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Abstract Background Low- and middle-income countries face a disproportionate impact of sexual health problems compared to high-income countries. To address this situation proper interpersonal communication skills are essential for clinician to gather necessary information during medical history-taking related to sexual health. This study aimed to evaluate the interrater reliability of ratings on sexual health-related interpersonal communication and medical history-taking between SPs and trained HCP faculty for health care professional students. Methods We conducted a cross-sectional comparative study to evaluate the interrater reliability of ratings for sexual health-related interpersonal communication and medical history-taking. The data were collected from medical and nursing students at Muhimbili University of Health and Allied Sciences, who interviewed 12 Standardized Patients (SPs) presenting with sexual health issues. The video-recorded interviews rated by SPs, were compared to the one rated by 5 trained Health Care Professional (HCP) faculty members. Inter-rater reliability was evaluated using percent agreement (PA) and kappa statistics (κ). Results A total of 412 students (mean age 24) were enrolled in the study to conduct interviews with two SPs presenting with sexual health concerns. For interpersonal communication (IC), the overall median agreement between raters was slight (κ2 0.0095; PA 48.9%) while the overall median agreement for medical sexual history-taking was deemed fair (κ2 0.139; PA 75.02%). Conclusion The use of SPs for training and evaluating medical and nursing students in Tanzania is feasible only if they undergo proper training and have sufficient time for practice sessions, along with providing feedback to the students.

Abstract Background Female genital mutilation/cutting (FGM/C) is tied to one of the most conservative cultures in the Mediterranean and Sub-Saharan Africa. More than 200 million girls and women in 30 African, Asian and the middle Eastern countries have undergone FGM/C. However, healthcare professionals are not adequately trained to prevent and manage FGM/C-related complications including sexual health problems. This study aimed to assess the need and acceptability of a curriculum to train nursing and medical students in the sexual healthcare of clients with FGM/C in Tanzania. Methods We used a descriptive and cross sectional study design to collect and analyse information from 271 medical and 137 nursing students in Tanzania. A Qualtrics online survey was used to obtain quantitative data on training interest, previous training received, and the curriculum delivery method. Open-ended questions were used to explore their insights on significance to obtain the necessary competencies to treat and prevent FGM/C. Descriptive statistics were used to analyze quantitative data while qualitative data were analyzed using a thematic approach. Results Almost half of the participants reported they had little to no training in sexual healthcare for women with FGM/C (47%). In all, 82.4% reported the training to be acceptable. Following thematic analysis of open-ended questions, participants expressed a desire to improve their competencies to meet the current and future sexual and psychological health needs of women and girls who have undergone FGM/C. Conclusion It is a necessary and acceptable to develop a curriculum to train healthcare students to diagnose, treat and prevent sexual health complications related to FGM/C. In our study, designing a culturally sensitive curriculum and its delivery method, that includes practical sessions with simulated patients, was considered the most beneficial and favorable.

Objectives: To explore nurse’s perceptions on barriers for Implementing Pressure Ulcers Preventive Measures among critically ill patients. Research Design: An exploratory qualitative study design was employed. In-depth interviews were conducted with 15 nurses to explore their perceptions on barriers for preventing pressure ulcers among critically ill patient. Qualitative thematic analysis approach was used to analyze data. Study settings: Medical high dependent unit, surgical high dependent unit, medical Intensive Care unit and surgical intensive care units at tertiary teaching hospital. Findings: Seven themes emerged that describes nurses’ perceptions on barriers for implementing pressure ulcers preventive measures among patients with critical illness. The themes are: Restrictions from therapeutic interventions and severity of patient condition; Shortage of staff and equipment for pressure ulcer prevention; Unbalanced shift staffing and competing need for patient care; health care providers’ incompetence and carelessness; Lack of guidelines on implementing best practice for pressure ulcer prevention; Lack of accountability measures for negligence; and Minimal management’s commitment for PUs prevention. Conclusion: This study reveals contextual based barriers for effective prevention of ppressure ulcers among critically ill patients. Nurses should be continuously empowered with competence for pressure ulcers prevention. This should go along with staffing and equipping critical care settings for pressure ulcers prevention. Managerial oversight is required for effective implementation of pressure ulcer preventive measures. This should include development of guidelines and policies, establishing accountability measures for negligence, malpractice as well as initiating clinical audit for reflective learning purpose.

ObjectiveMaintaining provision and utilization of maternal healthcare services is susceptible to external influences. This study describes how maternity care was provided during the COVID-19 pandemic and assesses patterns of service utilization and perinatal health outcomes in 16 referral hospitals (four each) in Benin, Malawi, Tanzania and Uganda.MethodsWe used an embedded case-study design and two data sources. Responses to open-ended questions in a health-facility assessment survey were analyzed with content analysis. We described categories of adaptations and care provision modalities during the pandemic at the hospital and maternity ward levels. Aggregate monthly service statistics on antenatal care, delivery, caesarean section, maternal deaths, and stillbirths covering 24 months (2019 and 2020; pre-COVID-19 and COVID-19) were examined.ResultsDeclines in the number of antenatal care consultations were documented in Tanzania, Malawi, and Uganda in 2020 compared to 2019. Deliveries declined in 2020 compared to 2019 in Tanzania and Uganda. Caesarean section rates decreased in Benin and increased in Tanzania in 2020 compared to 2019. Increases in maternal mortality ratio and stillbirth rate were noted in some months of 2020 in Benin and Uganda, with variability noted between hospitals. At the hospital level, teams were assigned to respond to the COVID-19 pandemic, routine meetings were cancelled, and maternal death reviews and quality improvement initiatives were interrupted. In maternity wards, staff shortages were reported during lockdowns in Uganda. Clinical guidelines and protocols were not updated formally; the number of allowed companions and visitors was reduced.ConclusionVarying approaches within and between countries demonstrate the importance of a contextualized response to the COVID-19 pandemic. Maternal care utilization and the ability to provide quality care fluctuated with lockdowns and travel bans. Women's and maternal health workers' needs should be prioritized to avoid interruptions in the continuum of care and prevent the deterioration of perinatal health outcomes.

