Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

A theory-based multi-component intervention to increase reactive balance measurement by physiotherapists in three rehabilitation hospitals: an uncontrolled single group study.

BACKGROUND: Most implementation interventions in rehabilitation, including physiotherapy, have used passive, non-theoretical approaches without demonstrated effectiveness. The goal of this study was to improve an important domain of physiotherapy practice - reactive balance measurement - with a targeted theory-based multi-component intervention developed using the Theoretical Domains Framework. The primary objective was to determine documented reactive balance measure use in a 12-month baseline, during, and for three months post- intervention. METHODS: An uncontrolled before-and-after study was completed with physiotherapists at three urban adult rehabilitation hospitals in Ontario, Canada. The 12-month intervention included group meetings, local champions, and health record modifications for a validated reactive balance measure. The primary outcome was the proportion of records with a documented reactive balance measure when balance was assessed pre-, during- and post-intervention. Secondary outcomes were changes in use, knowledge, and confidence post-intervention, differences across sites, and intervention satisfaction. RESULTS: Reactive balance was not measured in any of 211 eligible pre-intervention records. Thirty-three physiotherapists enrolled and 28 completed the study. Reactive balance was measured in 31% of 300 eligible records during-intervention, and in 19% of 90 eligible records post-intervention (p < 0.04). Knowledge and confidence significantly increased post-intervention (all p < 0.05). There were significant site differences in use during- and post-intervention (all p < 0.05). Most participants reported satisfaction with intervention content (71%) and delivery (68%). CONCLUSIONS: Reactive balance measurement was greater among participants during-intervention relative to the baseline, and use was partially sustained post-intervention. Continued study of intervention influences on clinical reasoning and exploration of site differences is warranted.

Development of a Theory-Based Intervention to Increase Clinical Measurement of Reactive Balance in Adults at Risk of Falls.

BACKGROUND: Effective balance reactions are essential for avoiding falls, but are not regularly measured by physical therapists. Physical therapists report wanting to improve reactive balance assessment, and theory-based approaches are recommended as the foundation for the development of interventions. This article describes how a behavior change theory for health care providers, the theoretical domains framework (TDF), was used to develop an intervention to increase reactive balance measurement among physical therapists who work in rehabilitation settings and treat adults who are at risk of falls. CASE DESCRIPTION: We employed published recommendations for using the TDF-guided intervention development. We identified what health care provider behavior is in need of change, relevant barriers and facilitators, strategies to address them, and how we would measure behavior change. In this case, identifying strategies required selecting both a reactive balance measure and behavior change techniques. Previous research had determined that physical therapists need to increase reactive balance measurement, and identified barriers and facilitators that corresponded to 8 TDF domains. A published review informed the selection of the Balance Evaluation Systems Test (Reactive Postural Responses Section) as addressing the barriers and facilitators, and existing research informed the selection of 9 established behavior change techniques corresponding to each identified TDF domain. OUTCOMES: The TDF framework were incorporated into a 12-month intervention with interactive group sessions, local champions, and health record modifications. Intervention effect can be evaluated using health record abstraction, questionnaires, and qualitative semistructured interviews. SUMMARY: Although future research will evaluate the intervention in a controlled study, the process of theory-based intervention development can be applied to other rehabilitation research contexts, maximizing the impact of this work.Video Abstract is available for more insights from the authors (see Supplemental Digital Content 1, http://links.lww.com/JNPT/A123).

Predicting cognitive decline in subjects at risk for Alzheimer disease by using combined cerebrospinal fluid, MR imaging, and PET biomarkers.

