A model of hope as a protective measure to lower burnout and secondary traumatic stress among employees working in developmental and intellectual disabilities services.

BACKGROUND: Burnout and secondary traumatic stress (STS) are problems for the workforce supporting people with developmental disabilities. This study investigated hope as a potential protective resource for burnout and STS among the developmental disability services workforce. METHOD: One hundred and fifty-two non-supervisor caseworkers from a state agency, developmental disabilities division were recruited to participate in an anonymous web-based survey. RESULTS: The analyses showed that hope was negatively associated with the three dimensions of STS (intrusion, avoidance, and arousal) and burnout. Controlling for tenure in the workforce and STS, the results of the hierarchical regression analyses showed that hope accounted for a significant incremental variance to burnout. CONCLUSION: These findings provide support for emerging literature showing hope as a protective resource to workforce burnout.

Incentivizing Students to Learn More About Gerontological Practice.

Enticing students to expand their knowledge of aging-related issues and careers can be fraught with challenges. Intrinsic and curricula-related factors associated with pursuit of aging-related careers have been identified, but little evidence exists demonstrating the effectiveness of external factors at motivating students to learn more about gerontological practice. This brief report presents findings from a survey of 214 students enrolled in at a single university in the mid-west to assess how likely they would be motivated to learn more about aging by twelve possible incentives with additional opportunities to write in other thoughts. Credit toward required field work, financial incentives such as stipends, scholarships, tuition waivers, and raffles were the most frequently mentioned incentives. Some variation was noted based on race/ethnicity, age, and program of study. Themes emerging from other suggestions provided by students included curricula enhancements, employment incentives, and the suggestion that nothing could entice some students. Findings can be used by scholars in program development and funding requests.

Clinical outcomes in the biomarkers of ketamine (Bio-K) study of open-label IV ketamine for refractory depression.

OBJECTIVE: We conducted an open-label clinical trial ("Bio-K") using IV ketamine for treatment-resistant depression to identify biomarkers linked to remission. Here, we report the clinical efficacy and side effect outcomes of Bio-K. METHODS: Across 4 US sites, 75 patients ages 18-65 with treatment-refractory unipolar or bipolar depression received 3 IV ketamine infusions over an 11-day period. Key exclusion criteria were psychotic symptoms, significant substance abuse, unstable medical conditions, and any use of cannabis. Pre-existing antidepressant medication was maintained. Primary outcome was remission as measured by Montgomery-Asberg Depression Rating Scale (MADRS), with secondary outcome of 50 % reduction in Beck Suicide Scale score. Safety monitoring and varying durations of infusions were also key parameters. RESULTS: Using remission as MADRS score <10, after 3 infusions 52 % achieved remission, with 67 % achieving response. Of those achieving response after a single infusion, 66 % (22 of 33) reached remission after 3 infusions, while 40 % (16 of 40) non-responders after the first infusion went on to achieve remission after 3 infusions. Only 20 % of non-responders after 2 infusions achieved remission. Most (81 %) participants had significant suicidal ideation at baseline; of these, two-thirds (67 %) experienced at least a 50 % reduction in suicidality. Side effects were minimal. Uniquely, we had three different types of infusion categories, with individuals receiving: (1) slow (100-min) infusions only or (2) regular (40-min) infusions only or (3) a mix of infusion durations. These three infusion groups showed comparable safety and efficacy. Exploration of clinical factors revealed no link between BMI, age, or gender to remission. CONCLUSIONS: The consistency of outcomes across 4 clinical sites and across multiple instruments, suggests high acute efficacy and safety of IV ketamine for serious depressive episodes. Duration of infusion did not alter outcomes. Meaningfully, 40 % of non-responders after a single infusion did reach remission subsequently, while only 20 % of non-responders after 2 infusions achieved remission, suggesting early response is suggestive for eventual remission. Our data on varying ketamine infusion duration adds novel insights into the clinical administration of this new treatment for refractory and severe patients. Our limitations included a lack of a control group, necessitating caution about conclusions of efficacy, balanced by the utility of reporting "real-world" outcomes across multiple clinical sites. We could also not separately analyze results for bipolar disorder due to small numbers. Together, the Bio-K clinical results are promising and provide significant sample sizes for forthcoming biological markers analyses.