Abstract Background Health professions training in Tanzania has gradually adopted the competency-based curricula (CBC) approach that focuses on learners’ acquisition of competencies in knowledge skills and aptitude. Feedback from the graduates is crucial for improving CBC implementation and review to ensure that graduates acquire clinical competencies and thus improve patient care. Objectives To explore the teaching–learning experiences of medical and nursing graduates during their internship and their supervisors in three teaching hospitals. Methods Exploratory qualitative data from in-depth interviews and focus group discussions were analysed using a content analysis framework. Medical and nursing graduates who were in an internship program in three teaching hospitals in Tanzania were conveniently recruited based on their availability during the data collection period. Results The use of clinical logbooks, guidelines and protocols, bedside teaching, mentorship, and supervision were important for the acquisition of clinical competencies. Graduates demonstrated inadequate clinical competency including confidence, commitment, and professionalism because of a lack of clear clinical guidelines and protocols, inadequate bedside teaching including supervision by faculty and ineffective communication and feedback. Conclusions Clinical teaching–learning tools and approaches facilitate students’ acquisition of clinical competencies. However, inadequate use of guidelines and protocols, bedside teaching, clinical supervision and effective communication was observed. Use of these clinical teaching approaches should be emphasized for students’ clinical competency acquisition.

IntroductionA thermoplastic mask is the most widely used immobilization device for head and neck cancer patients undergoing radiotherapy. The radiation therapist is the staff responsible to prepare these masks and set-up the patients for treatment, a procedure that requires time, patience, and precision. An understanding of Radiation therapists' perceptions regarding thermoplastic mask use will help design interventions to address challenges encountered in its use. This study explored Radiation therapists' perceptions of thermoplastic mask use for head and neck cancer patients undergoing radiotherapy at Ocean Road Cancer Institute in Tanzania.Material and methodsAn exploratory qualitative study design was used to explore thermoplastic mask use for head and neck cancer patients undergoing radiotherapy. Semi-structured in-depth interviews were conducted, involving fifteen Radiation therapists from Ocean Road Cancer Institute in Tanzania between March and May 2021. A thematic analysis method was used to identify themes from data scripts.ResultsFour themes emerged that reflected radiation therapists' perceptions of thermoplastic mask use for head and neck cancer immobilization among patients undergoing radiotherapy. Emerged themes were (1) Perceived benefits and limitations of thermoplastic mask use, (2) Refresher training and supervision requirements for effective use, (3) Proper storage for quality maintenance, and (4) Increased financial support and proper budgeting.ConclusionParticipants perceived better patient immobilization with a thermoplastic mask use. However, too often recycling of thermoplastic masks and the long waiting time between thermoplastic mask preparation and treatment delivery limits their effective use. For efficient use of thermoplastic masks, there is a need for Radiation therapists' refresher training and proper supervision, improving the storage system and increasing financial support for procuring new thermoplastic masks.

Abstract Background In order improve the quality of birth care and women satisfaction with birthing process it is recommended that every woman should be offered the option to experience labour and childbirth with a companion of her choice. Involving husbands who are decision makers in the household may a play role in reducing maternal mortality which is unacceptably high despite the targeted goal to reduce this mortality up to three quarters as targeted in the MDGs by 2015. This is still addressed in the Sustainable Development Goals (SDGs) of 2015/30. This study aimed to explore the experiences and perceptions of husbands’ support of their wives during pregnancy, labour and deliveries in Tanzania. Methods Qualitative descriptive study design was employed; involving men aged between 24 and 63 years. Participants were selected purposefully at the clinic and in labour ward of SekouToure Regional Referral Hospital (SRRH). The in-depth interview, guided by semi structured interview guide was used to collect the audio recorded and hand written information. Data were analysed using qualitative content analysis. Results Nine semi-structured interviews were conducted with husbands of women attended for antenatal care and those came for deliveries. Four themes emerged; Demonstrating care, love and affection, adopting modern life style, observing women’s right and meeting social economic difficulties. Husbands’ support to their partners is a good behaviour practiced during matrimonial lives. Husbands who support their partners during pregnancy and delivery consider themselves as being modern men as they at home take duties beside their usual tasks to let their wives have adequate time to rest during pregnancy. Poor road infrastructure makes difficult to get transport to the healthcare facility especially when labour is imminent. Also ward infrastructure is not supportive to accommodate husbands when they accompany their wives to the healthcare facility. Conclusions The healthcare settings in low income countries need to accommodate men during the routine antenatal and intranatal care for the positive outcome of labour and delivery. Educating men on importance of active involvement in reproductive and child health services is important. Exploratory research should be conducted to understand how education and urbanisation affects men involvement in maternal and child health specifically in the low income countries.

Background Attention-deficit hyperactivity disorder (ADHD) is the most common neurobehavioral childhood disorder. Children with ADHD are difficult to handle due to the symptoms causing great impairments such as inattention, hyperactivity compared to other childhood mental disorders. Having a child with ADHD is a stressful situation as it impacts the whole family. However, little is known about the experiences and challenges of parents caring for children with ADHD in low and middle-income countries such as Tanzania. Thus, this study explored the experiences and challenges of parents caring for children with ADHD in Dar es Salaam, Tanzania. Methods We conducted a qualitative descriptive study involving 16 parents of children with ADHD at Muhimbili National Hospital (MNH). We used the purposive sampling technique to obtain the participants. In-depth interviews, using a semi-structured interview guide, were used to collect data. Audio-recorded data were transcribed, translated, and analysed using qualitative content analysis. Results Parents experienced difficulties in handling the children whose level of functioning was impaired due to abnormal and disruptive behaviour such as not being able to follow parental instructions. Psychological problems were also experienced due to caring demands exacerbated by lack of support and stigma from the community. Moreover, there were disruptions in family functioning and social interactions among family members due to the children’s behaviour. Lastly, too much time and family resources spent to fulfil the needs of these children culminated into disruption in economic activities that negatively affected everyday life. Conclusion Parents struggle to meet and cope with care demands posed by children with ADHD. The disruptive nature of ADHD symptoms presents a unique caring challenge different from those experienced with other childhood mental illnesses. To address these challenges, a collaborative approach among key stakeholders such as the government, health care professionals, and non-governmental organizations, is needed.