PURPOSE: To assess the extent to which multiple Alzheimer disease (AD) biomarkers improve the ability to predict future decline in subjects with mild cognitive impairment (MCI) compared with predictions based on clinical parameters alone. MATERIALS AND METHODS: All protocols were approved by the institutional review board at each site, and written informed consent was obtained from all subjects. The study was HIPAA compliant. Alzheimer's Disease Neuroimaging Initiative (ADNI) baseline magnetic resonance (MR) imaging and fluorine 18 fluorodeoxyglucose (FDG) positron emission tomography (PET) studies for 97 subjects with MCI were used. MR imaging-derived gray matter probability maps and FDG PET images were analyzed by using independent component analysis, an unbiased data-driven method to extract independent sources of information from whole-brain data. The loading parameters for all MR imaging and FDG components, along with cerebrospinal fluid (CSF) proteins, were entered into logistic regression models (dependent variable: conversion to AD within 4 years). Eight models were considered, including all combinations of MR imaging, PET, and CSF markers with the covariates (age, education, apolipoprotein E genotype, Alzheimer's Disease Assessment Scale-Cognitive subscale score). RESULTS: Combining MR imaging, FDG PET, and CSF data with routine clinical tests significantly increased the accuracy of predicting conversion to AD compared with clinical testing alone. The misclassification rate decreased from 41.3% to 28.4% (P < .00001). FDG PET contributed more information to routine tests (P < .00001) than CSF (P = .32) or MR imaging (P = .08). CONCLUSION: Imaging and CSF biomarkers can improve prediction of conversion from MCI to AD compared with baseline clinical testing. FDG PET appears to add the greatest prognostic information.

A pattern-based approach to bowel obstruction in the newborn.

Intestinal obstruction is common in newborns, and the radiologist plays a critical role in the care of these children. Diagnosing and managing the potentially obstructed newborn can be challenging, especially given the myriad underlying pathologies that range from benign to acutely life-threatening. A familiarity with the most common diagnoses is essential, but equally important to the radiologist is a systematic approach to management of the child in this setting. We propose an approach based on the recognition of eight radiographic patterns, five upper gastrointestinal examination (UGI) patterns and four contrast enema patterns. Recognition of these patterns directs further imaging when necessary and allows triage of children who can be managed medically, those requiring elective or urgent surgery and those requiring emergent surgery.

The Development and Integration of a Safety Officer Role to Facilitate Prevention of COVID-19 Virus Transmission in an Adult Inpatient Rehabilitation Setting Using Collaborative Change Leadership Methodology.

BACKGROUND: With the onset of the COVID-19 pandemic, a large urban academic hospital responded by creating the temporary role of a "Safety Officer (SO)." The key task of the SO role was to supervise staff donning and doffing personal protective equipment (PPE) and provide real-time feedback on their performance. The support for safe donning and doffing would contribute to staff well-being by reducing their fear of infection transmission. METHODS: A Collaborative Change Leadership (CCL) approach was used to facilitate the development, implementation, and evaluation of the role. This included an iterative feedback process with clinicians and safety officers to continually refine the role. FINDINGS: Feedback indicated value in the initiative as increasing staff confidence about preventing virus transmission, as well as their sense of safety at work. Areas for future improvement included additional communication strategies for interprofessional teams and external partners, as well as planning around logistics to better support the safety officers in performing this new, temporary role. CONCLUSIONS/APPLICATION TO PRACTICE: The Safety Officer role was able to help alleviate concerns regarding potential infection transmission and contribute positively to staff well-being.

Family-Reflections.com: Creating a parent-to-parent web-based tool regarding pediatric home ventilation.

