Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA.

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1mΨ), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo . A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

Effects of weight loss and weight loss maintenance on cardiac autonomic function in obesity: a randomized controlled trial.

NOVELTY: Caloric restriction and exercise exert significant improvements in cardiac autonomic function as measured by HRV in overweight and obesity. Aerobic exercise training, within recommended guidelines coupled with weight loss maintenance, retains cardiac autonomic function benefits from weight loss in previously obese individuals.

The effect of exercise training level on arterial stiffness after clinically significant weight loss.

Arterial stiffness is improved by weight loss. However, no data exist on the impact of aerobic exercise levels on arterial stiffness during weight maintenance. Adults who were  overweight or with obesity (N = 39) participated in a 10-week weight loss program. Participants who achieved ≥7% weight loss were randomized to aerobic training at the minimum physical activity guidelines (PA-REC, 550 MET min/week) or weight maintenance guidelines (WM-REC, 970 MET min/week) for 18 additional weeks. Arterial stiffness (carotid-to-femoral pulse wave velocity [cfPWV], augmentation index normalized for 75 beats/min [AIX75]) and blood pressure [aortic and brachial]) were assessed at baseline, the end of the weight loss phase (week 10), and follow-up (week 28). There was a reduction in cfPWV in participants who met the weight loss goal (-0.34 m/s, p = .02) and approached significance for the entire sample (p = .051). Similarly, there were reductions in AIX75, brachial blood pressure, and aortic blood pressure (p < .05) in the full sample. In the weight maintenance phase, no differences were observed between the PA-REC and the WM-REC groups for change in arterial stiffness or blood pressure (p > .05). However, changes in cfPWV were independently associated with changes in LDL (r2 : 0.45, p = .004) and exercise intensity (r2 : 0.17, p = .033). Aerobic exercise level at the minimum physical activity guidelines or weight maintenance guidelines does not affect the change in PWV or the change in cfPWV after clinically significant weight loss. However, interventions which limit increases in LDL cholesterol and promote high-intensity aerobic exercise may prevent increases in stiffness during weight maintenance.

Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins.

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.

A capillary-based microfluidic device enables primary high-throughput room-temperature crystallographic screening.

A novel capillary-based microfluidic strategy to accelerate the process of small-molecule-compound screening by room-temperature X-ray crystallography using protein crystals is reported. The ultra-thin microfluidic devices are composed of a UV-curable polymer, patterned by cleanroom photolithography, and have nine capillary channels per chip. The chip was designed for ease of sample manipulation, sample stability and minimal X-ray background. 3D-printed frames and cassettes conforming to SBS standards are used to house the capillary chips, providing additional mechanical stability and compatibility with automated liquid- and sample-handling robotics. These devices enable an innovative in situ crystal-soaking screening workflow, akin to high-throughput compound screening, such that quantitative electron density maps sufficient to determine weak binding events are efficiently obtained. This work paves the way for adopting a room-temperature microfluidics-based sample delivery method at synchrotron sources to facilitate high-throughput protein-crystallography-based screening of compounds at high concentration with the aim of discovering novel binding events in an automated manner.

The Effect of Aerobic Training and Increasing Nonexercise Physical Activity on Cardiometabolic Risk Factors.

PURPOSE: Epidemiological studies suggest that sedentary behavior is an independent risk factor for cardiovascular mortality independent of meeting physical activity guidelines. However, limited evidence of this relationship is available from prospective interventions. The purpose of the present study is to evaluate the combined effect of aerobic training and increasing nonexercise physical activity on body composition and cardiometabolic risk factors. METHODS: Obese adults (N = 45) were randomized to 6 months of aerobic training (AERO), aerobic training and increasing nonexercise physical activity (~3000 steps above baseline levels; AERO-PA), or a control (CON) group. The AERO and AERO-PA groups performed supervised aerobic training (3-4 times per week). The AERO-PA group wore Fitbit One accelerometers and received behavioral coaching to increase nonexercise physical activity. RESULTS: There was a larger increase in fitness in the AERO-PA group (0.27 L·min-1; confidence interval (CI), 0.16 to 0.40 L·min-1) compared with the AERO group (0.09 L·min-1; CI, -0.04 to 0.22 L·min-1) and the CON group (0.01; CI, -0.11 to 0.12 L·min-1). Although significant findings were not observed in the entire study sample, when the analysis was restricted to participants compliant to the intervention (n = 33), we observed significant reductions in waist circumference, percent weight loss, body fat, 2-h glucose, and 2-h insulin in comparison to the CON group (P < 0.05), but not the AERO group. Furthermore, linear regression models showed that change in steps was associated with 21% and 26% of the variation in percent weight loss and percent fat loss, respectively. CONCLUSIONS: Increasing nonexercise physical activity with aerobic training may represent a viable strategy to augment the fitness response in comparison to aerobic training alone and has promise for other health indicators.

Prescribed exercise to Reduce Recidivism After Weight Loss-Pilot (PREVAIL-P): Design, methods and rationale.