Abstract Background Tanzania has ratified and abides to legal treaties indicating the obligation of the state to provide essential maternal health care as a basic human right. Nevertheless, the quality of maternal health care is disproportionately low. The current study sets to understand maternal health services’ delivery from the perspective of rural health workers’, and to understand barriers for and better strategies for realization of the right to quality maternal health care. Methods Semi-structured in-depth interviews were conducted, involving 11 health workers mainly; medical attendants, enrolled nurses and Assistant Medical Officers from primary health facilities in rural Tanzania. Structured observation complemented data from interviews. Interview data were analyzed using thematic analysis guided by the conceptual framework of the right to health. Results Three themes emerged that reflected health workers’ opinion towards the quality of health care services; “It’s hard to respect women’s preferences”, “Striving to fulfill women’s needs with limited resources”, and “Trying to facilitate women’s access to services at the face of transport and cost barriers”. Conclusion Health system has left health workers as frustrated right holders, as well as dis-empowered duty bearers. This was due to the unavailability of adequate material and human resources, lack of motivation and lack of supervision, which are essential for provision of quality maternal health care services. Pregnant women, users of health services, appeared to be also left as frustrated right holders, who incurred out-of-pocket costs to pay for services, which were meant to be provided free.

Background: Maternal health care provision remains a major challenge in developing countries. There is agreement that the provision of quality clinical services is essential if high rates of maternal death are to be reduced. However, despite efforts to improve access to these services, a high number of women in Tanzania do not access them. The aim of this study is to explore women's views about the maternal health services (pregnancy, delivery, and postpartum period) that they received at health facilities in order to identify gaps in service provision that may lead to low-quality maternal care and increased risks associated with maternal morbidity and mortality in rural Tanzania. Design: We gathered qualitative data from 15 focus group discussions with women attending a health facility after child birth and transcribed it verbatim. Qualitative content analysis was used for analysis. Results: ‘Three categories emerged that reflected women's perceptions of maternal health care services: “mothers perceive that maternal health services are beneficial,” “barriers to accessing maternal health services” such as availability and use of traditional birth attendants (TBAs) and the long distances between some villages, and “ambivalence regarding the quality of maternal health services” reflecting that women had both positive and negative perceptions in relation to quality of health care services offered’. Conclusions: Mothers perceived that maternal health care services are beneficial during pregnancy and delivery, but their awareness of postpartum complications and the role of medical services during that stage were poor. The study revealed an ambivalence regarding the perceived quality of health care services offered, partly due to shortages of material resources. Barriers to accessing maternal health care services, such as the cost of transport and the use of TBAs, were also shown. These findings call for improvement on the services provided. Improvements should address, accessibility of services, professionals' attitudes and stronger promotion of the importance of postpartum check-ups, both among health care professionals and women.

The combination of gemcitabine with platinum agents is a widely used chemotherapy regimen for a number of tumour types. Gemcitabine plus cisplatin remains the current therapeutic choice for biliary tract cancer. Gemcitabine is associated with multiple cellular drug resistance mechanisms and other limitations and has thereforelined in use. NUC-1031 (Acelarin) is a phosphorylated form of gemcitabine, protected by the addition of a phosphoramidate moiety, developed to circumvent the key limitations and generate high levels of the cytotoxic metabolite, dFdCTP. The rationale for combination of gemcitabine and cisplatin is determined by in vitro cytotoxicity. This, however, does not offer an explanation of how these drugs lead to cell death. In this study we investigate the mechanism of action for NUC-1031 combined with cisplatin as a rationale for treatment. NUC-1031 is metabolised to dFdCTP, detectable up to 72 h post-treatment and incorporated into DNA, to stall the cell cycle and cause DNA damage in biliary tract and ovarian cancer cell lines. In combination with cisplatin, DNA damage was increased and occurred earlier compared to monotherapy. The damage associated with NUC-1031 may be potentiated by a second mechanism, via binding the RRM1 subunit of ribonucleotide reductase and perturbing the nucleotide pools; however, this may be mitigated by increased RRM1 expression. The implication of this was investigated in case studies from a Phase I clinical trial to observe whether baseline RRM1 expression in tumour tissue at time of diagnosis correlates with patient survival., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: DP and GMZ were funded by Nucana plc https://www.nucana.com/. ALD and DJH partly employed by Nucana plc. OJR, JB were at time of research, fully employed by Nucana plc The sponsor did not have any role in the study design, collection or analysis. The funders approved theision to publish and provided feedback on the written manuscript Conflict of interest statement DP and GMZ were funded by Nucana plc https://www.nucana.com/. ALD and DJH partly employed by Nucana plc. OJR and JB were at time of research, fully employed by Nucana plc. The sponsor did not have any role in the study design, data collection or analysis. The funders approved theision to publish and provided feedback on the written manuscript., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: Phenotyping is a core task in observational health research utilizing electronic health records (EHRs). Developing an accurate algorithm demands substantial input from domain experts, involving extensive literature review and evidence synthesis. This burdensome process limits scalability and delays knowledge discovery. We investigate the potential for leveraging large language models (LLMs) to enhance the efficiency of EHR phenotyping by generating high-quality algorithm drafts., Materials and Methods: We prompted four LLMs-GPT-4 and GPT-3.5 of ChatGPT, Claude 2, and Bard-in October 2023, asking them to generate executable phenotyping algorithms in the form of SQL queries adhering to a common data model (CDM) for three phenotypes (ie, type 2 diabetes mellitus, dementia, and hypothyroidism). Three phenotyping experts evaluated the returned algorithms across several critical metrics. We further implemented the top-rated algorithms and compared them against clinician-validated phenotyping algorithms from the Electronic Medical Records and Genomics (eMERGE) network., Results: GPT-4 and GPT-3.5 exhibited significantly higher overall expert evaluation scores in instruction following, algorithmic logic, and SQL executability, when compared to Claude 2 and Bard. Although GPT-4 and GPT-3.5 effectively identified relevant clinical concepts, they exhibited immature capability in organizing phenotyping criteria with the proper logic, leading to phenotyping algorithms that were either excessively restrictive (with low recall) or overly broad (with low positive predictive values)., Conclusion: GPT versions 3.5 and 4 are capable of drafting phenotyping algorithms by identifying relevant clinical criteria aligned with a CDM. However, expertise in informatics and clinical experience is still required to assess and further refine generated algorithms., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A workflow has been evaluated that utilizes a single tissue section to obtain spatially co-registered, molecular, and phenotypical information suitable for AI-enabled image analysis. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to obtain molecular information followed by conventional histological staining and immunolabelling. The impact of varying DESI-MSI conditions (e.g., heated transfer line (HTL) temperature, scan rate, acquisition time) on the detection of small molecules and lipids as well as on tissue integrity crucial for integration into typical clinical pathology workflows was assessed in human kidney. Increasing the heated transfer line temperature from 150 to 450 °C resulted in a 1.8-fold enhancement in lipid signal at a scan rate of 10 scans/s, while preserving histological features. Moreover, increasing the acquisition speed to 30 scans/s yielded superior lipid signal when compared to 10 scans/s at 150 °C. Tissue morphology and protein epitopes remained intact allowing full histological assessment and further multiplex phenotyping by immunofluorescence (mIF) and immunohistochemistry (mIHC) of the same section. The successful integration of the workflow incorporating DESI-MSI, H&E, and immunolabelling on a single tissue section revealed an accumulation of ascorbic acid in regions of focal chronic inflammatory cell infiltrate within non-cancerous kidney tissue. Additionally, a strong positive correlation between PI 38:3 and proliferating cells was observed in clear cell renal cell carcinoma (ccRCC) showing the utility of this approach in uncovering molecular associations in disease pathology., (© 2024. The Author(s).)