BACKGROUND: The decision to initiate pediatric mechanical ventilation via tracheostomy ("home ventilation") is complex and parents often desire information from other parents who have faced this decision. However, parent-to-parent communication is challenging as it is difficult to connect new families to experienced families in ways that optimize informed, balanced decision-making. OBJECTIVE: Create a parent-to-parent web-based tool to support decision-making about pediatric home ventilation. PATIENT INVOLVEMENT: The tool was created based on interviews and feedback from parents. METHODS: We interviewed parents who previously chose for, or against, home ventilation for their child. Interview themes and family comments guided website development. Viewer feedback was solicited via an embedded survey in the tool. RESULTS: We created 6 composite character families to communicate 6 themes about home ventilation: 1) Considering treatment options, 2) Talking with medical team, 3) Impact on life at home, 4) Impact on relationships, 5) Experience for the child, and 6) If the child's life is short. Nine families who reviewed the draft tool felt it would have helped with their decision about home ventilation. Specifically, it supported families in thinking through what was "most important about their child's breathing problems" (7 of 9 parents) and feeling "more at peace with the decision" (8 of 9 parents). Between 6/1/20-12/31/22, nearly 5500 viewers have accessed the tool and 56 viewers completed the survey (including 13 families and 39 clinicians). Feedback from experienced families and clinicians reported the tool taught them something new. DISCUSSION: This novel parent-to-parent tool shows promise for expanding access to balanced, family-centered information about pediatric home ventilation. PRACTICAL VALUE: The diverse stories and decisions let parents access multiple family perspectives. The tool's focus is on family-centric information that parents reported was usually missing from clinician counseling. FUNDING: This work was supported by the National Palliative Care Research Center.

A prospective analysis of lymphocyte phenotype and function over the course of acute sepsis.

INTRODUCTION: Severe sepsis is characterized by an initial hyper-inflammatory response that may progress to an immune-suppressed state associated with increased susceptibility to nosocomial infection. Analysis of samples obtained from patients who died of sepsis has identified expression of specific inhibitory receptors expressed on lymphocytes that are associated with cell exhaustion. The objective of this study was to prospectively determine the pattern of expression of these receptors and immune cell function in patients with acute sepsis. METHODS: Twenty-four patients with severe sepsis were enrolled within 24 hours of the onset of sepsis, as were 12 age-matched healthy controls. Peripheral blood was obtained at enrollment and again seven days later. Immune cell subsets and receptor expression were extensively characterized by quantitative flow cytometry. Lymphocyte function was assayed by stimulated cytokine secretion and proliferation assays. Results were also correlated to clinical outcome. RESULTS: At the onset of severe sepsis, patients had decreased circulating innate and adaptive immune cells and elevated lymphocyte expression of receptors associated with cell activation compared to controls. Samples analyzed seven days later demonstrated increased expression of the inhibitory receptors CTLA4, TIM-3 and LAG-3 on T lymphocytes accompanied by decreased expression of the IL-7 receptor. Functional assays revealed impaired secretion of interferon γ following stimulation in vitro, which was reversible by incubation overnight in fresh media. Impaired secretion of IFNγ correlated with death or development of secondary infection. CONCLUSIONS: Lymphocytes from patients with acute sepsis upregulate expression of receptors associated with cell exhaustion, which may contribute to the immune suppressed state that occurs in protracted disease. Therapy that reverses T cell exhaustion may restore immune function in immunocompromised patients and improve survival in sepsis.

The cognitive augmented mobility program (CAMP): feasibility and preliminary efficacy.

BACKGROUND: Best evidence suggests incorporating task-specific training, aerobics, and strengthening to improve mobility and gait in persons with stroke (PWS). Related research suggests incorporating cognitive problem-solving strategy training may lead to better skill maintenance and transfer. The present study examined the feasibility and preliminary outcomes of an evidence-based cognitive augmented mobility program (CAMP) for PWS. METHODS: A single-arm pre-post and follow-up design was conducted, in two blocks (NCT03683160). Participants completed an exercise intervention with an integrated cognitive strategy component and had weekly 1:1 sessions with a physiotherapist trained in Cognitive Orientation to daily Occupational Performance (CO-OP), focusing on goal practice and cognitive strategy use. CAMP consisted of a pre-intervention education and goal-setting session and 16 90-minute sessions held in a group format, 2x/wk for 8 weeks. Assessments were conducted 1 week prior to beginning the intervention, 1 week post-intervention, and at a 4-5 week follow-up. RESULTS: CAMP was found to be feasible with 96% session attendance and strong participant satisfaction. Pre to post-CAMP intervention, a large effect was found for endurance (r = 0.53), balance (r = 0.59), mobility (r = 0.63) and goal attainment (r = 0.63) indicators, and large effects were maintained at follow-up for balance, mobility, and goal attainment. A medium effect was found for functional independence (r = 0.38), gait speed (r = 0.39), and balance confidence (r = 0.38). A small effect was found for participation (r = 0.27) and overall stroke recovery (r = 0.25). CONCLUSIONS: Preliminary results suggest CAMP is feasible; there may be a beneficial effect of combining best evidence for mobility and fitness with cognitive strategy training. The positive results for skill maintenance and transfer suggest further investigation is warranted.

