In vivo Reactions of Tetrazines Incorporated through Genetic Code Expansion

The inverse electron demand Diels Alder reaction between tetrazines and strained alkenes is an exceptionally useful tool in functionalizing to biomolecules since it is orthogonal to the chemistry of most living systems and have exceptionally high rate constants. In particular reactions between strained trans-cyclooctenes (sTCO) and tetrazines can achieve second order rate constants of 106 M-1s-1 or greater. This bioorthogonal coupling reaction can be used to quantitatively modify proteins with fluorescent labels, inhibitors, or dimerization agents in live cells with short reaction time. In this work, we explore the use of genetic code expansion to incorporate tetrazine containing amino acids into proteins for the purpose of conjugating sTCO-containing labeling agents to proteins in live cells In Escherichia coli, the tetrazine containing amino acid, 4-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v2.0) is incorporated into cells through genetic code expansion. An orthogonal amino acyl-tRNA synthetase is generated that can charge tRNACUA using Tet-v2.0 as a substrate allowing for the site-specific incorporation of Tet-v2.0 into proteins. Tet-v2.0 on proteins is shown to react with sTCO with a high rate of reaction of 87,000 M-1s-1 and minimal off-target reactions. To express tetrazine containing proteins in eukaryotic cells, a new set of amino acids based on 3-(6-methyl-s-tetrazin-3-yl)phenylalanine (Tet-v3.0), are developed that can be used as substrate by Methanosarcina. barkeri Pyrrolysine tRNA synthetases (PylRS). The orthogonal Tet-v3.0-amino acyl-tRNA synthetase/tRNA pair was used to incorporate Tet-v3.0 amino acids into proteins in HEK293T cells and shown to react with both fluorescent labels and dimerizing sTCO molecules. We then explore methods to improve the efficiency of the reaction between tetrazines and sTCO, through minimizing side reactions. These side reactions include the reduction of tetrazines to dihydro-tetrazines and irreversible degradation of tetrazines to a yet to be characterized product. To eliminate off-target reactions the reduction of tetrazines is monitored in cellulo and proteins are oxidized in cellulo through photooxidation. This oxidation reaction has the potential to be an effective method of establishing higher labeling yields. Finally, trapping the tetrazine in the dihydro-state and selectively oxidizing and labeling the tetrazine may be an effective method of minimizing off-target tetrazine reactions in cells by protecting the oxidized amino acid

Orbit Determination During Spacecraft Emergencies with Sparse Tracking Data - THEMIS and TDRS-3 Lessons Learned

This paper provides an overview of the lessons learned from the National Aeronautics and Space Administration (NASA) Goddard Space Flight Center s (GSFC) Flight Dynamics Facility s (FDF) support of the Time History of Events and Macroscale Interactions during Substorms (THEMIS) spacecraft emergency in February 2007, and the Tracking and Data Relay Satellite-3 (TDRS-3) spacecraft emergency in March 2006. A successful and timely recovery from both of these spacecraft emergencies depended on accurate knowledge of the orbit. Unfortunately, the combination of each spacecraft emergency with very little tracking data contributed to difficulties in estimating and predicting the orbit and delayed recovery efforts in both cases. In both the THEMIS and TDRS-3 spacecraft emergencies, numerous factors contributed to problems with obtaining nominal tracking data measurements. This paper details the various causative factors and challenges. This paper further enumerates lessons learned from FDF s recovery efforts involving the THEMIS and TDRS-3 spacecraft emergencies and scant tracking data, as well as recommendations for improvements and corrective actions. In addition, this paper describes the broad range of resources and complex navigation methods employed within the FDF for supporting critical navigation activities during all mission phases, including launch, early orbit, and on-orbit operations

Takeaway food consumption and its associations with diet quality and abdominal obesity: a cross-sectional study of young adults

