatoid arthritis: Targeting professional development needs among physiotherapists.

Introduction: Contemporary health policy promotes delivery of community-based health services to people with musculoskeletal conditions, including rheumatoid arthritis (RA). This emphasis requires a skilled workforce to deliver safe, effective care. We aimed to explore physiotherapy workforce readiness to co-manage consumers with RA by determining the RA-specific professional development (PD) needs in relation to work and educational characteristics of physiotherapists in Western Australia (WA). Methods. An e-survey was sent to physiotherapists regarding their confidence in co-managing people with RA and their PD needs. Data including years of clinical experience, current RA clinical caseload, professional qualifications, and primary clinical area of practice were collected. Results. 273 physiotherapists completed the survey. Overall confidence in managing people with RA was low (22.7–58.2%) and need for PD was high (45.1–95.2%). Physiotherapists with greater years of clinical experience, a caseload of consumers with RA, postgraduate qualifications in musculoskeletal physiotherapy, or who worked in the musculoskeletal area were more confident in managing people with RA and less likely to need PD. Online and face-to-face formats were preferred modes of PD delivery. Discussion. To enable community-based RA service delivery to be effectively established, subgroups within the current physiotherapy workforce require upskilling in the evidence-based management of consumers with RA

Quantifying speech intelligibility based on crowdsourcing

The intelligibility of speech within media content, e.g., audio or video streams, is an important factor that determines the reach and popularity of the media. Objective measures of audio and speech quality, e.g., PESQ and SII scores, correlate poorly with human assessment. MOS, a widely accepted intelligibility test, is subjective, expensive, and time consuming. Techniques disclosed herein provide an objective measure of the intelligibility of speech within video or audio content. Speech intelligibility scores are calculated based on the edit distance between human speech transcriptions of short clips and transcripts produced by an automatic speech recognizer. The speech intelligibility score is based on human rating and retains objectivity

Bridging the gap between evidence and what people value from osteoarthritis care in New Zealand using multi-criteria decision analysis (MCDA)

International clinical practice guidelines (CPG) for osteoarthritis (OA) consistently recommend core management strategies of exercise, weight-loss and self-management education. However, these interventions are not routinely delivered or taken up by people experiencing OA, resulting in a tendency to deliver low-value care. Tailoring the delivery of recommended OA care to the preferences of stakeholders in a health system may help support greater implementation of high-value OA care. However, little is known about the preferences of stakeholders for evidence and recommendations for OA care in a CPG. The aim of this research was to establish a framework to prioritise knee interventions for managing OA and evaluate the relative importance of these interventions across the healthcare sector in New Zealand (NZ) using multi-criteria decision analysis (MCDA). This research used a mixed-methods approach to develop the MCDA framework. The first stage involved focus group discussions to generate a thematic framework of what OA consumers, health care providers, policy-makers, Māori representatives and OA experts consider in their choice of knee OA interventions. A Delphi survey was used to verify the thematic analysis and rank the most important criteria concerning the characteristics of interventions; these findings informed the criteria selected in the second stage. In the second stage, interventions were indirectly prioritised by systematically combining preference data with intervention performance data on the criteria. First, a survey involving trade-offs was used to measure stakeholders’ preferences, represented as ‘weights’, within and between the criteria. Evidence for the performance of 15 recommended knee OA interventions were then extracted from a CPG for hip and knee OA, and rated on the criteria according to their level of achievement (e.g. high, medium, low). To prioritise the interventions, a total score for each intervention was calculated by summing the weights associated with the intervention ratings, after which they were ranked by importance. Associations between the weights and stakeholder groups were explored using regression analysis. Thematic analysis of data from six focus groups produced a framework comprising three overarching categories, consisting of characteristics of the: (i) intervention, (ii) consumer and (iii) health system. Participants identified and ranked nine characteristics of interventions; the most important eight were included in the MCDA framework. The choice-based survey revealed that stakeholders valued the intervention characteristics, in decreasing order of importance (weight): Recommendation (19.0%), Quality of evidence (17.7%), Effectiveness (15.0%), Duration of effect (13.2%), Risk of serious harm (12.8%), Risk of mild side-effects (9.4%), Cost (6.6%) and Accessibility (6.3%). Total scores for the 15 guideline-recommended interventions revealed that for first-, second- and third-line OA care respectively, all land-based exercise (total score= 71.7%), NSAIDs (topical) (74.2%) and total joint replacement (74.3%) were ranked first. For first-, second- and third-line OA care, the recommended core interventions of weight management and self-management education ranked between 11th and 15th (48.0% to 56.0%). Regression analysis identified only small differences in weights (≤5.7%; p<0.01) between stakeholder groups. These findings suggest that stakeholders’ preferences for the core interventions of weight management and self-management education represent a system-wide barrier to their implementation. This research addresses an important knowledge gap concerning cross-sectoral stakeholders’ preferences for knee OA interventions in a CPG. By inclusively, systematically and transparently incorporating what matters to people with evidence and recommendations in a CPG, the MCDA framework developed in this thesis can help support the design of patient-centred, high-value healthcare for people experiencing OA

H3K9me-Independent Gene Silencing in Fission Yeast Heterochromatin by Clr5 and Histone Deacetylases

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci