Preparation of Translationally Competent tRNA by Direct Chemical Acylation

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes

Mortality in Hip Fracture Patients During the COVID-19 Pandemic: A Retrospective Analysis in a District General Hospital in the United Kingdom

Introduction Hip fracture is commonly seen in elderly patients because of low-energy trauma. It carries significant morbidity and mortality. Scoring systems such as the Nottingham hip fracture score (NHFS) have shown a good correlation with increased mortality as the value of these scores increases. In our study, we aim to ascertain the hip fracture mortality in our population, compare the mortality in hip fractures compared to previously reported figures in literature and nationally reported figures during the first year of the COVID-19 pandemic, and also ascertain the usefulness of NHFS in predicting mortality in hip fractures. Methods We gathered mortality data on hip fracture patients admitted to our unit from January 1, 2020 to December 31, 2020.&nbsp;NHFS was calculated for all patients and the 30-day mortality rate was compared to previously reported hip fracture mortality rates using the standard mortality ratio (SMR). One-year mortality was stratified by placing patients in high and low NHFS groups. The log-rank test was used to compare hip fracture survival at one month and at one year in the high NHFS (NHFS &gt;4) group and low NHFS group (NHFS value 4 or below). Additionally, a log-rank test was used to compare one-month and one-year survival in hip fractures managed with hemiarthroplasty, dynamic hip screw and intramedullary nail. Results In 2020, 388 patients were admitted with hip fractures to our unit. The crude mortality rate was 3.9% at 30 days and 20.88% at one year. Compared to the National Hip Fracture Database report for 2020, the incidence risk ratio for mortality was 0.46 (p-value&lt;0.05). The SMR at 30 days was 0.34 (CI=0.17-0.51) and the SMR at one year was 0.63 (CI=0.49-0.77). The survival rate was higher at 30 days and one year in the low NHFS group compared to the high NHFS group (p-value&lt;0.01). The survival rate at one month and one year were similar in groups managed with hemiarthroplasty, dynamic hip screws, and intramedullary nails (p-value&gt;0.05). Conclusions Hip fracture mortality has been decreasing steadily and we noted a lower rate of hip fracture mortality compared to figures reported previously as per NHFS studies even though the study was conducted during the COVID-19 pandemic period. We also noted lower 30-day mortality in our hospital as compared to the national 30-day mortality rate for hip fracture patients in 2020. &nbsp;</p

Impact of COVID-19 Pandemic on the Length of Hospital Stay in Hip Fracture Patients: A Single Centre Study

Background Hip fracture is a debilitating injury, especially in older individuals, which is associated with significant morbidity and mortality. In recent decades, there has been a great focus on early rehabilitation and discharge after hip fractures. The aim of such efforts is to minimize the financial and clinical burden of this condition. We conducted our study during the COVID-19 pandemic and compared the length of hospital stay (LOS) in 2020 to the LOS in 2019. Additionally, we studied the factors which may impact the LOS, such as premorbid status according to established scoring systems, the type of fracture, an operation performed, and time to surgery. &nbsp; Methods We collected the data regarding the length of stay (in days) for all hip fracture patients admitted to our unit from 1st January 2019 until 31st December 2020. We then compared the mean LOS for both years using the t-test. We calculated the Nottingham Hip Fracture Score (NHFS) and American Society of Anaesthesiologists (ASA) scores for patients admitted in 2020 and calculated the correlation between increasing values of these scores and the LOS. We also compared the mean LOS for patients admitted in 2020 based on the type of fracture and type of management. We studied the correlation between the time to surgery and the LOS for patients admitted in 2020. &nbsp; Results Three hundred and eighty-eight patients were admitted with hip fractures in 2020, and 452 were admitted in 2019. LOS in 2020 was significantly lower (23.39 days) compared to 2019 (31.36 days) with p&lt;0.01. While evaluating data from 2019, it was noted that there was a small positive correlation between LOS and NHFS (r=0.231, p&lt;0.001) and LOS and ASA (r=0.18, p&lt;0.001). The mean LOS for intracapsular fractures was noted to be lower than that of extracapsular fractures, but this was not statistically significant (p=0.17). An ANOVA test showed that the mean LOS for patients undergoing hemiarthroplasty, dynamic hip screws (DHS), and intramedullary nails (IMN) was significantly longer than for patients managed with total hip replacement or patients managed non-operatively (F=3.551, p&lt;0.01). &nbsp; Conclusion Hip fracture patients admitted to our department were discharged quicker during the first year of the COVID-19 pandemic. The LOS for hip fractures increases with an increase in their NHFS or ASA scores. Extracapsular and intracapsular fractures lead to roughly the same periods of inpatient stay. Patients undergoing hemiarthroplasty, DHS, or IMN stay longer in the hospital compared to other treatment modalities.</p

The development of a HAMstring InjuRy (HAMIR) index to mitigate injury risk through innovative imaging, biomechanics, and data analytics : Protocol for an observational cohort study

Background The etiology of hamstring strain injury (HSI) in American football is multi-factorial and understanding these risk factors is paramount to developing predictive models and guiding prevention and rehabilitation strategies. Many player-games are lost due to the lack of a clear understanding of risk factors and the absence of effective methods to minimize re-injury. This paper describes the protocol that will be followed to develop the HAMstring InjuRy (HAMIR) index risk prediction models for HSI and re-injury based on morphological, architectural, biomechanical and clinical factors in National Collegiate Athletic Association Division I collegiate football players. Methods A 3-year, prospective study will be conducted involving collegiate football student-athletes at four institutions. Enrolled participants will complete preseason assessments of eccentric hamstring strength, on-field sprinting biomechanics and muscle–tendon volumes using magnetic-resonance imaging (MRI). Athletic trainers will monitor injuries and exposure for the duration of the study. Participants who sustain an HSI will undergo a clinical assessment at the time of injury along with MRI examinations. Following completion of structured rehabilitation and return to unrestricted sport participation, clinical assessments, MRI examinations and sprinting biomechanics will be repeated. Injury recurrence will be monitored through a 6-month follow-up period. HAMIR index prediction models for index HSI injury and re-injury will be constructed. Discussion The most appropriate strategies for reducing risk of HSI are likely multi-factorial and depend on risk factors unique to each athlete. This study will be the largest-of-its-kind (1200 player-years) to gather detailed information on index and recurrent HSI, and will be the first study to simultaneously investigate the effect of morphological, biomechanical and clinical variables on risk of HSI in collegiate football athletes. The quantitative HAMIR index will be formulated to identify an athlete’s propensity for HSI, and more importantly, identify targets for injury mitigation, thereby reducing the global burden of HSI in high-level American football players. Trial Registration The trial is prospectively registered on ClinicalTrials.gov (NCT05343052; April 22, 2022)

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Studies of the Serotonin Type 3A Receptor and the Chemical Preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT3AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis. In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT3A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT3AR, which establishes a binding orientation for these drugs. In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT3A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2. In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes. Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, KB, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT3A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.</p