An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis.

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

Effects of large exercise-induced weight loss on insulin sensitivity and metabolic risk factors in young males with obesity.

BACKGROUND: Obesity around the world is increasing at an alarming rate. One of the issues with obesity is whether exercise with large energy expenditure have any effect on obesity risk factors such as insulin resistance and coronary heart diseases. METHODS: Twenty participants (mean age: 19.5±1.09 years) with Body Mass Index (BMI) of >30 kg/m2, and body fat percentage (BF%) of >25% completed an institutionalized regimented training (IRT) for 16 weeks. 12-hour fasting blood samples were collected at least 48 hours after the last exercise session. Glucose and insulin variables were determined through an oral glucose tolerance test. Participants underwent 446 hours of IRT and ate from four standardized meal menus per day with a caloric intake of 3066 kcal. RESULTS: IRT resulted in a significant weight loss of 13.48±1.97 kg. Pre- and Post-Training total cholesterol (4.80±0.92 vs. 4.12±0.82 mmol/L) (P<0.01), low-density lipoprotein cholesterol (3.04±0.83 vs. 2.51±0.74 mmol/L) (P<0.01), triglycerides (1.19±0.57 vs. 0.74±0.30 mmol/L) (P<0.01) and apolipoprotein levels (Apo-A: 133.30±13.10 vs. 120.40±14.54 mg/dL; Apo-B: 88.08±25.72 vs. 70.12±18.21 mg/dL) (P<0.01) were significantly reduced, and glucose tolerance and insulin sensitivity were also improved. CONCLUSIONS: Large exercise-induced weight loss can be achieved through IRT and may be a solution for weight loss for individuals with obesity to reduce obesity related complications.

Blueprints for the rational design of therapeutic mutacin 1140 variants.

Lantibiotics represent a large untapped pipeline of attractive scaffolds for the development of novel antibiotics. Saturation mutagenesis was employed to substitute every amino acid of a lantibiotic called mutacin 1140 (MU1140), creating an unbiased expression library of 418 variants that was used to study the permissiveness to mutagenesis and the "drugability" of several compounds. Contrasting previous reports, the results from this study supported that not all residues involved in lanthionine bridge formation were critical for maintaining optimal activity. While substitutions in lanthionine bridges in Ring A, C, and D invariably lead to inactive variants, permissive substitutions in Abu8 and Ala11 (Ring B) were observed, albeit infrequently. Further, the data generated suggested that the unsaturated bond from Dha5 (Ser5) may not be critically involved in Lipid-II binding but still important for conferring optimal activity. This study identified additional permissive mutations of Ser5, including Ser5His, Ser5Met, Ser5Gln, and Ser5Leu. In contrast, no permissive substitutions were identified for Dhb14, which suggested that this residue may be critical for optimal activity. Novel blueprints are proposed for directing further development of MU1140 variants and other lantibiotics, which may enable the rational design, development, manufacture, and formulation of an entirely new class of anti-infectives.

Exercise-induced weight loss preferentially reduces abdominal fat.

PURPOSE: To investigate whether abdominal fat is reduced in response to substantial weight loss induced by exercise in young obese men. METHODS: Thirty obese men (mean age 19.8 +/- 0.6 yr) were evaluated before (pretraining) and after (posttraining) 4 months of regimented training in the Singapore Armed Forces. There were 30 obese male subjects (mean age 19.2 +/- 1.3 yr) without training who were monitored as control subjects. Fat free mass (FFM), fat mass, and percent body fat were determined from skinfold measurements. Differences between pre- and posttraining responses were analyzed with a paired -test. RESULTS: Subjects lost 12.0 +/- 3.6 kg (P < 0.001) from pre- to posttraining, which was attributable to a reduction in fat mass (P < 0.001), as FFM was unchanged. Both waist circumference (WC) and hip circumference (HC) decreased (P < 0.01), the reduction in WC (13.7%) being greater than the reduction in HC (7.7%) as reflected by the decrease in waist-to-hip ratio (WHR; P < 0.001). These data reveal that large exercise-induced weight losses are associated with maintenance of FFM. The significant reduction in WHR indicates a greater mobilization of abdominal fat and a preferential loss of fat from this region. CONCLUSIONS: Large exercise-induced weight loss is associated with a preferential reduction in abdominal fat and a corresponding maintenance of FFM. Such an effect on body composition should reduce disease risk and the eventual weight regain that typically follows diet-induced weight losses with obese subjects.