Limited utility of exercise-stress testing to prevent T-wave oversensing in pediatric internal cardioverter defibrillator recipients.

BACKGROUND: Inappropriate internal cardioverter defibrillator (ICD) therapies may result from T-wave oversensing (TWOS) during exertion in children. The aim of this study was to evaluate the utility of an exercise treadmill test to predict inappropriate ICD therapies secondary to TWOS. METHODS: Eligible pediatric ICD recipients underwent exercise-stress testing with concomitant evaluation of all intracardiac electrograms. Double counting at a programmed sensitivity of 0.3 mV was considered indicative of TWOS. Patients were prospectively followed for 2 years and censored at either the development of an inappropriate ICD therapy secondary to TWOS or at the time of ICD revision. RESULTS: Nineteen patients (age: 13.8 ± 3.2 years) underwent exercise testing (median time from ICD implant: 1.5 years, range 2-4.3 years). Two patients were identified with TWOS during the stress test and had a clinically inappropriate ICD discharge within 2 weeks despite a sensitivity adjustment to 0.6 mV. One individual had an inappropriate ICD discharge from TWOS 11 months following an initial uneventful exercise-stress test. CONCLUSIONS: Inappropriate ICD therapies from TWOS relate to a reduction in the intrinsic R wave or augmentation of the T wave during exertion. While intracardiac electrogram assessment during stress testing may aid in the early recognition of TWOS, it did not absolutely translate to a reduction in the incidence of inappropriate ICD shocks.

Antitarget Selectivity and Tolerability of Novel Pyrrolo[2,3-d]pyrimidine RET Inhibitors.

The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

Efficacy and Tolerability of Pyrazolo[1,5-a]pyrimidine RET Kinase Inhibitors for the Treatment of Lung Adenocarcinoma.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS).

Previous kinetic studies demonstrated that nucleotide-derived conformational changes regulate function in the COOH-terminal Src kinase. We have employed enhanced methods of hydrogen-deuterium exchange-mass spectrometry (DXMS) to probe conformational changes on CSK in the absence and presence of nucleotides and thereby provide a structural framework for understanding phosphorylation-driven conformational changes. High quality peptic fragments covering approximately 63% of the entire CSK polypeptide were isolated using DXMS. Time-dependent deuterium incorporation into these probes was monitored to identify short peptide segments that exchange differentially with solvent. Regions expected to lie in loops exchange rapidly, whereas other regions expected to lie in stable secondary structure exchange slowly with solvent implying that CSK adopts a modular structure. The ATP analog, AMPPNP, protects probes in the active site and distal regions in the large and small lobes of the kinase domain, the SH2 domain, and the linker connecting the SH2 and kinase domains. The product ADP protects similar regions of the protein but the extent of protection varies markedly in several crucial areas. These areas correspond to the activation loop and helix G in the kinase domain and several inter-domain regions. These results imply that delivery of the gamma phosphate group of ATP induces unique local and long-range conformational changes in CSK that may influence regulatory motions in the catalytic pathway.

Dynamic coupling between the SH2 domain and active site of the COOH terminal Src kinase, Csk.