Clinically significant weight loss is associated with health benefits for overweight and obese adults. Participation in adequate amounts of physical activity is critical for weight maintenance. However, the recommended amount of physical activity needed to promote weight maintenance is based primarily on retrospective studies that quantified physical activity levels through questionnaires which tend to overestimate physical activity levels. In addition, the present literature has provided little data on the impact of these physical activity levels on cardiovascular and diabetes risk factors, which may have equal or more clinical importance than weight changes. The Prescribed Exercise to Reduce Recidivism After Weight Loss-Pilot (PREVAIL-P) study will evaluate the effect of aerobic exercise training amount on weight maintenance following clinically significant weight loss in overweight and obese adults (BMI 25-40 kg/m2) age 30-65 years. Participants (N = 39) will complete a 10-week OPTIFAST® weight loss program with supervised aerobic exercise training. Individuals who achieve ≥7% weight loss from baseline will be subsequently randomized to levels of aerobic training consistent with physical activity recommendations (PA-REC) or weight maintenance recommendations (WM-REC) for 18 additional weeks. The primary outcome of the PREVAIL-P study will be change in weight from the completion of OPTIFAST® program to the end of the study. Notable secondary measures include changes in clinically relevant cardiometabolic risk factors between study groups (e.g. blood lipids concentrations, oral glucose tolerance, arterial stiffness). This pilot study will be used to estimate the effect sizes needed for a randomized controlled trial on this topic.

High-intensity exercise to promote accelerated improvements in cardiorespiratory fitness (HI-PACE): study protocol for a randomized controlled trial.

BACKGROUND: African Americans have a disproportionate prevalence and incidence of type 2 diabetes compared with Caucasians. Recent evidence indicates that low cardiorespiratory fitness (CRF) level, an independent risk factor for type 2 diabetes, is also more prevalent in African Americans than Caucasians. Numerous studies in Caucasian populations suggest that vigorous exercise intensity may promote greater improvements in CRF and other type 2 diabetes risk factors (e.g., reduction of glucose/insulin levels, pulse wave velocity, and body fat) than moderate intensity. However, current evidence comparing health benefits of different aerobic exercise intensities on type 2 diabetes risk factors in African Americans is negligible. This is clinically important as African Americans have a greater risk for type 2 diabetes and are less likely to meet public health recommendations for physical activity than Caucasians. The purpose of the HI-PACE (High-Intensity exercise to Promote Accelerated improvements in CardiorEspiratory fitness) study is to evaluate whether high-intensity aerobic exercise elicits greater improvements in CRF, insulin action, and arterial stiffness than moderate-intensity exercise in African Americans. METHODS/DESIGN: A randomized controlled trial will be performed on overweight and obese (body mass index of 25-45 kg/m2) African Americans (35-65 years) (n = 60). Participants will be randomly assigned to moderate-intensity (MOD-INT) or high-intensity (HIGH-INT) aerobic exercise training or a non-exercise control group (CON) for 24 weeks. Supervised exercise will be performed at a heart rate associated with 45-55% and 70-80% of VO2 max in the MOD-INT and HIGH-INT groups, respectively, for an exercise dose of 600 metabolic equivalents of task (MET)-minutes per week (consistent with public health recommendations). The primary outcome is change in CRF. Secondary outcomes include change in insulin sensitivity (measured via an intravenous glucose tolerance test), skeletal muscle mitochondrial oxidative capacity (via near-infrared spectroscopy), skeletal muscle measurements (i.e., citrate synthase, COX IV, GLUT-4, CPT-1, and PGC1-α), arterial stiffness (via carotid-femoral pulse wave velocity), body fat, C-reactive protein, and psychological outcomes (quality of life/exercise enjoyment). DISCUSSION: The anticipated results of the HI-PACE study will provide vital information on the health effects of high-intensity exercise in African Americans. This study will advance health disparity research and has the potential to influence future public health guidelines for physical activity. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02892331 . Registered on September 8, 2016.

The Effects of Exercise and Physical Activity on Weight Loss and Maintenance.

Obesity represents a major health problem in the United States and is associated with increased prevalence of cardiovascular (CV) disease risk factors. Physical activity (PA) and exercise training (ET) are associated with reduced CV risk, improved cardiometabolic risk factors, and facilitated weight loss through creating a negative energy balance. Clinicians need to counsel overweight and obese patients on how much PA/ET is needed to promote weight loss and weight loss maintenance. This will help establish realistic expectations and maximize improvements in CV risk factors. Although the minimum guidelines for aerobic PA (150 min of moderate or 75 min of vigorous physical activity per week) can improve CV health, these levels are generally inadequate for clinically significant weight loss or weight maintenance without caloric restriction. The purpose of this review is to evaluate the role of ET to promote clinically significant weight loss and promote weight maintenance. In particular, we will evaluate the likelihood of weight loss from ET programs composed of aerobic training only, resistance training only and programs that combine diet and ET. We will also explore the role of PA in promoting short-term and long-term weight maintenance.

Cardiorespiratory Fitness and Exercise Training in African Americans.

African Americans (AAs) have a higher risk for cardiovascular disease (CVD) compared to their Caucasian American (CA) counterparts, which represents a major health disparity. Low cardiorespiratory fitness (CRF) is a well-established independent risk factor for all-cause and CVD mortality, which has been shown across many epidemiological and clinical trials to be lower in AAs compared to CAs. While much attention has been given to traditional health disparity risk factors (e.g. blood pressure, obesity, insulin resistance), the impact of racial differences in CRF on CVD mortality has not been widely considered. Thus, the purpose of this paper is to review the literature on: 1) the magnitude of racial differences in CRF and the potential clinical significance, 2) examine the relationships between CRF and mortality in AAs and CAs, 3) Potential physiological and behavioral etiologies for racial difference in CRF, and 4) the impact of exercise training on CRF and other cardiometabolic risk factors in AAs compared to CAs. Given that both CRF and change in CRF are associated with reduced CVD mortality, advocating aerobic exercise training or moderate to vigorous activities in AAs has the potential to reduce racial cardiovascular health disparities.