Hypertriglyceridemia (HTG) is a common cardiovascular risk factor characterized by elevated triglyceride (TG) levels. Researchers have assessed the genetic factors that influence HTG in studies focused predominantly on individuals of European ancestry. However, relatively little is known about the contribution of genetic variation of HTG in people of African ancestry (AA), potentially constraining research and treatment opportunities. Our objective was to characterize genetic profiles among individuals of AA with mild-to-moderate HTG and severe HTG versus those with normal TGs by leveraging whole-genome sequencing data and longitudinal electronic health records available in the All of Us program. We compared the enrichment of functional variants within five canonical TG metabolism genes, an AA-specific polygenic risk score for TGs, and frequencies of 145 known potentially causal TG variants between HTG patients and normal TG among a cohort of AA patients (N = 15,373). Those with mild-to-moderate HTG (N = 342) and severe HTG (N ≤ 20) were more likely to carry APOA5 p.S19W (odds ratio = 1.94, 95% confidence interval = [1.48-2.54], P = 1.63 × 10 -6 and OR = 3.65, 95% confidence interval: [1.22-10.93], P = 0.02, respectively) than those with normal TG. They were also more likely to have an elevated (top 10%) polygenic risk score, elevated carriage of potentially causal variant alleles, and carry any genetic risk factor. Alternative definitions of HTG yielded comparable results. In conclusion, individuals of AA with HTG were enriched for genetic risk factors compared to individuals with normal TGs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Polygenic variation unrelated to disease contributes to interindividual variation in baseline white blood cell (WBC) counts, but its clinical significance is uncharacterized. We investigated the clinical consequences of a genetic predisposition toward lower WBC counts among 89,559 biobank participants from tertiary care centers using a polygenic score for WBC count (PGS WBC ) comprising single nucleotide polymorphisms not associated with disease. A predisposition to lower WBC counts was associated with a decreased risk of identifying pathology on a bone marrow biopsy performed for a low WBC count (odds-ratio = 0.55 per standard deviation increase in PGS WBC [95%CI, 0.30-0.94], p = 0.04), an increased risk of leukopenia (a low WBC count) when treated with a chemotherapeutic (n = 1724, hazard ratio [HR] = 0.78 [0.69-0.88], p = 4.0 × 10 -5 ) or immunosuppressant (n = 354, HR = 0.61 [0.38-0.99], p = 0.04). A predisposition to benign lower WBC counts was associated with an increased risk of discontinuing azathioprine treatment (n = 1,466, HR = 0.62 [0.44-0.87], p = 0.006). Collectively, these findings suggest that there are genetically predisposed individuals who are susceptible to escalations or alterations in clinical care that may be harmful or of little benefit., (© 2024. The Author(s).)

Drug repurposing represents an attractive alternative to the costly and time-consuming process of new drug development, particularly for serious, widespread conditions with limited effective treatments, such as Alzheimer's disease (AD). Emerging generative artificial intelligence (GAI) technologies like ChatGPT offer the promise of expediting the review and summary of scientific knowledge. To examine the feasibility of using GAI for identifying drug repurposing candidates, we iteratively tasked ChatGPT with proposing the twenty most promising drugs for repurposing in AD, and tested the top ten for risk of incident AD in exposed and unexposed individuals over age 65 in two large clinical datasets: (1) Vanderbilt University Medical Center and (2) the All of Us Research Program. Among the candidates suggested by ChatGPT, metformin, simvastatin, and losartan were associated with lower AD risk in meta-analysis. These findings suggest GAI technologies can assimilate scientific insights from an extensive Internet-based search space, helping to prioritize drug repurposing candidates and facilitate the treatment of diseases., (© 2024. The Author(s).)

Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2'-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2'-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2'-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2'-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis ( Ct NDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5-9.5, with two very distinct p K a values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2'-fluoro-2'-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2'-difluoro-2'-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2'-deoxy)ribonucleoside selection, as Ct NDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of Ct NDT in biocatalysis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Gabapentin is prescribed for pain and is perceived as safe generally. However, gabapentin can cause respiratory depression, exacerbated by concomitant central nervous system depressants (e.g., opioids), a concern for vulnerable populations. We compared mortality rates among new users of either gabapentin or duloxetine with or without concurrent opioids in the 20% Medicare sample. We conducted a new-user design retrospective cohort study, in Medicare enrollees ages 65-89 years with noncancer chronic pain and no severe illness who filled prescriptions between 2015 and 2018 for gabapentin (n = 233,060) or duloxetine (n = 34,009). Daily opioid doses, estimated in morphine milligram equivalents (MMEs), were classified into none, low (0 < MME < 50), and high (≥ 50 MME), based on Centers for Disease Control and Prevention (CDC) recommendations. The outcomes were all-cause mortality (primary) and out-of-hospital mortality (secondary). We used inverse probability of treatment weighting to adjust for differences between gabapentin and duloxetine users. During 116,707 person-years of follow-up, 1,379 patients died. All-cause mortality rate in gabapentin users was 12.16 per 1,000 person-years vs. 9.94 per 1,000 in duloxetine users. Risks were similar for users with no concurrent opioids (adjusted hazard ratio (aHR) = 1.03, 95% confidence interval (CI): 0.80-1.31) or low-dose daily opioids (aHR = 1.06, 95% CI: 0.63-1.76). However, gabapentin users receiving concurrent high-dose daily opioids had an increased rate of all-cause mortality compared with duloxetine users on high-dose opioids (aHR = 2.03, 95% CI: 1.19-3.46). Out-of-hospital mortality yielded similar results. In this retrospective cohort study of Medicare beneficiaries, concurrent use of high-dose opioids and gabapentin was associated with a higher all-cause mortality risk than that for concurrent use of high-dose opioids and duloxetine., (© 2023 The Authors. Clinical Pharmacology & Therapeutics © 2023 American Society for Clinical Pharmacology and Therapeutics.)

Background: Two risk variants in the apolipoprotein L1 gene ( APOL1 ) have been associated with increased susceptibility to sepsis in Black patients. However, it remains unclear whether APOL1 high-risk genotypes are associated with occurrence of either sepsis or sepsis-related phenotypes in patients hospitalized with infections, independent of their association with pre-existing severe renal disease., Methods: A retrospective cohort study of 2242 Black patients hospitalized with infections. We assessed whether carriage of APOL1 high-risk genotypes was associated with the risk of sepsis and sepsis-related phenotypes in patients hospitalized with infections. The primary outcome was sepsis; secondary outcomes were short-term mortality, and organ failure related to sepsis., Results: Of 2242 Black patients hospitalized with infections, 565 developed sepsis. Patients with high-risk APOL1 genotypes had a significantly increased risk of sepsis (odds ratio [OR]=1.29 [95% CI, 1.00-1.67; p=0.047]); however, this association was not significant after adjustment for pre-existing severe renal disease (OR = 1.14 [95% CI, 0.88-1.48; p=0.33]), nor after exclusion of those patients with pre-existing severe renal disease (OR = 0.99 [95% CI, 0.70-1.39; p=0.95]). APOL1 high-risk genotypes were significantly associated with the renal dysfunction component of the Sepsis-3 criteria (OR = 1.64 [95% CI, 1.21-2.22; p=0.001]), but not with other sepsis-related organ dysfunction or short-term mortality. The association between high-risk APOL1 genotypes and sepsis-related renal dysfunction was markedly attenuated by adjusting for pre-existing severe renal disease (OR = 1.36 [95% CI, 1.00-1.86; p=0.05]) and was nullified after exclusion of patients with pre-existing severe renal disease (OR = 1.16 [95% CI, 0.74-1.81; p=0.52])., Conclusions: APOL1 high-risk genotypes were associated with an increased risk of sepsis; however, this increased risk was attributable predominantly to pre-existing severe renal disease., Funding: This study was supported by R01GM120523 (QF), R01HL163854 (QF), R35GM131770 (CMS), HL133786 (WQW), and Vanderbilt Faculty Research Scholar Fund (QF). The dataset(s) used for the analyses described were obtained from Vanderbilt University Medical Center's BioVU which is supported by institutional funding, the 1S10RR025141-01 instrumentation award, and by the CTSA grant UL1TR0004from NCATS/NIH. Additional funding provided by the NIH through grants P50GM115305 and U19HL065962. The authors wish to acknowledge the expert technical support of the VANTAGE and VANGARD core facilities, supported in part by the Vanderbilt-Ingram Cancer Center (P30 CA068485) and Vanderbilt Vision Center (P30 EY08126). The funders had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication., Competing Interests: LJ, GL, AO, AI, AD, LD, AH, NC, CC, WW, CS, QF No competing interests declared, (© 2023, Jiang et al.)

