Multi-omic analysis of SDHB-deficient pheochromocytomas and paragangliomas identifies metastasis and treatment-related molecular profiles.

Hereditary SDHB-mutant pheochromocytomas (PC) and paragangliomas (PG) are rare tumours with a high propensity to metastasize although their clinical behaviour is unpredictable. To characterize the genomic landscape of these tumours and identify metastasis biomarkers, we performed multi-omic analysis on 94 tumours from 79 patients using seven molecular methods. Sympathetic (chromaffin cell) and parasympathetic (non-chromaffin cell) PCPG had distinct molecular profiles reflecting their cell-of-origin and biochemical profile. TERT and ATRX-alterations were associated with metastatic PCPG and these tumours had an increased mutation load, and distinct transcriptional and telomeric features. Most PCPG had quiet genomes with some rare co-operative driver events observed, including EPAS1/HIF-2α mutations. Two mechanisms of acquired resistance to DNA alkylating chemotherapies were also detected - MGMT overexpression and mismatch repair-deficiency causing hypermutation. Our comprehensive multi-omic analysis of SDHB-mutant PCPG therefore identified features of metastatic disease and treatment response, expanding our understanding of these rare neuroendocrine tumours.

Allenamides as a Powerful Tool to Incorporate Diversity: Thia-Michael Lipidation of Semisynthetic Peptides and Access to ß-Ketoamides.

Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semi-synthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22 lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a ß-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.

Role of pharmacists in the care of adult asthma patients: A scoping review.

BACKGROUND: Asthma is a common long-term condition that affects people of all ages. Evidence suggests that a significant proportion of asthma patients in the Gulf Cooperation Council (GCC) do not receive appropriate diagnosis, monitoring and/or treatment. When inadequately treated, asthma can negatively affect quality of life and may lead to hospitalisation and death. Although pharmacists play a role in asthma care globally, there appears to be no defined role for pharmacists in providing care to patients with asthma in the GCC countries. AIM: This scoping review aims to review and summarise studies conducted in the GCC countries involving pharmacists in the management of adults with asthma or evaluating pharmacists' asthma care knowledge and/or skills. METHOD: A systematic scoping review was undertaken. Seven databases were searched using relevant search terms for articles published up to May 2023. Studies that evaluated pharmacists roles, knowledge and skills in providing asthma care to adults in the United Arab Emirates (UAE), Qatar, Kuwait, Oman, Saudi Arabia, and Bahrain were considered eligible for inclusion. Extracted data were collated using tables and used to produce narrative descriptive summaries. RESULTS: Out of the 1588 search results, only seven studies met the inclusion criteria. Of those, only one developed and tested a pharmacist-led inhaler technique educational intervention in the UAE within community pharmacy setting for asthma patients. The remaining six studies assessed community pharmacists knowledge in providing asthma management and patient education in UAE, Saudi Arabia and Qatar. The quality of the included studies varied with four relying on simulated patients to assess pharmacists knowledge. The study that tested the intervention suggested improvement in inhaler technique and asthma symptoms control after receiving the intervention. The findings suggest a need to improve pharmacists knowledge of inhaler technique demonstration (mainly Metered Dose Inhalers), asthma management advice and assessment of asthma control and medication use. CONCLUSION: This review highlights a lack of research on pharmacist-led asthma interventions and identifies training needs to enable pharmacists to be involved in asthma care in the GCC countries. Future research could develop approaches involving pharmacists to improve asthma care and outcomes in the region.

Bound star clusters observed in a lensed galaxy 460 Myr after the Big Bang.

The Cosmic Gems arc is among the brightest and highly magnified galaxies observed at redshift z ∼ 10.21. However, it is an intrinsically UV faint galaxy, in the range of those now thought to drive the reionization of the universe2-4. Hitherto the smallest features resolved in a galaxy at a comparable redshift are between a few hundreds and a few tens of parsecs5,6. Here we report JWST observations of the Cosmic Gems. The light of the galaxy is resolved into five star clusters located in a region smaller than 70 parsec. They exhibit minimal dust attenuation and low metallicity, ages younger than 50 Myr and intrinsic masses of ∼ 106 M⊙. Their lensing-corrected sizes are approximately 1 pc, resulting in stellar surface densities near 105 M⊙ /pc2, three orders of magnitude higher than typical young star clusters in the local universe7. Despite the uncertainties inherent to the lensing model, they are consistent with being gravitationally bound stellar systems, i.e., proto-globular clusters (proto-GCs). We conclude that star cluster formation and feedback likely contributed to 3 shape the properties of galaxies during the epoch of reionization.

