Massively parallel base editing to map variant effects in human hematopoiesis.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

DNA origami signposts for identifying proteins on cell membranes by electron cryotomography.

Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce "molecular signposts" that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.

Trauma-informed physiotherapy and the principles of safety, trustworthiness, choice, collaboration, and empowerment: a qualitative study.

INTRODUCTION: Trauma is common and may lead to lasting adverse effects on health. Trauma-informed practice does not treat trauma but uses a strengths-based approach to encourage engagement in services. OBJECTIVE: To understand how physiotherapy attends to trauma-informed principles. METHODS: This qualitative ethnographic study was set in an Australian hospital. Three data collection methods were used, including observations of clinical practice, interactive reflexive group discussions with physiotherapists, and interviews with patients. Data analysis included an initial inductive phase followed by thematic mapping to trauma-informed principles. Critical reflexivity was used throughout to examine how the authors' perspectives and assumptions affected the analysis. RESULTS: Twelve observations of consultations, ten interviews with people receiving physiotherapy, and five group discussions with physiotherapists were conducted. Themes produced within each of five principles of trauma-informed care included: Safety: not just a number, uncertainty beyond managing physical risks, upbeat approach as default needs balance, pragmatic environments inadequate; Trustworthiness: touch needs further consideration, assumed consent; Choice: limited options; Collaboration: let's do it together, variable consideration of the patient as expert, task focus, pushing the "right" treatment, missing insight into power imbalance; Empowerment: extending function and independence, building nonphysical skills but lack of clarity. CONCLUSION: Physiotherapy incorporates crucial aspects of trauma-informed care, but opportunities exist to enhance physiotherapists' skills and knowledge, particularly in relation to non-physical safety considerations.

A noncoding regulatory variant in IKZF1 increases acute lymphoblastic leukemia risk in Hispanic/Latino children.

Hispanic/Latino children have the highest risk of acute lymphoblastic leukemia (ALL) in the US compared to other racial/ethnic groups, yet the basis of this remains incompletely understood. Through genetic fine-mapping analyses, we identified a new independent childhood ALL risk signal near IKZF1 in self-reported Hispanic/Latino individuals, but not in non-Hispanic White individuals, with an effect size of ∼1.44 (95% confidence interval = 1.33-1.55) and a risk allele frequency of ∼18% in Hispanic/Latino populations and <0.5% in European populations. This risk allele was positively associated with Indigenous American ancestry, showed evidence of selection in human history, and was associated with reduced IKZF1 expression. We identified a putative causal variant in a downstream enhancer that is most active in pro-B cells and interacts with the IKZF1 promoter. This variant disrupts IKZF1 autoregulation at this enhancer and results in reduced enhancer activity in B cell progenitors. Our study reveals a genetic basis for the increased ALL risk in Hispanic/Latino children.

Factors influencing selection for rehabilitation after stroke: a questionnaire using case scenarios to investigate physician perspectives and level of agreement.

OBJECTIVES: To explore the key factors involved in decision making when selecting patients for rehabilitation after stroke and to examine the level of agreement among physician assessors regarding admission to rehabilitation. DESIGN: Questionnaire. SETTING: Health services with rehabilitation units in Victoria, Australia. PARTICIPANTS: Rehabilitation unit physicians. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURE: Questionnaire with 2 components: the clinical and nonclinical factors that influence decision making and clinical case scenarios. RESULTS: Responses were received from 17 physicians from 12 of the 18 health services in Victoria. The most influential clinical factors listed by the respondents were prognosis, social supports, anticipated discharge destination, age, and anticipated length of stay. Key nonclinical factors were prioritization of internal health service referrals, patient's residence, and workforce capacity. Analysis of the clinical scenarios of patients with severe stroke showed that there was variability in the responses, with high levels of agreement for some cases and low levels for others. Almost all respondents agreed that prognosis was a key factor, yet, within the case scenarios, the reasons given for accepting or not accepting the patient demonstrated different opinions on the prognosis of the case presented. CONCLUSIONS: The decision-making processes in selection for rehabilitation and the factors that influence that decision require further investigation to optimize the use and outcomes from rehabilitation resources.

How much and where: assessment of knowledge level of the application of cricoid pressure.