The enzyme 2'-deoxynucleoside 5'-phosphate N -hydrolase 1 (DNPH1) catalyzes the N -ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum k cat / K M , two groups with an average p K a of 6.4 must be deprotonated, while two groups with an average p K a of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to k cat / K M . Solvent deuterium isotope effects on k cat / K M and k cat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Background: Amiodarone, the most effective antiarrhythmic drug in atrial fibrillation, inhibits apixaban and rivaroxaban elimination, thus possibly increasing anticoagulant-related risk for bleeding., Objective: For patients receiving apixaban or rivaroxaban, to compare risk for bleeding-related hospitalizations during treatment with amiodarone versus flecainide or sotalol, antiarrhythmic drugs that do not inhibit these anticoagulants' elimination., Design: Retrospective cohort study., Setting: U.S. Medicare beneficiaries aged 65 years or older., Patients: Patients with atrial fibrillation began anticoagulant use between 1 January 2012 and 30 November 2018 and subsequently initiated treatment with study antiarrhythmic drugs., Measurements: Time to event for bleeding-related hospitalizations (primary outcome) and ischemic stroke, systemic embolism, and death with or without recent (past 30 days) evidence of bleeding (secondary outcomes), adjusted with propensity score overlap weighting., Results: There were 91 590 patients (mean age, 76.3 years; 52.5% female) initiating use of study anticoagulants and antiarrhythmic drugs, 54 977 with amiodarone and 36 613 with flecainide or sotalol. Risk for bleeding-related hospitalizations increased with amiodarone use (rate difference [RD], 17.5 events [95% CI, 12.0 to 23.0 events] per 1000 person-years; hazard ratio [HR], 1.44 [CI, 1.27 to 1.63]). Incidence of ischemic stroke or systemic embolism did not increase (RD, -2.1 events [CI, -4.7 to 0.4 events] per 1000 person-years; HR, 0.80 [CI, 0.62 to 1.03]). The risk for death with recent evidence of bleeding (RD, 9.1 events [CI, 5.8 to 12.3 events] per 1000 person-years; HR, 1.66 [CI, 1.35 to 2.03]) was greater than that for other deaths (RD, 5.6 events [CI, 0.5 to 10.6 events] per 1000 person-years; HR, 1.15 [CI, 1.00 to 1.31]) (HR comparison: P  = 0.003). The increased incidence of bleeding-related hospitalizations for rivaroxaban (RD, 28.0 events [CI, 18.4 to 37.6 events] per 1000 person-years) was greater than that for apixaban (RD, 9.1 events [CI, 2.8 to 15.3 events] per 1000 person-years) ( P  = 0.001)., Limitation: Possible residual confounding., Conclusion: In this retrospective cohort study, patients aged 65 years or older with atrial fibrillation treated with amiodarone during apixaban or rivaroxaban use had greater risk for bleeding-related hospitalizations than those treated with flecainide or sotalol., Primary Funding Source: National Heart, Lung, and Blood Institute., Competing Interests: Disclosures: Disclosures can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M22-3238.

The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N 1 -(5-phospho-β-D-ribosyl)-ATP (PRATP) to N 1 -(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N -(5'-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall k cat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on k cat and k cat / K M ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N 1 -(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Objective: Duloxetine is a serotonin-norepinephrine reuptake inhibitor prescribed for musculoskeletal and other forms of chronic pain. Its dual pharmacologic properties have the potential to either raise or lower cardiovascular risk: adrenergic activity may increase the risk for acute myocardial infarction (AMI) and stroke, but antiplatelet activity may decrease risk. Gabapentin is another nonopioid medication used to treat pain, which is not thought to have adrenergic/antiplatelet effects. With the current emphasis on the use of nonopioid medications to treat patients with chronic pain, assessing cardiovascular risks associated with these medications among high-risk patients is important., Materials and Methods: We conducted a retrospective cohort study among a 20% sample of Medicare enrollees, aged 65 to 89, with chronic pain who were new users between 2015 and 2018 of either duloxetine (n = 34,009) or gabapentin (n = 233,060). We excluded individuals with cancer or other life-threatening conditions at study drug initiation. The primary outcome was a composite of AMI, stroke, and out-of-hospital mortality. We adjusted for comorbidity differences with time-dependent inverse probability of treatment weighting., Results: During 115,668 person-years of follow-up, 2361 patients had the composite primary outcome; the rate among new users of duloxetine was 16.7/1000 person-years compared with new users of gabapentin (21.1/1000 person-years), adjusted hazard ratio = 0.98 (95% CI: 0.83, 1.16). Results were similar for the individual components of the composite outcome as well as in analyses stratified by demographic and clinical characteristics., Discussion: In summary, cohort Medicare patients with non-cancer pain beginning treatment with duloxetine had rates of AMI, stroke, and out-of-hospital mortality comparable to those who initiated gabapentin., Competing Interests: Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland of the National Institutes of Health under award number R01AR073764. M.A.C. has started working for Vertex Pharmaceuticals. The remaining authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU., Methods: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC 50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage., Results: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU., Conclusion: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients., (© 2023. The Author(s).)

Sepsis accounts for one in three hospital deaths. Higher concentrations of high-density lipoprotein cholesterol (HDL-C) are associated with apparent protection from sepsis, suggesting a potential therapeutic role for HDL-C or drugs, such as cholesteryl ester transport protein (CETP) inhibitors that increase HDL-C. However, these beneficial clinical associations might be due to confounding; genetic approaches can address this possibility. We identified 73,406 White adults admitted to Vanderbilt University Medical Center with infection; 11,612 had HDL-C levels, and 12,377 had genotype information from which we constructed polygenic risk scores (PRS) for HDL-C and the effect of CETP on HDL-C. We tested the associations between predictors (measured HDL-C, HDL-C PRS, CETP PRS, and rs1800777) and outcomes: sepsis, septic shock, respiratory failure, and in-hospital death. In unadjusted analyses, lower measured HDL-C concentrations were significantly associated with increased risk of sepsis (p = 2.4 × 10 -23 ), septic shock (p = 4.1 × 10 -12 ), respiratory failure (p = 2.8 × 10 -8 ), and in-hospital death (p = 1.0 × 10 -8 ). After adjustment (age, sex, electronic health record length, comorbidity score, LDL-C, triglycerides, and body mass index), these associations were markedly attenuated: sepsis (p = 2.6 × 10 -3 ), septic shock (p = 8.1 × 10 -3 ), respiratory failure (p = 0.11), and in-hospital death (p = 4.5 × 10 -3 ). HDL-C PRS, CETP PRS, and rs1800777 significantly predicted HDL-C (p < 2 × 10 -16 ), but none were associated with sepsis outcomes. Concordant findings were observed in 13,254 Black patients hospitalized with infections. Lower measured HDL-C levels were significantly associated with increased risk of sepsis and related outcomes in patients with infection, but a causal relationship is unlikely because no association was found between the HDL-C PRS or the CETP PRS and the risk of adverse sepsis outcomes., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The aim of this study was to identify oxysterols and any down-stream metabolites in placenta, umbilical cord blood plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S-hydroxycholesterol in human placenta, previously reported in a single publication, and propose a pathway from 22R-hydroxycholesterol to a C 27 bile acid of probable structure 3β,20R,22R-trihydroxycholest-5-en-(25R)26-oic acid. The pathway is evident not only in placenta, but pathway intermediates are also found in umbilical cord plasma, maternal plasma and amniotic fluid but not non-pregnant women., Competing Interests: WG and YW are listed as inventors on the patent “Kit and method for quantitative detection of steroids” US9851368B2. WG, EY, and YW are shareholders in CholesteniX Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dickson, Yutuc, Thornton, Wang and Griffiths.)