Lesions to the mediodorsal thalamus, but not orbitofrontal cortex, enhance volatility beliefs linked to paranoia.

Beliefs-attitudes toward some state of the environment-guide action selection and should be robust to variability but sensitive to meaningful change. Beliefs about volatility (expectation of change) are associated with paranoia in humans, but the brain regions responsible for volatility beliefs remain unknown. The orbitofrontal cortex (OFC) is central to adaptive behavior, whereas the magnocellular mediodorsal thalamus (MDmc) is essential for arbitrating between perceptions and action policies. We assessed belief updating in a three-choice probabilistic reversal learning task following excitotoxic lesions of the MDmc (n = 3) or OFC (n = 3) and compared performance with that of unoperated monkeys (n = 14). Computational analyses indicated a double dissociation: MDmc, but not OFC, lesions were associated with erratic switching behavior and heightened volatility belief (as in paranoia in humans), whereas OFC, but not MDmc, lesions were associated with increased lose-stay behavior and reward learning rates. Given the consilience across species and models, these results have implications for understanding paranoia.

Clinical Implementation of DIGEST as an Evidence-Based Practice Tool for Videofluoroscopy in Oncology: A Six-Year Single Institution Implementation Evaluation.

Clinical implementation of evidence-based practice (EBP) tools is a healthcare priority. The Dynamic Grade of Swallowing Toxicity (DIGEST) is an EBP tool developed in 2016 for videofluoroscopy in head and neck (H&N) oncology with clinical implementation as a goal. We sought to examine: (1) feasibility of clinical implementation of DIGEST in a national comprehensive cancer center, and (2) fidelity of DIGEST adoption in real-world practice. A retrospective implementation evaluation was conducted in accordance with the STARI framework. Electronic health record (EHR) databases were queried for all consecutive modified barium swallow (MBS) studies conducted at MD Anderson Cancer Center from 2016 to 2021. Implementation outcomes included: feasibility as measured by DIGEST reporting in EHR (as a marker of clinical use) and fidelity as measured by accuracy of DIGEST reporting relative to the decision-tree logic (penetration-aspiration scale [PAS], residue, and Safety [S] and Efficiency [E] grades). Contextual factors examined included year, setting, cancer type, MBS indication, and provider. 13,055 MBS were conducted by 29 providers in 7,842 unique patients across the lifespan in diverse oncology populations (69% M; age 1-96 years; 58% H&N cancer; 10% inpatient, 90% outpatient). DIGEST was reported in 12,137/13,088 exams over the 6-year implementation period representing 93% (95% CI: 93-94%) adoption in all exams and 99% (95% CI: 98-99%) of exams excluding the total laryngectomy population (n = 730). DIGEST reporting varied modestly by year, cancer type, and setting/provider (> 91% in all subgroups, p < 0.001). Accuracy of DIGEST reporting was high for overall DIGEST (incorrect SE profile 1.6%, 200/12,137), DIGEST-safety (incorrect PAS 0.4% 51/12,137) and DIGEST-efficiency (incorrect residue 1.2%, 148/12,137). Clinical implementation of DIGEST was feasible with high fidelity in a busy oncology practice across a large number of providers. Adoption of the tool across the lifespan in diverse cancer diagnoses may motivate validation beyond H&N oncology.

National Breast, Cervical, and Colorectal Cancer Screening Use in Federally Qualified Health Centers.