Measurement of Information Transfer During Simulated Sequential Complete Shift-to-Shift Intraoperative Handoffs.

Objective: To determine information transfer during simulated shift-to-shift intraoperative anesthesia handoffs and the benefits of using a handoff tool. Patients and Methods: Anesthesiology residents and faculty participating in simulation-based education in a simulation center on April 6 and 20, 2017, and April 11 and 25, 2019. We used a fixed clinical scenario to compare information transfer in multiple sequential simulated handoff chains conducted from memory or guided by an electronic medical record generated tool. For each handoff, 25 informational elements were assessed on a discrete 0-2 scale generating a possible information retention score of 50. Time to handoff completion and number of clarifications requested by the receiver were also determined. Results: We assessed 32 handoff chains with up to 4 handoffs per chain. When both groups were combined, the mean information retention score was 31 of 50 (P<.001) for the first clinician and declined by an average of 4 points per handoff (P<.001). The handoff tool improved information retention by almost 7 points (P=.002), but did not affect the rate of information degradation (P=.38). Handoff time remained constant for the intervention group (P=.67), but declined by 2 minutes/handoff (P<.001) in the control group, which required 7 more clarifications/handoff (P=.003). In the control group, 7 of 16 (44%) handoff chains contained one or more information retention scores below the lowest score of the entire intervention group (P=.007). Conclusion: Clinical handoffs are accompanied by degradation of information that is only partially reduced by use of a handoff tool, which appears to prevent extremes of information degradation.

Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

SIGNIFICANCE: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. AIM: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. APPROACH: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825's impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. RESULTS: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. CONCLUSIONS: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Examining novice anaesthesia trainee simulation performance: a tale of two clusters.

Introduction: Understanding performance differences between learners may provide useful context for optimising medical education. This pilot study aimed to explore a technique to contextualise performance differences through retrospective secondary analyses of two randomised controlled simulation studies. One study focused on speaking up (non-technical skill); the other focused on oxygen desaturation management (technical skill). Methods: We retrospectively analysed data from two independent simulation studies conducted in 2017 and 2018. We used multivariate hierarchical cluster analysis to explore whether participants in each study formed homogenous performance clusters. We then used mixed-design analyses of variance and χ2 analyses to examine whether reported task load differences or demographic variables were associated with cluster membership. Results: In both instances, a two-cluster solution emerged; one cluster represented trainees exhibiting higher performance relative to peers in the second cluster. Cluster membership was independent of experimental allocation in each of the original studies. There were no discernible demographic differences between cluster members. Performance differences between clusters persisted for at least 8 months for the non-technical skill but quickly disappeared following simulation training for the technical skill. High performers in speaking up initially reported lower task load than standard performers, a difference that disappeared over time. There was no association between performance and task load during desaturation management. Conclusion: This pilot study suggests that cluster analysis can be used to objectively identify high-performing trainees for both a technical and a non-technical skill as observed in a simulated clinical setting. Non-technical skills may be more difficult to teach and retain than purely technical ones, and there may be an association between task load and initial non-technical performance. Further study is needed to understand what factors may confer inherent performance advantages, whether these advantages translate to clinical performance and how curricula can best be designed to drive targeted improvement for individual trainees.

Evaluation of 3 Cognitive Load Measures During Repeated Simulation Exercises for Novice Anesthesiology Residents.