Introduction: Infectious diseases are common causes of morbidity and mortality worldwide. Susceptibility to infection is highly heritable; however, little has been done to identify the genetic determinants underlying common infectious diseases. One GWAS was performed using 23andMe information about self-reported infections; we set out to confirm previous loci and identify new ones using medically diagnosed infections., Methods: We used the electronic health record (EHR)-based biobank at Vanderbilt and diagnosis codes to identify cases of 12 infectious diseases in white patients: urinary tract infection, pneumonia, chronic sinus infections, otitis media, candidiasis, streptococcal pharyngitis, herpes zoster, herpes labialis, hepatitis B, infectious mononucleosis, tuberculosis (TB) or a positive TB test, and hepatitis C. We selected controls from patients with no diagnosis code for the candidate disease and matched by year of birth, sex, and calendar year at first and last EHR visits. We conducted GWAS using SAIGE and transcriptome-wide analysis (TWAS) using S-PrediXcan. We also conducted phenome-wide association study to understand associations between identified genetic variants and clinical phenotypes., Results: We replicated three 23andMe loci (p ≤ 0.05): herpes zoster and rs7047299-A (p = 2.6 × 10 -3 ) and rs2808290-C (p = 9.6 × 10 -3 ;); otitis media and rs114947103-C (p = 0.04). We also identified 2 novel regions (p ≤ 5 × 10 -8 ): rs113235453-G for otitis media (p = 3.04 × 10 -8 ), and rs10422015-T for candidiasis (p = 3.11 × 10 -8 ). In TWAS, four gene-disease associations were significant: SLC30A9 for otitis media (p = 8.06 × 10 -7 ); LRP3 and WDR88 for candidiasis (p = 3.91 × 10 -7 and p = 1.95 × 10 -6 ); and AAMDC for hepatitis B (p = 1.51 × 10 -6 )., Conclusion: We conducted GWAS and TWAS for 12 infectious diseases and identified novel genetic contributors to the susceptibility of infectious diseases., (© 2022. The Author(s).)

Background: Thiopurines are an important class of immunosuppressants despite their risk for hematopoietic toxicity and narrow therapeutic indices. Benign neutropenia related to an ACKR1 variant (rs2814778-CC) is common among persons of African ancestries., Objective: To test whether rs2814778-CC was associated with azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine dosing., Design: Retrospective cohort study., Setting: Two tertiary care centers., Patients: Thiopurine users with White or Black race., Measurements: Azathioprine discontinuation attributed to hematopoietic toxicity. Secondary outcomes included weight-adjusted final dose, leukocyte count, and change in leukocyte count., Results: The rate of azathioprine discontinuation attributed to hematopoietic toxicity was 3.92 per 100 person-years among patients with the CC genotype ( n  = 101) and 1.34 per 100 person-years among those with the TT or TC genotype ( n  = 1365) (hazard ratio [HR] from competing-risk model, 2.92 [95% CI, 1.57 to 5.41]). The risk remained significant after adjustment for race (HR, 2.61 [CI, 1.01 to 6.71]). The risk associated with race alone (HR, 2.13 [CI, 1.21 to 3.75]) was abrogated by adjustment for genotype (HR, 1.13 [CI, 0.48 to 2.69]). Lower last leukocyte count and lower dosing were significant among patients with the CC genotype. Lower dosing was validated in an external cohort of 94 children of African ancestries prescribed the thiopurine 6-mercaptopurine (6-MP) for acute lymphoblastic leukemia. The CC genotype was independently associated with lower 6-MP dose intensity relative to the target daily dose of 75 mg/m 2 (median, 0.83 [IQR, 0.70 to 0.94] for the CC genotype vs. 0.94 [IQR, 0.72 to 1.13] for the TT or TC genotype; P  = 0.013)., Limitations: Unmeasured confounding; data limited to tertiary centers., Conclusion: Patients with the CC genotype had higher risk for azathioprine discontinuation attributed to hematopoietic toxicity and lower thiopurine doses. Genotype was associated with those risks, even after adjustment for race., Primary Funding Source: National Institutes of Health.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab . Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