The SH2 domain is required for high catalytic activity in the COOH-terminal Src kinase (Csk). Previous solution studies suggest that a short peptide sequence, the SH2-kinase linker, provides a functional connection between the active site and the distal SH2 domain that could underlie this catalytic phenomenon. Substitutions in Phe183 (tyrosine, alanine, and glycine), a critical hydrophobic residue in the linker, result in large decreases in substrate turnover and large increases in the K(m) for ATP. Indeed, F183G possesses kinetic parameters that are similar to that for a truncated form of Csk lacking the SH2 domain, suggesting that a single mutation disrupts communication between this domain and the active site. Based on equilibrium and stopped-flow fluorescence experiments, the elevated K(m) values for the mutants are due to changes in the rates of phosphoryl transfer and not to reduced ATP-binding affinities. Based on hydrogen-deuterium exchange experiments, glycine substitution reduces flexibility in several polypeptide regions in Csk, tyrosine substitution increases flexibility, and alanine substitution leads to mixed effects compared to wild-type. Normal mode analysis indicates that Phe183 and its environment are under strain, a theoretical finding that supports the results of mutations. Overall, the data indicate that domain-domain interactions, controlled through the SH2-kinase linker, provide a dynamic balance within the Csk framework that is ideal for efficient phosphoryl transfer in the active site.

Use of Nintendo Wii Fit™ in the rehabilitation of outpatients following total knee replacement: a preliminary randomised controlled trial.

OBJECTIVES: To determine whether Nintendo Wii Fit™ is an acceptable adjunct to physiotherapy treatment in the rehabilitation of balance, lower extremity movement, strength and function in outpatients following total knee replacement. DESIGN: Preliminary randomised controlled trial. SETTING: Outpatient department of a rehabilitation hospital. PARTICIPANTS: Adult outpatients following total knee replacement. INTERVENTIONS: The study group received a physiotherapy session followed by 15minutes of Wii Fit gaming activities. The games encouraged lateral and multidirectional weight shifting, and provided visual feedback regarding postural balance. The control group received a physiotherapy session followed by 15minutes of lower extremity strengthening and balance training exercises. MAIN OUTCOME MEASURES: Length of outpatient rehabilitation, 2-minute walk test, knee range of motion, timed standing, Activity-specific Balance Confidence Scale, Lower Extremity Functional Scale and Numeric Pain Rating Scale, all measured on admission and every 2 weeks until discharge. A patient satisfaction survey was completed at discharge. RESULTS: Seventeen males (34%) and 33 females (66%) with a mean age of 68 (standard deviation 11) years participated in the study. No significant differences in age, gender, days since surgery or length of outpatient rehabilitation were found between the groups. In addition, there were no significant differences in pain (P=0.220), knee flexion (P=0.951), knee extension (P=0.492), walking speed (P=0.855), timed standing tasks (P=0.289), Lower Extremity Functional Scale (P=0.079), Activity-specific Balance Confidence Scale (P=0.523) or patient satisfaction with therapy services (P=0.201) between the groups. CONCLUSIONS: Wii Fit is potentially acceptable as an adjunct to physiotherapy intervention for outpatients following total knee replacement, provided the games chosen challenge balance and postural control, and use the lower extremities. Further research is needed to establish whether video games as a therapy adjunct increase patient motivation and compliance with rehabilitation goals. ClinicalTrials.gov ID: NCT01548664.

Structure of Ddn, the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis involved in bioreductive activation of PA-824.

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.

Easy access to firearms: juveniles' risks for violent offending and violent victimization.

Keeping firearms at home may increase personal safety but it may also increase the risk of injury. This study uses data from three waves of the National Longitudinal Study of Adolescent Health to assess the extent to which adolescents' easy access to firearms at home increases the risk of violent offending and violent victimization. Access to firearms was higher for males, Whites, and adolescents having two parents, especially fathers. Current access to firearms at home significantly increased the odds of both violent offending and violent victimization, even after controlling for prior access, prior offending, and prior victimization. This relationship persisted into early adulthood; access to firearms still significantly increased the odds of violent offending and violent victimization.