Importance: Federally qualified health centers (FQHCs) deliver health care to nearly 30 million underserved persons across the US, yet nationwide and state-level breast, cervical, and colorectal cancer screening use in FQHCs is not described. Furthermore, it is unknown how the underscreened FQHC population contributes to the total underscreened population at national and state levels. Objective: To describe national- and state-level breast, cervical, and colorectal cancer screening use among individuals served by FQHCs in the US and to estimate the percentage of underscreened individuals in the general population served by FQHCs. Design, Setting, and Participants: This cross-sectional analysis of cancer screening used data from January 1 through December 31, 2020, from the FQHC Uniform Data System, reported by 1364 FQHCs across the US, and self-reported estimates from the Behavioral Risk Factor Surveillance System. Participants were 16 696 692 US adults served by FQHCs who were eligible for breast (age, 50-74 years), cervical (age, 21-64 years), and colorectal (age, 50-75 years) cancer screening. Analyses were conducted between January 1 and June 30, 2023. Exposures: Breast, cervical, and colorectal cancer screening. Main Outcomes and Measures: Percentages of breast, cervical, and colorectal cancer screening-eligible individuals up to date on screening. Results: A total of 3 162 882 breast, 7 444 465 cervical, and 6 089 345 colorectal screening-eligible individuals were served by FQHCs in 2020. Nationally, screening use in FQHCs was 45.4% (95% CI, 45.4%-45.5%) for breast cancer, 51.0% (95% CI, 51.0%-51.1%) for cervical cancer, and 40.2% (95% CI, 40.1%-40.2%) for colorectal cancer. Screening use among the US general population was 78.2% (95% CI, 77.6%-78.9%) for breast cancer, 82.9% (95% CI, 82.3%-83.4%) for cervical cancer, and 72.3% (95% CI, 71.7%-72.8%) for colorectal cancer. The contribution of the underscreened population served by FQHCs to the national underscreened general population was 16.9% (95% uncertainty interval [UI], 16.4%-17.4%) for breast cancer, 29.7% (95% UI, 28.8%-30.7%) for cervical cancer, and 14.7% (95% UI, 14.4%-15.0%) for colorectal cancer. Conclusions and Relevance: Findings from this national cross-sectional study indicated major gaps in cancer screening use in FQHCs in the US. Improved prevention is urgently needed to address screening disparities.

Using the Dynamic Well-Stirred Model to Extrapolate Hepatic Clearance of Organic Anion-Transporting Polypeptide Transporter Substrates without Assuming Albumin-Mediated Hepatic Drug Uptake.

Extrapolating in vivo hepatic clearance from in vitro uptake data is a known challenge, especially for organic anion-transporting polypeptide transporter (OATP) substrates, and the well-stirred model (WSM) commonly yields systematic underpredictions for those anionic drugs, hypothetically due to "albumin-mediated hepatic drug uptake". In the present study, we demonstrate that the WSM incorporating the dynamic free fraction (f D), a measure of drug protein binding affinity, performs reasonably well in predicting hepatic clearance of OATP substrates. For a selection of anionic drugs, including atorvastatin, fluvastatin, pravastatin, rosuvastatin, pitavastatin, cerivastatin, and repaglinide, this dynamic well-stirred model (dWSM) correctly predicts hepatic plasma clearance within 2-fold error for six out of seven OATP substrates examined. The geometric mean of clearance ratios between the predicted and the observed values falls in the range of 1.21-1.38. As expected, the WSM with unbound fraction (f u) systematically underpredicts hepatic clearance with greater than 2-fold error for five out of seven drugs, and the geometric mean of clearance ratios between the predicted and the observed values is in the range of 0.20-0.29. The results suggest that, despite its simplicity, the dWSM operates well for transporter-mediated uptake clearance, and that clearance under-prediction of OATP substrates may not necessarily be associated with the chemical class of the anionic drugs, nor is it a result of albumin-mediated hepatic drug uptake as currently hypothesized. Instead, the superior prediction power of the dWSM confirms the utility of the dynamic free fraction in clearance prediction and the importance of drug plasma binding kinetics in hepatic uptake clearance. SIGNIFICANCE STATEMENT: The traditional well-stirred model (WSM) consistently underpredicts organin anion-transporting polypeptide transporter (OATP)-mediated hepatic uptake clearance, hypothetically due to the albumin-mediated hepatic drug uptake. In this manuscript, we apply the dynamic WSM to extrapolate hepatic clearance of the OATP substrates, and our results show significant improvements in clearance prediction without assuming albumin-mediated hepatic drug uptake.

Nativity Disparities in Colorectal Cancer Screening Among Hispanics in the United States.

Hispanics in the United States (U.S.) have previously exhibited lower guideline-concordant colorectal cancer (CRC) screening uptake than non-Hispanic (NH) Whites, with disparities accentuated in foreign-born Hispanics, however it is unclear whether nativity-related CRC screening disparities have changed in the last two decades and whether these disparities are attenuated after adjusting for socioeconomic and demographic characteristics. We evaluated CRC screening adherence in foreign- and U.S.-born Hispanics compared to U.S.-born NH Whites. We used 2019 National Health Interview Survey data to compare the prevalence of up-to-date CRC screening per the 2019 U.S. Preventive Services Task Force recommendations among Hispanic nativity subgroups (i.e., foreign- and U.S.-born) and U.S.-born NH Whites using unadjusted and adjusted weighted log-linked binomial regression. Foreign- and U.S.-born Hispanics had a significantly lower unadjusted prevalence of up-to-date screening than U.S.-born NH Whites (47.18% and 64.18% versus 70.70%; p < 0.0001 and p = 0.0109, respectively). After adjusting for socioeconomic and demographic differences, the prevalence of up-to-date screening was lower in foreign-born Hispanics compared to U.S.-born NH Whites [adjusted prevalence ratio 0.80 (95% confidence interval 0.70-0.91)]; however, no statistically significant difference was observed between U.S.-born Hispanics and NH Whites. Our results suggest a low screening uptake in foreign-born Hispanics independent of socioeconomic and demographic differences. Future interventions should target foreign-born Hispanics to address disparities and promote early detection and prevention of CRC regardless of socioeconomic factors.

A programmatic update on COVID-19 vaccination in rural communities in the United States.

When public health experts think of rural barriers to vaccines, they often initially focus on access, which makes sense with a new vaccine during a pandemic. This commentary highlights that there can be more complexity to vaccine uptake in rural communities. What follows are some examples of CDC's efforts to better understand rural health and learnings to inform ongoing vaccination efforts in rural communities.

Risk factors for Cryptosporidium contamination in Minnesota public supply wells.

In a recent monitoring study of Minnesota's public supply wells, Cryptosporidium was commonly detected with 40% of the wells having at least one detection. Risk factors for Cryptosporidium occurrence in drinking water supply wells, beyond surface water influence, remain poorly understood. To address this gap, physical and chemical factors were assessed as potential predictors of Cryptosporidium occurrence in 135 public supply wells in Minnesota. Univariable analysis, regression techniques, and classification trees were used to analyze the data. Many variables were identified as significant risk factors in univariable analysis and several remained significant throughout the succeeding analysis techniques. These factors fell into general categories of well use and construction, aquifer characteristics, and connectedness to the land surface, well capture zones, and land use therein, existence of potential contaminant sources within 200-feet of the well, and variability in the chemical and isotopic parameters measured during the study. These risk categories, and the specific variables and threshold values we have identified, can help guide future research on factors influencing Cryptosporidium contamination of wells and can be used by environmental health programs to develop risk-based sampling plans and design interventions that reduce associated health risks.

Frequent and strong cold-air pooling drives temperate forest composition.

Cold-air pooling is an important topoclimatic process that creates temperature inversions with the coldest air at the lowest elevations. Incomplete understanding of sub-canopy spatiotemporal cold-air pooling dynamics and associated ecological impacts hinders predictions and conservation actions related to climate change and cold-dependent species and functions. To determine if and how cold-air pooling influences forest composition, we characterized the frequency, strength, and temporal dynamics of cold-air pooling in the sub-canopy at local to regional scales in New England, USA. We established a network of 48 plots along elevational transects and continuously measured sub-canopy air temperatures for 6-10 months (depending on site). We then estimated overstory and understory community temperature preferences by surveying tree composition in each plot and combining these data with known species temperature preferences. We found that cold-air pooling was frequent (19-43% seasonal occurrences) and that sites with the most frequent inversions displayed inverted forest composition patterns across slopes with more cold-adapted species, namely conifers, at low instead of high elevations. We also observed both local and regional variability in cold-air pooling dynamics, revealing that while cold-air pooling is common, it is also spatially complex. Our study, which uniquely focused on broad spatial and temporal scales, has revealed some rarely reported cold-air pooling dynamics. For instance, we discovered frequent and strong temperature inversions that occurred across seasons and in some locations were most frequent during the daytime, likely affecting forest composition. Together, our results show that cold-air pooling is a fundamental ecological process that requires integration into modeling efforts predicting future forest vegetation patterns under climate change, as well as greater consideration for conservation strategies identifying potential climate refugia for cold-adapted species.

