Evolution of a fluorinated green fluorescent protein

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved {approx}650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents

Ground state and glass transition of the RNA secondary structure

RNA molecules form a sequence-specific self-pairing pattern at low temperatures. We analyze this problem using a random pairing energy model as well as a random sequence model that includes a base stacking energy in favor of helix propagation. The free energy cost for separating a chain into two equal halves offers a quantitative measure of sequence specific pairing. In the low temperature glass phase, this quantity grows quadratically with the logarithm of the chain length, but it switches to a linear behavior of entropic origin in the high temperature molten phase. Transition between the two phases is continuous, with characteristics that resemble those of a disordered elastic manifold in two dimensions. For designed sequences, however, a power-law distribution of pairing energies on a coarse-grained level may be more appropriate. Extreme value statistics arguments then predict a power-law growth of the free energy cost to break a chain, in agreement with numerical simulations. Interestingly, the distribution of pairing distances in the ground state secondary structure follows a remarkable power-law with an exponent -4/3, independent of the specific assumptions for the base pairing energies

COVID-19 knowledge and prevention behaviors in rural Zambia: a qualitative application of the information-motivation-behavioral skills model

In early 2020, the Zambian Ministry of Health instituted prevention guidelines to limit spread of COVID-19. We assessed community knowledge, motivations, behavioral skills, and perceived community adherence to prevention behaviors (i.e., hand hygiene, mask wearing, social distancing, and limiting gatherings). Within a cluster-randomized controlled trial in four rural districts, in November 2020 and May 2021, we conducted in-depth interviews with health center staff (N = 19) and community-based volunteers (N = 34) and focus group discussions with community members (N = 281). A content analysis was conducted in Nvivo v12. Data were interpreted using the Information-Motivation-Behavioral Skills Model. Generally, respondents showed good knowledge of COVID-19 symptoms, spread, and high-risk activities, with some gaps. Prevention behavior performance was driven by personal and social factors. Respondents described institutional settings (e.g., clinics and church) having higher levels of perceived adherence due to stronger enforcement measures and clear leadership. Conversely, informal community settings (e.g., weddings, funerals, football matches) lacked similar social and leadership expectations for adherence and had lower perceived levels of adherence. These settings often involved higher emotions (excitement or grief), and many involved alcohol use, resulting in community members "forgetting" guidelines. Doubt about disease existence or need for precautions persisted among some community members and drove non-adherence more generally. Although COVID-19 information successfully penetrated these very remote rural communities, more targeted messaging may address persistent COVID-19 doubt and misinformation. Engaging local leaders in religious, civic, and traditional leadership positions could improve community behaviors without adding additional monitoring duties on an already overburdened, resource-limited health system

Outcomes of Durable Mechanical Circulatory Support in Myocarditis: Analysis of the International Society for Heart and Lung Transplantation Registry for Mechanically Assisted Circulatory Support Registry

Myocarditis can be refractory to medical therapy and require durable mechanical circulatory support (MCS). The characteristics and outcomes of these patients are not known. We identified all patients with clinically-diagnosed or pathology-proven myocarditis who underwent mechanical circulatory support in the International Society for Heart and Lung Transplantation Registry for Mechanically Assisted Circulatory Support registry (2013-2016). The characteristics and outcomes of these patients were compared to those of patients with nonischemic cardiomyopathy (NICM). Out of 14,062 patients in the registry, 180 (1.2%) had myocarditis and 6,602 (46.9%) had NICM. Among patients with myocarditis, duration of heart failure was22%, 1-12 months in 22.6%, and \u3e1 year in 55.4%. Compared with NICM, patients with myocarditis were younger (45 vs. 52 years, P \u3c 0.001) and were more often implanted with Interagency Registry for Mechanically Assisted Circulatory Support profile 1 (30% vs. 15%, P \u3c 0.001). Biventricular mechanical support ( biventricular ventricular assist device [BIVAD] or total artificial heart) was implanted more frequently in myocarditis (18% vs. 6.7%, P \u3c 0.001). Overall postimplant survival was not different between myocarditis and NICM (left ventricular assist device: P = 0.27, BIVAD: P = 0.50). The proportion of myocarditis patients that have recovered by 12 months postimplant was significantly higher in myocarditis compared to that of NICM (5% vs. 1.7%, P = 0.0003). Adverse events (bleeding, infection, and neurologic dysfunction) were all lower in the myocarditis than NICM. In conclusion, although myocarditis patients who receive durable MCS are sicker preoperatively with higher needs for biventricular MCS, their overall MCS survival is noninferior to NICM. Patients who received MCS for myocarditis are more likely than NICM to have MCS explanted due to recovery, however, the absolute rates of recovery were low

Net clinical benefit of dabigatran vs. warfarin in venous thromboembolism: analyses from RE-COVER®, RE-COVER™ II, and RE-MEDY™

The direct oral anticoagulants, e.g., dabigatran etexilate (DE), are effective and well tolerated treatments for venous thromboembolism (VTE). Net clinical benefit (NCB) is a useful concept in weighing potential benefits against potential harm of comparator drugs. The NCB of DE vs. warfarin in VTE treatment was compared. Post-hoc analyses were performed on pooled data from the 6-month RE-COVER® and RE-COVER™ II trials, and data from the RE-MEDY™ trial (up to 36 months), to compare the NCB of DE (150 mg twice daily) and warfarin [target international normalized ratio (INR) 2.0-3.0]. Patients (≥18 years old) had symptomatic proximal deep vein thrombosis and/or pulmonary embolism. NCB was the composite of cardiovascular endpoints (non-fatal events of recurrent VTE, myocardial infarction, stroke or systemic embolism), all-cause death, and bleeding outcomes, all weighted equally. A broad definition of NCB included major bleeding events (MBE) and clinically relevant non-major bleeding events as bleeding outcomes, while a narrow definition included just MBE. The pooled dataset totalled 5107 patients from RE-COVER/RE-COVER II and 2856 patients from RE-MEDY. When NCB was narrowly defined, NCB was similar between DE and warfarin. When broadly defined, NCB was superior with DE vs. warfarin [RE-COVER/RE-COVER II, hazard ratio (HR) 0.80; 95% confidence interval (CI), 0.68-0.95 and RE-MEDY, HR 0.73; 95% CI 0.59-0.91]. These findings were unaffected by warfarin time in therapeutic range. The NCB of DE was similar or superior to warfarin, depending on the NCB definition used, regardless of the quality of INR control