INTRODUCTION: Application of cricoid pressure is a frequently used technique in both rapid sequence intubation in multiple settings and in a more controlled setting in the operating room. In a survey of emergency department personnel performed at the University of Michigan, it was found that there is a knowledge deficit in the recommended force and the anatomic localization of cricoid pressure. Participants in the original study, which included emergency nurses, medical residents, and attending physicians, rated their training in cricoid pressure as poor or nonexistent. A review of the literature shows that, although cricoid pressure is used during endotracheal intubation to protect against regurgitation of gastric contents, many people applying cricoid pressure do not have a good knowledge of where to apply the pressure or how much pressure to apply to be effective. Because cricoid pressure is applicable in areas other than the emergency department, our study surveys personnel in emergency medical services/flight crew; emergency, intensive care unit, and operating room nurses; and respiratory therapists. Even though the use of cricoid pressure is no longer recommended, it is still routinely used. Although applying cricoid pressure is a simple procedure, persons using it must be thoroughly trained and retrained to prevent complications. METHODS: When we replicated the University of Michigan study at a 254-bed tertiary care facility, a potential of 325 staff members were given access to an online survey using the questions in the original survey. Staff were assigned to a HealthStream module and sent an invitation through their employee e-mail account. The module included a link to the questionnaire, and demographic data were gathered. The module was optional and results confidential. RESULTS: Operating room nurses were most likely to receive supervised instruction on anesthetized patients. These operating room nurses also showed the highest overall knowledge level about the application technique of cricoid pressure. DISCUSSION: There continues to be a lack of knowledge about the application of cricoid pressure during intubation. There is an opportunity for collaboration between staff and academic educators to allow for additional theoretical as well as hands-on practice.

The utility of short-term goal achievement as an early indicator of discharge destination in people admitted to neurological rehabilitation with severe functional deficits.

PURPOSE: This study investigates whether short-term goal achievement in the early phase of neurological rehabilitation is an accurate indicator of discharge destination in patients with severe disability in comparison to change in scores in the motor domain of the Functional Independence Measure (FIM motor). METHOD: A prospective observational cohort study. PARTICIPANTS: A consecutive sample of 53 patients admitted to rehabilitation with a neurological diagnosis and FIM motor score below 47.Measures: Short-term goal achievement and FIM motor change in the first 2 weeks following admission and discharge destination. RESULTS: Short-term goal achievement showed good prognostic utility [area under the curve (AUC) of 0.75; 95% confidence intervals (CI) 0.6, 0.89] for discharge destination, dichotomized as home or semi-independent living versus nursing home care, similar to that demonstrated by change in FIM motor scores (AUC of 0.69; 95% CI 0.55, 0.84), p = 0.55. A cut-off was established for short-term goal achievement at more than half of goals achieved, with an AUC of 0.73 (95% CI 0.58, 0.87); sensitivity 71.4% and specificity 74.4%. CONCLUSIONS: Short-term goal achievement in the early phase post-admission is a good indicator of the person's potential to return home.Implications for RehabilitationShort-term goal setting should be a key practice feature of neurological rehabilitation.Goal achievement is an indicator of a person's potential to return home or to semi-independent living in people with severe neurological deficits.Evaluation of short-term goal achievement may inform the ongoing rehabilitation program and discharge planning.

Tryptophan in the pore of the mechanosensitive channel MscS: assessment of pore conformations by fluorescence spectroscopy.

Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions. We show that Trp can be inserted in place of Leu at the two pore seal positions, Leu(105) and Leu(109), resulting in functional channels. Using Trp(105) as a probe, we demonstrate that the A106V mutation causes a modified conformation in the purified channel protein consistent with a more open state in solution. Moreover, we show that solubilized MscS changes to a more open conformation in the presence of phospholipids or their lysoforms.

The critical role of S-lactoylglutathione formation during methylglyoxal detoxification in Escherichia coli.

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.

Sensing bilayer tension: bacterial mechanosensitive channels and their gating mechanisms.

Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL. Consequently, in cells, gating has severe consequences for energetics and homoeostasis, since membrane depolarization and modification of cytoplasmic ionic composition is an immediate consequence. Protection against disruption of cellular integrity, which is the function of the major channels, provides a strong evolutionary rationale for possession of such disruptive channels. The elegant crystal structures for these channels has opened the way to detailed investigations that combine molecular genetics with electrophysiology and studies of cellular behaviour. In the present article, the focus is primarily on the structure of MscS, the small mechanosensitive channel. The description of the structure is accompanied by discussion of the major sites of channel-lipid interaction and reasoned, but limited, speculation on the potential mechanisms of tension sensing leading to gating.

Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript.

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2). THC2 is generally caused by single nucleotide variants that prevent silencing of ANKRD26 expression during hematopoietic differentiation. Short-read whole-exome and genome sequencing approaches were unable to identify a causal variant in this family. Using long-read whole-genome sequencing, a large complex structural variant involving a paired-duplication inversion was identified. Through functional studies, we show that this structural variant results in a pathogenic gain-of-function WAC-ANKRD26 fusion transcript. Our findings illustrate how complex structural variants that may be missed by conventional genome sequencing approaches can cause human disease.

Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel.

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.

Is achievement of short-term goals a valid measure of patient progress in inpatient neurological rehabilitation?

OBJECTIVE: To determine whether the use of short-term goal setting is effective in monitoring patient progress, with regard to achievement of rehabilitation goals for discharge and predicted length of hospital stay. DESIGN: A prospective observational cohort study. SETTING: An inpatient rehabilitation unit co-located with a large tertiary teaching hospital. SUBJECTS: Consecutive cases admitted to rehabilitation with a neurological condition and a planned length of stay of three weeks or longer. PROCEDURE: Discharge and short-term goals and predicted length were set in the initial team meeting. Goals were set, and achievement scored, in the domains of health, social functioning, communication and cognition, mobility and activities of daily living (ADLs) and a composite goal of global function. Actual length of stay and discharge destination were recorded. RESULTS: Data from 53 consecutive cases were examined, with 45 cases returning home. The median length of stay was 49 days (interquartile (IQ) 30-74). Significant correlations (P<0.05) were demonstrated between short-term goal achievement and discharge goals for continence (rho = 0.62), transfers (rho = 0.53), personal activities of daily living (rho = 0.47) and global function (rho = 0.62). For those that returned home, there were significant correlations (P<0.05) between adherence to predicted length of stay and achieving their initial goals in transfers (rho = 0.44), walking (rho = 0.51) and global function (rho = 0.55). CONCLUSION: Short-term goal setting is a valid measure of patient progress in inpatient neurological rehabilitation and can be used to identify patients who are not progressing as anticipated, facilitating review of the rehabilitation plan.

Recruitment of Youth Living With HIV to Optimize Adherence and Virologic Suppression: Testing the Design of Technology-Based Community Health Nursing to Improve Antiretroviral Therapy (ART) Clinical Trials.

BACKGROUND: Despite advances in HIV diagnosis and treatment, adolescents and young adults 12-25 years old have high HIV incidence, poor engagement and retention in treatment, and low rates of adherence and virologic suppression when compared to their older counterparts. HIV has emerged as a chronic disease for which antiretroviral therapy (ART) adherence is critical for virologic suppression and long-term survival. Virologic suppression has been elusive for many youth with HIV (YHIV). Novel strategies designed to facilitate health care systems' support for YHIV between medical visits are essential for improving ART adherence, virologic suppression, and long-term survival. OBJECTIVE: The aim of this study is to compare the effectiveness of a technology-enhanced community health nursing intervention (TECH2CHECK) to a standard of care (SOC) control group for improving ART adherence and subsequent viral suppression using a randomized trial design. The objectives are to assess the feasibility, acceptability, and cost-effectiveness of TECH2CHECK as compared to SOC for management of HIV in the outpatient setting and to examine the sustainability of self-care behavior, adherence, and virologic suppression among youth following the intervention period. METHODS: We will recruit 120 adherence-challenged YHIV being followed at clinics specializing in HIV care in the Baltimore-Washington metropolitan area and in Jacksonville. Eligible participants complete an audio, computer-assisted self-interview and are randomized to either TECH2CHECK intervention or the SOC (60 participants in each arm). The primary outcome of interest is virologic suppression (viral load <20 copies/mL) and improved treatment adherence. Participants in the intervention arm receive community health nursing visits at 2 weeks, 6 weeks, 10 weeks, 14 weeks, and 26 weeks. The intervention arm also receives SMS messaging comprising daily adherence and appointment reminders and positive reinforcement for medication adherence daily for 2 weeks, on alternate days for 2 weeks, thrice weekly for 1 month, weekly for 3 months, and every 2 weeks for the rest of the study duration. The control group receives appointment reminders and SOC per clinic protocol. Exploratory analysis will be conducted to determine differences in medication adherence and virologic suppression in the 2 arms and to assess cost-effectiveness and study feasibility and acceptability. RESULTS: In the first 23 months of the study (July 2018-April 2020), 56 (55%) of 102 eligible patients were enrolled and randomized. At present, participating youths are primarily African American (53/56, 95%), male (37/56, 66%), and ≥18 years old (53/56, 95%). Follow-up study visits, as required per the protocol, have been completed by 77% (43/56), 94% (45/48), 95% (37/39), 96% (24/25), and 100% (10/10) of participants at the 1-month, 3-month, 6-month, 12-month, and 18-month follow-ups, respectively. CONCLUSIONS: Preliminary accrual and retention data suggest that TECH2CHECK is feasible and acceptable. TRIAL REGISTRATION: ClinicalTrials.gov NCT03600103 https://clinicaltrials.gov/ct2/show/NCT03600103. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/23480.