Validation of a Novel Neurologic Assessment Test for Healthcare Staff.

ABSTRACT: BACKGROUND: To measure the effectiveness of an educational intervention, it is essential to develop high-quality, validated tools to assess a change in knowledge or skills after an intervention. An identified gap within the field of neurology is the lack of a universal test to examine knowledge of neurological assessment. METHODS: This instrument development study was designed to determine whether neuroscience knowledge as demonstrated in a Neurologic Assessment Test (NAT) was normally distributed across healthcare professionals who treat patients with neurologic illness. The variables of time, knowledge, accuracy, and confidence were individually explored and analyzed in SAS. RESULTS: The mean (standard deviation) time spent by 135 participants to complete the NAT was 12.9 (3.2) minutes. The mean knowledge score was 39.5 (18.2), mean accuracy was 46.0 (15.7), and mean confidence was 84.4 (24.4). Despite comparatively small standard deviations, Shapiro-Wilk scores indicate that the time spent, knowledge, accuracy, and confidence are nonnormally distributed ( P < .0001). The Cronbach α was 0.7816 considering all 3 measures (knowledge, accuracy, and confidence); this improved to an α of 0.8943 when only knowledge and accuracy were included in the model. The amount of time spent was positively associated with higher accuracy ( r2 = 0.04, P < .05), higher knowledge was positively associated with higher accuracy ( r2 = 0.6543, P < .0001), and higher knowledge was positively associated with higher confidence ( r2 = 0.4348, P < .0001). CONCLUSION: The scores for knowledge, confidence, and accuracy each had a slightly skewed distribution around a point estimate with a standard deviation smaller than the mean. This suggests initial content validity in the NAT. There is adequate initial construct validity to support using the NAT as an outcome measure for projects that measure change in knowledge. Although improvements can be made, the NAT does have adequate construct and content validity for initial use.

Factors associated with parental human papillomavirus vaccination intentions among adolescents from socioeconomically advantaged versus deprived households: a nationwide, cross-sectional survey.

Background: In the USA, HPV vaccine coverage is substantially lower among adolescents from high-income households compared to their low-income counterparts. We examined and compared the factors associated with parental HPV vaccination intentions between socioeconomically divergent groups. Methods: Data from unvaccinated and not fully HPV-vaccinated adolescents from the 2017-2021 National Immunization Survey (NIS)-Teen were analyzed. Socioeconomically advantaged vs. deprived groups were identified based on dichotomized income (material capital) and education (social capital). Parental intent to initiate and complete the HPV vaccine series was compared using bivariable analysis and the factors associated with lacking intent were identified. Findings: The 2017-2021 NIS-Teen included a total of 212,643 participants; the final analytical sample consisted of 105,958 adolescents (an estimated 10.3 million adolescents) who were unvaccinated or not fully vaccinated. In the advantaged group, 64.7% of parents of unvaccinated adolescents (equating to 2.4 million US adolescents) had no intention to initiate the HPV vaccine compared to 40.9% of parents in the deprived group (equating to 0.2 million adolescents) (P < 0.0001; S > 13.29). The most frequent reason for lacking intent in the advantaged group was 'safety concerns' (25.5%). In the deprived group, 'lack of knowledge', 'not recommended', and 'not needed' were common reasons (nearly 15% each). Lack of intent to complete the HPV vaccine series was higher in the advantaged group (43.9%; 1.1 million adolescents) compared to the deprived group (25.2%; 0.08 million adolescents) (P < 0.0001; S > 13.29). More than half in the advantaged group (58.4%) and over a third (37.1%) in the deprived group cited 'already up to date' as the main reason for not completing the HPV vaccine series. Interpretation: Lack of intent to initiate and complete the HPV vaccination series, particularly among socioeconomically advantaged parents is a significant barrier to achieving the national goal in the USA. Funding: The US National Institute on Minority Health and Health Disparities, the National Center for Advancing Translational Sciences, MUSC Hollings Cancer Center Seed funding, and the US National Cancer Institutes.