INTRODUCTION: Maintaining an appropriate level of cognitive load during simulations is crucial to optimize learning. We evaluated 3 subjective measures of cognitive load in a simulated operating room (OR) context across multiple scenarios of varying complexity. METHODS: This observational study of 25 first-year anesthesiology residents took place during a 5-day simulation "Boot Camp." Each day, participants completed 2 different high-fidelity scenarios in a fully equipped simulated OR. After each simulation, participants completed 3 cognitive load measures: the Paas scale, NASA Task Load Index (TLX), and Cognitive Load Component (CLC) questionnaire. Two-way repeated-measures and mixed-design analyses of variance, with the cognitive load measures and scenarios as independent factors, were used to determine the effect of using different measures to report cognitive load. RESULTS: Cognitive load scores reported by all measures correlated significantly with one another (P < 0.01): TLX and Paas (r = 0.65); Paas and CLC (r = 0.63); and TLX and CLC (r = 0.61). The CLC subscale scores (intrinsic, extraneous, germane) also correlated significantly with composite TLX and Paas scores (P < 0.01). Scenarios and measures displayed significant interaction: F(10, 210) = 3.01, P = 0.001. Participants reported highest overall cognitive load using the Paas scale. CONCLUSIONS: All cognitive load measures were sensitive to scenario variability and showed similar fluctuation patterns across the 10 scenarios. The findings suggest that cognitive load measures can help create benchmarks based on learner perceptions of cognitive burden for different simulation scenarios.

The sim must go on: adapting resident education to the COVID-19 pandemic using telesimulation.

The COVID-19 pandemic and social distancing rules necessitated the suspension of all in-person learning activities at our institution. Consequently, distance learning became essential. We adapted a high-fidelity immersive case-based simulation scenario for telesimulation by using the virtual meeting platform Zoom® to meet our curricular needs. The use of telesimulation to teach a complex case-based scenario is novel. Two cohorts of anesthesiology residents participated 2 weeks apart. All learners were located at home. Four faculty members conducted the telesimulation from different locations within our simulation center in the roles of director, simulation operator, confederate anesthesiologist, and confederate surgeon. The anesthesiologist performed tasks as directed by learners. The scenario was divided into four scenes to permit reflection on interventions/actions by the participants based on the clinical events as the scenario progressed, to facilitate intermittent debriefing and learner engagement. All residents were given a medical knowledge pretest before the telesimulation and a posttest and learner satisfaction survey at the conclusion. The scenario was authentic and immersive, represented an actual case, and provided the opportunity to practice lessons that could be applied in the clinical setting. Participants rated telesimulation a reasonable substitution for in-person learning and expressed gratitude for continuation of their simulation-based education in this format during the pandemic. Participants in the second cohort reported feeling more engaged (p = 0.008) and stimulated to think critically (p = 0.003). Audio quality was the most frequently noted limitation. Fifty-three residents completed both pre- and posttests. The two cohorts did not differ in knowledge pretest scores (62% vs 60%, p = 0.80) or posttest scores (78% vs. 77%, p = 0.87). Overall, knowledge scores improved with the telesimulation intervention (pretest mean = 61% [SD = 14%]; posttest mean = 78% [SD = 12%]; t (41) = - 7.89, p < 0.001). Thus, using a Zoom format, we demonstrated the feasibility of adapting a complex case for telesimulation and effective knowledge gain. Furthermore, we improved our process in real time based on participant feedback. Participants were satisfied with their learning experience, suggesting that this format may be used in other distance learning situations.

Factors that influence flow through intravascular catheters: the clinical relevance of Poiseuille's law.

BACKGROUND: The physics of ideal fluid flow is well characterized. However, the effect of catheter size, tubing types, injection port adjuncts, and viscosity on flow is not well described. We used a simulated environment to determine how various permutations of common elements affect fluid flow. STUDY DESIGN AND METHODS: We tested 16 peripheral and central venous catheters to assess flow through several standard infusion sets and a rapid infuser set; tested flow through standard and blood infusion sets with the addition of intravenous extension tubing, stopcocks, and a needleless connector; and compared the relative viscosity of commonly used blood products and colloids to that of normal saline. RESULTS: The maximal flow rate was 200 mL/min for the standard infusion set but 800 mL/min for the rapid infusion set. Choice of infusion tubing was the rate-limiting component for many larger catheters. A 14-gauge, single-lumen central venous catheter (CVC) and 18-gauge peripheral intravenous catheter (PIV) had equivalent flow rates with all infusion sets. A 16-gauge single-lumen CVC allowed a flow rate that was slower than that of a 20-gauge PIV, and faster than that of a 22-gauge PIV. The addition of adjuncts slowed flow rate. Needleless connectors had the greatest impact, reducing flow by 75% for the blood infusion set. Packed red blood cells had a viscosity 4.5 times that of normal saline and thereby reduced flow. CONCLUSION: Catheter and tubing choice, adjuncts, and fluid viscosity influence flow rates. Our results will help inform adequate vascular access planning in the perioperative environment.