Goal achievement in the six months after inpatient rehabilitation for stroke.

PURPOSE: The purpose of the project was to identify characteristics associated with successful re-integration into the community post-inpatient rehabilitation after stroke. A key issue was determining re-integration from the person's perspective, taking into account the person's preferred lifestyle choices. RESEARCH DESIGN: A prospective exploratory follow up study. PARTICIPANTS: A consecutive sample of 45 participants discharged from IP rehabilitation following stroke and 23 carers associated with the participants. MEASURES: Goal attainment scaling was utilised to determine successful community integration. Factors that may have contributed to goal achievement were measured prior to discharge and at 6 months post-discharge. Scales used include the Functional Independence Measure, Mini Mental test, the CES-D depression scale and a self-efficacy scale, Strategies Used by People to Promote Health. London Handicap Scale scores and Carer Strain Index were collected at 6 months. RESULTS: Twenty percent of participants achieved all their goals. Significant correlations were observed between goal achievement score and concurrent measures of physical function, depression and self efficacy at 6 months post-discharge. CONCLUSIONS: Stroke survivors who achieved their goals were less likely to be depressed, showed stronger self efficacy beliefs and more positive perceptions of their participation in everyday and community life.

Using Lean Performance Improvement for Patient-Centered Medical Home Transformation at an Academic Public Hospital.

Although Lean performance improvement (PI) has been used in health care for more than 15 years, little is known about how Lean has been used in Patient-Centered Medical Home (PCMH) transformation. We describe our experience implementing Lean in our safety-net, primary care teaching clinic. To advance high value care, a culture of systematic, sustainable PI methods needed to be integrated into primary care and taught to resident physicians. Clinic leadership were trained in Lean methods, protected time was dedicated to PI for a stable, interdisciplinary team, then visual management was introduced, and resident physicians were integrated into the clinic's PI initiatives. Self-assessment using the PCMH Assessment tool demonstrated improvement in core features of the PCMH model. Process outcomes also revealed successful, sustainable integration of Lean into our primary care clinic and resident training, and early findings show improvements in clinical quality outcomes. Patient survey outcomes demonstrate improvement in patient experience. Lean can be used successfully to promote PCMH transformation and create a culture of continuous PI in an academic, safety-net primary care setting.

Interaction of the Mechanosensitive Channel, MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets.

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein-lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even "conservative" hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4.

Determining discharge destination in geriatric evaluation and management units: Is progressive goal attainment a better early indicator of discharge destination than improvement in functional independence measure scores?

AIM: To determine whether progress in goal attainment is a more responsive measure of functional improvement than the Functional Independence Measure, and whether it is a more accurate predictor of discharge destination in the early phase of a Geriatric Evaluation and Management episode. METHODS: The present study was a prospective audit of goal achievement, functional ability and discharge destination. A total of 83 patients were included in this study. Logistic regression was carried out with the outcome variable discharge destination (home vs residential care), and the predictors being goal attainment, age at admission and change in the Functional Independence Measure motor function scale from admission to 2 weeks. RESULTS: Of the predictors, goal attainment was the only significant predictor. The overall amount of variability in the discharge destination explained by the predictors was 25% (Nagelkerke pseudo R2 ). CONCLUSIONS: Progressive goal attainment might be a better predictor of improvement in function and discharge destination than change in the Functional Independence Measure. Geriatr Gerontol Int 2018; 18: 1058-1063.