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The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli , the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.

The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d- alanine. The basis of this strain will support future clinical studies of this strain in humans. Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis , for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d- alanine. The S. epidermidis NRRL B-4268 Δ alr1 Δ alr2 Δ dat strain (SE ΔΔΔ ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d- alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d- alanine-deficient medium, pooled human blood, and skin. In the presence of d- alanine, SE ΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SE ΔΔΔ showed a low propensity to revert to d- alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE ΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d- alanine. IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d- alanine. The basis of this strain will support future clinical studies of this strain in humans.

Background To compare validity evidence for dichotomous and trichotomous versions of a neonatal intubation (NI) procedural skills checklist. Methods NI skills checklists were developed utilizing an existing framework. Experts were trained on scoring using dichotomous and trichotomous checklists, and rated recordings of 23 providers performing simulated NI. Videolaryngoscope recordings of glottic exposure were evaluated using Cormack-Lehane (CL) and Percent of Glottic Opening scales. Internal consistency and reliability of both checklists were analyzed, and correlations between checklist scores, airway visualization, entrustable professional activities (EPA), and global skills assessment (GSA) were calculated. Results During rater training, raters gave significantly higher scores on better provider performance in standardized videos (both p  < 0.001). When utilized to evaluate study participants’ simulated NI attempts, both dichotomous and trichotomous checklist scores demonstrated very good internal consistency (Cronbach’s alpha 0.868 and 0.840, respectively). Inter-rater reliability was higher for dichotomous than trichotomous checklists [Fleiss kappa of 0.642 and 0.576, respectively ( p  < 0.001)]. Sum checklist scores were significantly different among providers in different disciplines ( p  < 0.001, dichotomous and trichotomous). Sum dichotomous checklist scores correlated more strongly than trichotomous scores with GSA and CL grades. Sum dichotomous and trichotomous checklist scores correlated similarly well with EPA. Conclusions Neither dichotomous or trichotomous checklist was superior in discriminating provider NI skill when compared to GSA, EPA, or airway visualization assessment. Sum scores from dichotomous checklists may provide sufficient information to assess procedural competence, but trichotomous checklists may permit more granular feedback to learners and educators. The checklist selected may vary with assessment needs.

US drug companies turn taxpayer-funded innovation into astronomical profits. A new agency focused on health innovation could shift the rewards of medical advances to benefit public health.

Years before the pandemic began, the US government was a major investor in what would become COVID-19 vaccines. It supplied nearly $350 million to build technologies crucial to mRNA vaccines. Later, as coronavirus infections surged, it spent some $2 billion to support vaccine clinical trials. Ultimately, the US government put more than $30 billion into research, development, and procurement of the vaccines. Many biopharma companies have piggybacked on government-funded projects to bring in record profits, set astronomical executive pay, boost share prices, and pressure officials for funding favors. As scholars who study the economics of innovation, we think that the Advanced Research Projects Agency for Health (ARPA-H), a newly created US agency devoted to health innovation, provides an opportunity to rethink how medical advances are funded so that they benefit all stakeholders fairly: not just innovators, but also taxpayers and the broader public. Well-crafted government investment policies could help bolster the creation of accessible products that improve health and well-being, rather than simply channeling returns toward shareholders.

IntroductionEmergency department (ED) visits for behavioural conditions are rising, with 1.7 million associated episodes of patient agitation occurring annually in acute care settings. When de-escalation techniques fail during agitation management, patients are subject to use of physical restraints and sedatives, which are associated with up to 37% risk of hypotension, apnoea and physical injuries. At the same time, ED staff report workplace violence due to physical assaults during agitation events. We recently developed a theoretical framework to characterise ED agitation, which identified teamwork as a critical component to reduce harm. Currently, no structured team response protocol for ED agitation addressing both patient and staff safety exists.Methods and analysisOur proposed study aims to develop and implement the agitation code team (ACT) response intervention, which will consist of a standardised, structured process with defined health worker roles/responsibilities, work processes and clinical protocols. First, we will develop the ACT response intervention in a two-step design loop; conceptual design will engage users in the creation of the prototype, and iterative refinement will occur through in situ simulated agitated patient encounters in the ED to assess and improve the design. Next, we will pilot the intervention in the clinical environment and use a controlled interrupted time series design to evaluate its effect on our primary outcome of patient restraint use. The intervention will be considered efficacious if we effectively lower the rate of restraint use over a 6-month period.Ethics and disseminationEthical approval by the Yale University Human Investigation Committee was obtained in 2019 (HIC #2000025113). Results will be disseminated through peer-reviewed publications and presentations at scientific meetings for each phase of the study. If this pilot is successful, we plan to formally integrate the ACT response intervention into clinical workflows at all EDs within our entire health system.