Training novice anaesthesiology trainees to speak up for patient safety.

BACKGROUND: Effectively communicating patient safety concerns in the operating theatre is crucial, but novice trainees often struggle to develop effective speaking up behaviour. Our primary objective was to test whether repeated simulation-based practice helps trainees speak up about patient management concerns. We also tested the effect of an additional didactic intervention over standard simulation education. METHODS: This prospective observational study with a nested double-blind, randomised controlled component took place during a week-long simulation boot camp. Participants were randomised to receive simulation education (SE), or simulation education plus a didactic session on speaking up behaviour (SE+). Outcome measures were: changes in intrapersonal factors for speaking up (self-efficacy, social outcome expectations, and assertiveness), and speaking up performance during four simulated scenarios. Participants self-reported intrapersonal factors and blinded observers scored speaking up behaviour. Cognitive burden for each simulation was also measured using the National Aeronautics and Space Administration Task Load Index. Mixed-design analysis of variance was used to analyse scores. RESULTS: Twenty-two participants (11 per group) were included. There was no significant interaction between group and time for any outcome measure. There was a main effect for time for self-efficacy (P<0.001); for social outcome expectations (P<0.001); for assertive attitude (P=0.003); and for speaking up scores (P=0.001). The SE+ group's assertive attitude scores increased at follow-up whereas the SE group reverted to near baseline scores (P=0.025). CONCLUSIONS: In novice anaesthesia trainees, intrapersonal factors and communication performance benefit from repeated simulation training. Focused teaching may help trainees develop assertive behaviours.

Adverse Childhood Experiences and Trust in the Medical Profession among Young Adults.

Attachment theory suggests that adverse childhood experiences (ACEs) should predict lower trust in the medical profession. To test this theory, a cross-sectional survey was administered to young adults self-identifying as homeless. The purpose of the survey was to examine the relationship of ACEs, attachment style, and trust in the medical profession. Hierarchical linear regression was used to determine if ACEs predict variance in trust in the medical profession after controlling for participants' attachment style and demographics. Results indicated ACEs were a robust predictor of lower trust in the medical profession over the controls. Since lower trust in the medical profession is associated with less willingness to seek care and follow provider recommendations, the established link between poorer health and ACEs may partially result from lower trust in the medical profession. The paper concludes with a discussion of the results' implications for future research and practice with ACE survivors.

Predicting the need for nonstandard tracheostomy tubes in critically ill patients.

PURPOSE: Few guidelines exist regarding the selection of a particular type or size of tracheostomy tube. Although nonstandard tubes can be placed over the percutaneous kit dilator, clinicians often place standard tracheostomy tubes and change to nonstandard tubes only after problems arise. This practice risks early tracheostomy tube change, possible bleeding, or loss of the airway. We sought to identify predictors of nonstandard tracheostomy tubes. MATERIALS AND METHODS: In this matched case-control study at an urban, academic, tertiary care medical center, we reviewed 1220 records of patients who received a tracheostomy. Seventy-seven patients received nonstandard tracheostomy tubes (cases), and 154 received standard tracheostomy tubes (controls). RESULTS: Sex, endotracheal tube size, severity of illness, and computed tomography scan measurement of the distance from the trachea to the skin at the level of the superior aspect of the anterior clavicle were significant predictors of nonstandard tracheostomy tubes. Specifically, trachea-to-skin distance >4.4 cm and endotracheal tube sizes ≥8.0 were associated with nonstandard tracheostomy. CONCLUSIONS: The findings suggest that clinicians should consider using nonstandard tracheostomy tubes as the first choice if the patient is male with an endotracheal tube size ≥8.0 and has a trachea-to-skin distance >4.4 cm on the computed tomography scan.