Primary care provision for young people with ADHD: a multi-perspective qualitative study.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) is a highly prevalent neurodevelopmental disorder. UK guidance states that primary care has a vital role in effective ADHD management, including referral, medication prescribing and monitoring, and providing broader mental health and wellbeing support. However, many GPs feel unsupported to provide health care for young people with ADHD. Inadequate health care is associated with rising costs for patients and society. AIM: To investigate the experiences of young people with ADHD accessing primary care in England, from the perspectives of people with lived experience of ADHD and healthcare professionals (HCPs). DESIGN AND SETTING: A qualitative study. Interviews were conducted with HCPs (GPs, practice managers, and a wellbeing worker) and people with lived experience of ADHD (young people aged 16-25 years and their supporters) located in integrated care systems across England. METHOD: Semi-structured interviews were conducted with participants at five purposively selected general practices (varying by deprivation, ethnicity, and setting). Questions focused on experiences of accessing/providing health care for ADHD. Reflexive thematic analysis was undertaken within a critical realist framework to understand how provision works in practice and to explore potential improvements. RESULTS: In total, 20 interviews were completed with 11 HCPs and nine people with lived experience. Three themes were generated: a system under stress, incompatibility between ADHD and the healthcare system, and strategies for change in ADHD primary care provision. CONCLUSION: Standardisation of ADHD management in primary care, providing better information and support for HCPs, and advising on reasonable adjustments for people with lived experience could help improve access to effective treatments for young people living with ADHD.

A GIPR antagonist conjugated to GLP-1 analogues promotes weight loss with improved metabolic parameters in preclinical and phase 1 settings.

Obesity is a major public health crisis. Multi-specific peptides have emerged as promising therapeutic strategies for clinical weight loss. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are endogenous incretins that regulate weight through their receptors (R). AMG 133 (maridebart cafraglutide) is a bispecific molecule engineered by conjugating a fully human monoclonal anti-human GIPR antagonist antibody to two GLP-1 analogue agonist peptides using amino acid linkers. Here, we confirm the GIPR antagonist and GLP-1R agonist activities in cell-based systems and report the ability of AMG 133 to reduce body weight and improve metabolic markers in male obese mice and cynomolgus monkeys. In a phase 1, randomized, double-blind, placebo-controlled clinical study in participants with obesity ( NCT04478708 ), AMG 133 had an acceptable safety and tolerability profile along with pronounced dose-dependent weight loss. In the multiple ascending dose cohorts, weight loss was maintained for up to 150 days after the last dose. These findings support continued clinical evaluation of AMG 133.

Enzyme Kinetics Analysis: An online tool for analyzing enzyme initial rate data and teaching enzyme kinetics.

Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time-consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web-tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis-Menten, Hill, simple reversible inhibition models, ternary-complex, and ping-pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g., K M or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements.

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.

Introducing the Dynamic Well-Stirred Model for Predicting Hepatic Clearance and Extraction Ratio.

The well-stirred model (WSM) incorporating the fraction of unbound drug (fu) to account for the effect of plasma binding on intrinsic clearance has been widely used for predicting hepatic clearance under the assumption that drug protein binding reaches equilibrium instantaneously. Our theoretical analysis reveals that the effect of protein binding on intrinsic clearance is better accounted for with the dynamic free fraction (fD), a measure of drug protein binding affinity, which leads to a putative dynamic well-stirred model (dWSM) without the instantaneous equilibrium assumption. Using recombinant CYP3A4 as the in vitro clearance system, we demonstrate that the binding effect of albumin on the intrinsic clearance of both highly bound midazolam and highly free verapamil is fully corrected by their corresponding fD values, respectively. On the other hand, fu only corrects the binding effect of albumin on the intrinsic clearance of verapamil, and yields severe over-correction of the intrinsic clearance of midazolam. The results suggest that the traditional WSM is suitable for highly free drugs like verapamil but not necessarily for highly bound drugs such as midazolam due to the violation of the instantaneous equilibrium assumption or under-estimating the true free drug concentration. In comparison, the dWSM incorporating fD holds true as long as drug elimination follows steady-state kinetics, and hence, it is more broadly applicable to drugs with different protein binding characteristics. Here we demonstrate with 36 diverse drugs, that the dWSM significantly improves the accuracy of predicting human hepatic clearance and liver extraction ratio from in vitro microsomal clearance data, highlighting the importance of drug plasma protein binding kinetics in addressing the under-prediction of hepatic clearance by the WSM.