Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site

Use of mHealth Technology for Patient-Reported Outcomes in Community-Dwelling Adults with Acquired Brain Injuries: A Scoping Review.

The purpose of our scoping review was to describe the current use of mHealth technology for long-term assessment of patient-reported outcomes in community-dwelling individuals with acquired brain injury (ABI). Following PRISMA guidelines, we conducted a scoping review of literature meeting these criteria: (1) civilians or military veterans, all ages; (2) self-reported or caregiver-reported outcomes assessed via mobile device in the community (not exclusively clinic/hospital); (3) published in English; (4) published in 2015-2019. We searched Ovid MEDLINE(R) \u3c 1946 to 16 August 2019, MEDLINE InProcess, EPub, Embase, and PsycINFO databases for articles. Thirteen manuscripts representing 12 distinct studies were organized by type of ABI [traumatic brain injury (TBI) and stroke] to extract outcomes, mHealth technology used, design, and inclusion of ecological momentary assessment (EMA). Outcomes included post-concussive, depressive, and affective symptoms, fatigue, daily activities, stroke risk factors, and cognitive exertion. Overall, collecting patient-reported outcomes via mHealth was feasible and acceptable in the chronic ABI population. Studies consistently showed advantage for using EMA despite variability in EMA timing/schedules. To ensure best clinical measurement, research on post-ABI outcomes should consider EMA designs (versus single time-point assessments) that provide the best timing schedules for their respective aims and outcomes and that leverage mHealth for data collection

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

Workplace harassment patterning, gender, and utilization of professional services: Findings from a US national study

This study constitutes the first national longitudinal survey to address the relationship between workplace harassment and service utilization. We examine how patterns of sexual harassment and generalized workplace harassment are linked to utilization of mental health, health, legal, spiritual, and work-related services, and whether and how gender influences these relationships. Data derive from a random digit dial telephone survey with a continental US sample of employed adults. Eligibility criteria were being 18 years of age or over, and being employed at least 20 h per week at some time in the 12 months prior to the wave 1 survey. Out of 4116 households with eligible individuals, 2151 agreed to participate at wave 1. At wave 2, 1418 participated, thus, the overall response rate was 34.5%. We show that the patterning of workplace harassment over two time points (chronic, remission, onset, never harassed) is associated with the use of different types of services. Gender partially moderated the relationship between workplace harassment and services.USA Workplace harassment Work stress Gender Service utilization

Targeting Pyk2 to Beta1-Integrin containing Focal Contacts Rescues Fibronectin stimulated Signaling and Haptotactic Motility Defects of Focal Adhesion Kinase null Cells

Focal adhesion kinase null (FAK-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK-/- cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK-/- cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a ß1-integrin containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK-/- cells, exhibit elevated FN-stimulated extracellular signal regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to ß-integrin containing focal contact sites via interactions mediated by the FAK-CT domain

CT of hepatocellular carcinoma in non-alcoholic fatty liver disease: imaging characteristics and inter-rater agreement

Aim: To determine the computed tomography (CT) features of non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC).Methods: In this institutional review board approved study, we reviewed 38 patients with NAFLD (68.4% male; mean age 63 years) with histology confirmed HCC and triphasic liver CT. CT images were independently reviewed by four readers blinded to clinical and pathology data. The reviewers assessed HCC for arterial phase hyper enhancement (APHE), portal venous phase washout (PVWO), delayed phase washout (DPWO), and enhancing capsule. Features of cirrhotic morphology and portal hypertension (PH) were also evaluated. The final CT features were determined by majority and a fifth reader reviewed cases lacking majority. Inter-rater agreement was determined by prevalence-adjusted kappa.Results: Mean HCC size was 3.6 ± 2.8 cm (range, 1.1-16.0 cm). The HCCs showed APHE in 92.1%, PVWO in 55.3%, DPWO in 81.6%, and enhancing capsule in 44.7%. Cirrhotic morphology was present in 65.8% and PH in 63.2%. Inter-rater agreement was moderate to almost perfect for APHE (0.74-1.0), cirrhosis (0.79-0.89), and PH (0.79-0.95), weak to perfect for DPWO (0.47-0.95) and poor for PVWO (0-0.42).Conclusion: NAFLD associated HCC demonstrate less frequent portal venous washout on CT which may affect their imaging diagnosis