Difficult airway response team: a novel quality improvement program for managing hospital-wide airway emergencies.

BACKGROUND: Difficult airway cases can quickly become emergencies, increasing the risk of life-threatening complications or death. Emergency airway management outside the operating room is particularly challenging. METHODS: We developed a quality improvement program-the Difficult Airway Response Team (DART)-to improve emergency airway management outside the operating room. DART was implemented by a team of anesthesiologists, otolaryngologists, trauma surgeons, emergency medicine physicians, and risk managers in 2005 at The Johns Hopkins Hospital in Baltimore, Maryland. The DART program had 3 core components: operations, safety, and education. The operations component focused on developing a multidisciplinary difficult airway response team, standardizing the emergency response process, and deploying difficult airway equipment carts throughout the hospital. The safety component focused on real-time monitoring of DART activations and learning from past DART events to continuously improve system-level performance. This objective entailed monitoring the paging system, reporting difficult airway events and DART activations to a Web-based registry, and using in situ simulations to identify and mitigate defects in the emergency airway management process. The educational component included development of a multispecialty difficult airway curriculum encompassing case-based lectures, simulation, and team building/communication to ensure consistency of care. Educational materials were also developed for non-DART staff and patients to inform them about the needs of patients with difficult airways and ensure continuity of care with other providers after discharge. RESULTS: Between July 2008 and June 2013, DART managed 360 adult difficult airway events comprising 8% of all code activations. Predisposing patient factors included body mass index >40, history of head and neck tumor, prior difficult intubation, cervical spine injury, airway edema, airway bleeding, and previous or current tracheostomy. Twenty-three patients (6%) required emergent surgical airways. Sixty-two patients (17%) were stabilized and transported to the operating room for definitive airway management. There were no airway management-related deaths, sentinel events, or malpractice claims in adult patients managed by DART. Five in situ simulations conducted in the first program year improved DART's teamwork, communication, and response times and increased the functionality of the difficult airway carts. Over the 5-year period, we conducted 18 airway courses, through which >200 providers were trained. CONCLUSIONS: DART is a comprehensive program for improving difficult airway management. Future studies will examine the comparative effectiveness of the DART program and evaluate how DART has impacted patient outcomes, operational efficiency, and costs of care.

Combining natural sequence variation with high throughput mutational data to reveal protein interaction sites.

Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2-RRM1) or protein-RNA (RRM2-poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single amino acid resolution.

A novel role for otolaryngologists in the multidisciplinary Difficult Airway Response Team.

OBJECTIVES/HYPOTHESIS: The Difficult Airway Response Team (DART) was implemented in July 2008 to address emergent difficult airway situations. The main objective of this study was to highlight the unique role and skill set that otolaryngologists bring and their impact on patient outcomes. STUDY DESIGN: Retrospective review of prospectively collected data from the hospital's airway registry. METHODS: We collected data on demographics, airway characteristics, airway management techniques used by each specialty, and clinical outcomes (such as cricothyrotomies) for patients for whom a code was activated between July 2006 and June 2010. We compared data between pre- and post-DART cohorts and between DART and non-DART patients using a matched case-control approach. RESULTS: Of the 2,826 codes, 90 patients required DART management between July 2008 and June 2010. Body mass index, cervical spine injury/fixation, history of difficult airway, head and neck mass, and oropharyngeal and/or supraglottic angioedema were identified as significant predictors for DART activation. Forty-nine (60%) patients' airways were secured by anesthesiologists, 30 (36%) by otolaryngologists, and three (4%) by trauma surgeons. Otolaryngologists were able to use specialized techniques such as Holinger and Dedo laryngoscopes to significantly decrease the number of cricothyrotomies from seven (0.73%) pre-DART implementation to four (0.21%) post-DART implementation. CONCLUSIONS: Otolaryngologists were able to decrease the need for cricothyrotomies using specialized techniques for patients with difficult airways. Otolaryngologists bring a special skill set to the DART that is beyond the scope of anesthesiologists and trauma surgeons and that can improve patient outcomes by preventing unnecessary emergency surgical airways.