ABSTRACT IMPACT: Within three EDs in a regional health system in Connecticut, African American race, male gender, non-Hispanic ethnicity, lack of private insurance, and homelessness were associated with significant odds of being physically restrained during a visit. OBJECTIVES/GOALS: Agitated patient encounters in the Emergency Department (ED) are on the rise, and physical restraints are used to protect staff and prevent self-harm. However, these are associated with safety risks and potential stigmatization of vulnerable individuals. We aim to determine factors that are associated with odds of being restrained in the ED. METHODS/STUDY POPULATION: We conducted a retrospective cohort analysis of all patients (≥18 yo) placed in restraints during an ED visit to three hospitals within a large tertiary health system from Jan 2013-Aug 2018. We undertook descriptive analysis of the data and created a generalized linear mixed model with a binary logistic identity link to model restraint use and determine odds ratios for various clinically significant demographic factors. These include gender, race, ethnicity, insurance status, alcohol use, illicit drug use, and homelessness. Our model accounted for patients nested across the three EDs and also accounted for multiple patient visits. RESULTS/ANTICIPATED RESULTS: In 726,417 total ED visits, 7,090 (1%) had associated restraint orders. Restrained patients had an average age of 45, with 64% male, 54% Caucasian and 29% African American. 17% had private insurance, 36% endorsed illicit substances, 51.4% endorsed alcohol use and 2.3% were homeless. African Americans had statistically significant odds of being restrained compared to Caucasians with adjusted odds ratio (AOR) of 1.14 (1.08,1.21). Females (AOR 0.75 [0.71, 0.79] had lower odds of being restrained compared to males while patients with Medicaid (AOR 1.57 [1.46, 1.68]) and Medicare (AOR 1.70 [1.57, 1.85]) had increased odds compared to the privately insured. Illicit substance use (AOR 1.55 [1.46, 1.64]), alcohol use (AOR 1.13 [1.07, 1.20] and homelessness (AOR 1.35 [1.14, 1.16]) had increased odds of restraint use. DISCUSSION/SIGNIFICANCE OF FINDINGS: We showed statistically significant effects of patient demographics on odds of restraint use in the ED. The increased odds based on race, insurance status, and substance use highlight the potential effects of implicit bias on the decision to physically restrain patients and underscores the importance of objective assessments of these patients.

OBJECTIVES/GOALS: Agitation has high prevalence in the emergency department (ED), but limited evidence exists regarding clinical decisions to use sedatives and physical restraint. We examined clinical factors and agitation attributes impacting thresholds for sedative and restraint use in the emergency setting. METHODS/STUDY POPULATION: We conducted a prospective cohort study of adult patients ( 3 18 yo) with acute or escalating agitation during their ED visit at an urban tertiary care referral center. Consecutive patients requiring security presence or scoring >1 on an agitation scale were enrolled during randomized 8-h blocks. We recorded patient characteristics, staff/team factors, and environmental/systems data as well as scores on 3 validated agitation scales: Agitated Behavior Scale, Overt Aggression Scale, and Severity Scale. We performed descriptive analyses, bivariable analyses, and logistic regression modeling of factors with relation to sedative/restraint use. We observed 95 agitation events on unique patients over 2 months. RESULTS/ANTICIPATED RESULTS: Median age was 42, and 62.1% were male. Most frequent chief complaints were alcohol/drug use (37.9%) and psychiatric (23.2%). Majority of events (73.7%) were associated with sedative/restraint use. Factors related to treatment course or staff interactions were the primary reasons for agitation in 56.8% of events. A logistic regression model found no association between demographics and odds of sedative/restraint use. Overt Aggression Scale scores were associated with significantly higher odds of sedative use (AOR 1.62 [1.13–2.32]), while Severity Scale scores had significantly higher odds of restraint use (AOR 1.39 [1.12–1.73]) but significantly lower odds of sedative use (AOR 0.79 [0.64–0.98]). DISCUSSION/SIGNIFICANCE OF IMPACT: External factors may be important targets for behavioral techniques in ED agitation management. Further study of the Severity Scale may allow for earlier detection of agitation and identify causal links between agitation severity and use of sedatives and restraints.

IntroductionDirect laryngoscopy (DL) and airway intubation are critical for neonatal resuscitation. A challenge in teaching DL is that the instructor cannot assess the learners’ airway view. Videolaryngoscopy (VL), which allows display of a patient's airway on a monitor, enables the instructor to view the airway during the procedure. This pilot study compared deliberate practice using either VL with instruction (I-VL) or traditional DL. We hypothesised that I-VL would improve the efficiency and effectiveness of neonatal intubation (NI) training.MethodsParticipants (students, paediatric interns and neonatal fellows) were randomised to I-VL or DL. Baseline technical skills were assessed using a skills checklist and global skills assessment. Following educational sessions, deliberate practice was performed on mannequins using the Storz C-MAC. With I-VL, the instructor could guide training using a real-time airway monitor view. With DL, feedback was based solely on technique or direct visual confirmation, but the instructor and learner views were not concurrent. During summative assessment, procedural skills checklists were used to evaluate intubation ability on a neonatal airway trainer. The duration of attempts was recorded, and recorded airway views were blindly reviewed for airway grade. ‘Effectiveness’ reflected achievement of the minimum passing score (MPS). ‘Efficiency’ was the duration of training for learners achieving the MPS.Results58 learners were randomised. Baseline demographics were similar. All participants had a significant improvement in knowledge, skills and comfort/confidence following training. There were no significant differences between randomised groups in efficiency or effectiveness, but trends towards improvement in each were noted. Fellows were more likely to achieve ‘competency’ postinstruction compared to non-fellows (p<0.001).ConclusionsThis educational intervention to teach NI increased the learner's knowledge, technical skills and confidence in procedural performance in both groups. I-VL did not improve training effectiveness. The small sample size and participant diversity may have limited findings, and future work is indicated.