Background : Few studies have investigated the associations of takeaway food consumption with overall diet quality and abdominal obesity. Young adults are high consumers of takeaway food so we aimed to examine these associations in a national study of young Australian adults.Methods : A national sample of 1,277 men and 1,585 women aged 26&ndash;36 completed a self-administered questionnaire on demographic and lifestyle factors, a 127 item food frequency questionnaire, usual daily frequency of fruit and vegetable consumption and usual weekly frequency of takeaway food consumption. Dietary intake was compared with the dietary recommendations from the Australian Guide to Healthy Eating. Waist circumference was measured for 1,065 men and 1,129 women. Moderate abdominal obesity was defined as &ge; 94 cm for men and &ge; 80 cm for women. Prevalence ratios (PR) were calculated using log binomial regression. Takeaway food consumption was dichotomised, with once a week or less as the reference group.Results : Consumption of takeaway food twice a week or more was reported by more men (37.9%) than women (17.7%, P &lt; 0.001). Compared with those eating takeaway once a week or less, men eating takeaway twice a week or more were significantly more likely to be single, younger, current smokers and spend more time watching TV and sitting, whereas women were more likely to be in the workforce and spend more time watching TV and sitting. Participants eating takeaway food at least twice a week were less likely (P &lt; 0.05) to meet the dietary recommendation for vegetables, fruit, dairy, extra foods, breads and cereals (men only), lean meat and alternatives (women only) and overall met significantly fewer dietary recommendations (P &lt; 0.001). After adjusting for confounding variables (age, leisure time physical activity, TV viewing and employment status), consuming takeaway food twice a week or more was associated with a 31% higher prevalence of moderate abdominal obesity in men (PR: 1.31; 95% CI: 1.07, 1.61) and a 25% higher prevalence in women (PR: 1.25; 95% CI: 1.04, 1.50).Conclusion : Eating takeaway food twice a week or more was associated with poorer diet quality and a higher prevalence of moderate abdominal obesity in young men and women.<br /

The association between grip strength measured in childhood, young- and mid-adulthood and prediabetes or type 2 diabetes in mid-adulthood

Background: Although low child and adult grip strength is associated with adverse cardiometabolic health, how grip strength across the life course associates with type 2 diabetes is unknown. This study identified the relative contribution of grip strength measured at specific life stages (childhood, young adulthood, mid-adulthood) with prediabetes or type 2 diabetes in mid-adulthood. Methods: Between 1985 and 2019, 263 participants had their grip strength measured using an isometric dynamometer in childhood (9-15 years), young adulthood (28-36 years) and mid-adulthood (38-49 years). In mid-adulthood, a fasting blood sample was collected and tested for glucose and glycated haemoglobin (HbA1c). Participants were categorized as having prediabetes or type 2 diabetes if fasting glucose levels were ≥ 5.6 mmol or if HbA1c levels were ≥ 5.7% (≥ 39 mmol/mol). A Bayesian relevant life course exposure model examined the association between lifelong grip strength and prediabetes or type 2 diabetes. Results: Grip strength at each time point was equally associated with prediabetes or type 2 diabetes in mid-adulthood (childhood: 37%, young adulthood: 36%, mid-adulthood: 28%). A one standard deviation increase in cumulative grip strength was associated with 34% reduced odds of prediabetes or type 2 diabetes in mid-adulthood (OR 0.66, 95% credible interval 0.40, 0.98). Conclusions: Greater grip strength across the life course could protect against the development of prediabetes and type 2 diabetes. Strategies aimed at increasing muscular strength in childhood and maintaining behaviours to improve strength into adulthood could improve future cardiometabolic health. The Association Between Grip Strength Measured in Childhood, Young- and Mid-adulthood and Prediabetes or Type 2 Diabetes in Mid-adulthood

Masculinity as Governance: police, public service and the embodiment of authority, c. 1700-1850

About the book: Public Men offers an introduction to an exciting new field: the history of masculinities in the political domain and will be essential reading for students and specialists alike with interests in gender or political culture. By building upon new work on gender and political culture, these new case studies explore the gendering of the political domain and the masculinities of the men who have historically dominated it. As such, Public Men is a major contribution to our understanding of the history of Britain between the Eighteenth and the Twentieth centuries