Utilization of a standardized tracheostomy capping and decannulation protocol to improve patient safety.

OBJECTIVES/HYPOTHESIS: To develop and assess the feasibility of a new standardized protocol to guide tracheostomy decannulation. STUDY DESIGN: Descriptive review of quality improvement project. METHODS: A quality improvement project was conducted in the inpatient setting of a tertiary urban academic hospital. Adult patients who had received a tracheostomy and for whom the indication for tracheostomy had resolved were included. A multidisciplinary task force reviewed input from clinicians caring for tracheostomy patients and developed a protocol for screening, capping, and decannulation. The primary outcome measured was successful decannulation. RESULTS: Fifty-seven patients were screened for a capping trial over a 12-month period; 54 were capped. Six patients were lost to follow-up. Fifty patients passed the capping trial, and all 50 were decannulated successfully. When decannulation was pursued in one patient who had twice failed the screening criteria and subsequent capping trials, the patient failed decannulation and ultimately required reintubation for the management of secretions. The screening tool had high sensitivity (90%) and positive predictive value (100%) for successful decannulation. Additionally, the number of reported patient safety concerns decreased from seven in the 6 months preceding implementation of the program to one report in the 6 months after implementation. CONCLUSION: The new tracheostomy capping and decannulation protocol assisted in predicting both successful and failed decannulation. Although several patients failed certain capping criteria initially, the protocol stipulated modifications of care that enabled successful decannulation. The screening tool had high sensitivity and promoted communication, standardization of practice, and patient safety.

Deep mutational scanning of an RRM domain of the Saccharomyces cerevisiae poly(A)-binding protein.

The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of ∼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.

High-throughput analysis of in vivo protein stability.

Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with high-throughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein. The variants are fused to a metabolic enzyme, which here is the yeast Leu2 protein. Plasmids encoding these Leu2 fusion proteins are transformed into yeast, with the resultant fusion proteins accumulating to different levels based on their stability and leading to different doubling times when the yeast are grown in the absence of leucine. Sequencing of an input population of variants of a protein and the population of variants after leucine selection allows the stability of tens of thousands of variants to be scored in parallel. By applying the Stable-seq method to variants of the protein degradation signal Deg1 from the yeast Matα2 protein, we generated a high-resolution map that reveals the effect of ∼30,000 mutations on protein stability. We identified mutations that likely affect stability by changing the activity of the degron, by leading to translation from new start codons, or by affecting N-terminal processing. Stable-seq should be applicable to other organisms via the use of suitable reporter proteins, as well as to the analysis of complex mixtures of fusion proteins.

Multidisciplinary team approach in the management of tracheostomy patients.

OBJECTIVE: To examine whether the implementation of a multidisciplinary percutaneous tracheostomy team decreases complications, improves efficiency in patient care, and reduces length of stay and cost in patients undergoing percutaneous tracheostomy. STUDY DESIGN: Case series with planned data collection. SETTING: Urban, academic, tertiary care medical center. SUBJECTS AND METHODS: Patients who underwent a percutaneous tracheostomy in 2004 and 2008, before and after the formation of a multidisciplinary percutaneous tracheostomy team, were included in the study. Data for the study were retrieved from a tracheostomy database. Measured outcomes include complications, efficiency, length of stay, and cost. RESULTS: Complications such as airway bleeding and physiological disturbances decreased significantly in 2008 as compared with 2004. The percentage of patients who received a tracheostomy within 2 days increased from 42.3% to 92% (2004 vs 2008), showing improvement in efficiency of care. There was no significant difference between the groups in terms of infection rate, length of stay, or mortality. However, in a subanalysis, the length of stay was found to be decreased in patients whose primary diagnosis was a neurological disorder. Finally, despite the necessity of a hospital-based subsidy, the team approach yielded substantial financial benefit to the medical center. CONCLUSIONS: Airway bleeding, physiological disturbances, and efficiency of care improved after the institution of a multidisciplinary percutaneous tracheostomy team approach and may have a favorable impact on health care costs.