TPMT and NUDT15 variants explain less than 25% of azathioprine-associated myelotoxicity. There are 25 additional genes in the thiopurine pathway that could also contribute to azathioprine myelotoxicity. We hypothesized that among TPMT and NUDT15 normal metabolizers, a score combining the genetically predicted expression of other proteins in the thiopurine pathway would be associated with a higher risk for azathioprine discontinuation due to myelotoxicity. We conducted a retrospective cohort study of new users of azathioprine who were normal TPMT and NUDT15 metabolizers. In 1201 White patients receiving azathioprine for an inflammatory disease, we used relaxed Least Absolute Shrinkage and Selection Operator (LASSO) regression to select genes that built a score for discontinuing azathioprine due to myelotoxicity. The score incorporated the predicted expression of AOX1 and NME1. Patients in the highest score tertile had a higher risk of discontinuing azathioprine compared to those in the lowest tertile (hazard ratio [HR] = 2.15, 95% confidence interval [CI] = 1.11-4.19, p = 0.024). Results remained significant after adjusting for a propensity score, including sex, tertile of calendar year at initial dose, initial dose, age at baseline, indication, prior TPMT testing, and the first 10 principal components of the genetic data (HR = 2.11, 95% CI = 1.08-4.13, p = 0.030). We validated the results in a cohort (N = 517 non-White patients and those receiving azathioprine to prevent transplant rejection) that included all other patients receiving azathioprine (HR = 2.00, (95% CI = 1.09-3.65, p = 0.024). In conclusion, among patients who were TPMT and NUDT15 normal metabolizers, a score combining the predicted expression of AOX1 and NME1 was associated with an increased risk for discontinuing azathioprine due to myelotoxicity., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Azathioprine is used frequently to treat several inflammatory conditions. However, treatment is limited by adverse events-in particular, myelotoxicity. Thiopurine-S-methyltransferase (TPMT) and nudix hydrolase-15 (NUDT15) are enzymes involved in azathioprine metabolism; variants in the genes encoding these enzymes increase the risk for azathioprine myelotoxicity. The Clinical Pharmacogenetics Implementation Consortium (CPIC) has recommended dose adjustments based on the results of TPMT and NUDT15 genotyping. However, little is known about the importance of this genetic information in routine clinical care. We hypothesized that in patients with inflammatory diseases, TPMT and NUDT15 genotype data predict the risk of discontinuing azathioprine due to myelotoxicity. This was a retrospective cohort study in 1,403 new adult azathioprine users for the management of inflammatory conditions for whom we had genetic information and clinical data. Among patients who discontinued azathioprine, we adjudicated the reason(s). Genotyping was performed using the Illumina Infinium Expanded Multi-Ethnic Genotyping Array plus custom content. We used CPIC guidelines to determine TPMT and NUDT15 metabolizer status; patients were grouped as either: (i) poor/intermediate, or (ii) normal/indeterminate metabolizers. We classified 110 patients as poor/intermediate, and 1,293 patients as normal/indeterminate metabolizers. Poor/intermediate status was associated with a higher risk for azathioprine discontinuation due to myelotoxicity compared to normal/indeterminate metabolizers (hazard ratio (HR) = 2.90, 95% confidence interval (CI): 1.58-5.31, P = 0.001). This association remained significant after adjustment for race, age at initiation, sex, primary indication, and initial daily dose of azathioprine (adjusted HR (aHR) = 2.67, 95% CI: 1.44-4.94, P = 0.002). In conclusion, TPMT and NUDT15 metabolizer status predicts discontinuation due to myelotoxicity for patients taking azathioprine for inflammatory conditions., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Cytochrome P450 2D6 (CYP2D6) O-demethylates codeine to the active drug, morphine. However, the utility of testing for CYP2D6 metabolizer status in patients receiving codeine in real-world clinical practice is poorly defined. Using data from a DNA bank linked to de-identified electronic health records, we studied 157 patients with a baseline pain score higher than 4 (0-10 scale) who received codeine. Based on CYP2D6 genotyping, 69 were classified as poor/intermediate and 88 as normal/ultrarapid CYP2D6 metabolizers. Pain response was defined as a score of 4 or lower while receiving codeine. In a propensity-score adjusted model, poor/intermediate metabolizers had lower odds (OR = 0.35, p = 0.02) of achieving a pain response than normal/ultrarapid metabolizers. To discriminate between codeine responders and nonresponders, a score including CYP2D6 phenotype and clinical variables was built. The response rate was 38.5% among patients in the high, 17.3% in the intermediate, and 9.4% in the low-score groups, respectively (p = 0.001)., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature.)

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: WJG and YW are listed as inventors on the patent “Kit and method for quantitative detection of steroids” US9851368B2. WJG, EY and YW are shareholders in CholesteniX Ltd., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ 5 -unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7β-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7β-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7β-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ability of zebrafish to heal their heart after injury makes them an attractive model for investigating the mechanisms governing the regenerative process. In this study, we show that the gene cellular communication network factor 2a ( ccn2a ), previously known as ctgfa , is induced in endocardial cells in the injured tissue and regulates CM proliferation and repopulation of the damaged tissue. We find that, whereas in wild-type animals, CMs track along the newly formed blood vessels that revascularize the injured tissue, in ccn2a mutants CM proliferation and repopulation are disrupted, despite apparently unaffected revascularization. In addition, we find that ccn2a overexpression enhances CM proliferation and improves the resolution of transient collagen deposition. Through loss- and gain-of-function as well as pharmacological approaches, we provide evidence that Ccn2a is necessary for and promotes heart regeneration by enhancing the expression of pro-regenerative extracellular matrix genes, and by inhibiting the chemokine receptor gene cxcr3.1 through a mechanism involving Tgfβ/pSmad3 signaling. Thus, Ccn2a positively modulates the innate regenerative response of the adult zebrafish heart., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Precision medicine aims to personalize treatment for both effectiveness and safety. As a critical component of this emerging initiative, pharmacogenomics seeks to guide drug treatment based on genetics. In this review article, we give an overview of pharmacogenomics in the setting of an immunosuppressant frequently prescribed by rheumatologists, azathioprine. Azathioprine has a narrow therapeutic index and a high risk of adverse events. By applying candidate gene analysis and unbiased approaches, researchers have identified multiple variants associated with an increased risk for adverse events associated with azathioprine, particularly bone marrow suppression. Variants in two genes, TPMT and NUDT15, are widely recognized, leading drug regulatory agencies and professional organizations to adopt recommendations for testing before initiation of azathioprine therapy. As more gene-drug interactions are discovered, our field will continue to face the challenge of balancing benefits and costs associated with genetic testing. However, novel approaches in genomics and the integration of clinical and genetic factors into risk scores offer unprecedented opportunities for the application of pharmacogenomics in routine practice. Key Points • Pharmacogenomics can help us understand how individuals' genetics may impact their response to medications. • Azathioprine is a success story for the clinical implementation of pharmacogenomics, particularly the effects of TPMT and NUDT15 variants on myelosuppression. • As our knowledge advances, testing and dosing recommendations will continue to evolve, with our field striving to balance costs and benefits to patients. • As we aim toward the goals of precision medicine, future research may integrate increasingly individualized traits-including clinical and genetic characteristics-to predict the safety and efficacy of particular medications for individual patients.

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection., Competing Interests: The authors have declared that no competing interests exist.

Leukopenia is a serious, frequent side effect associated with azathioprine use. Currently, we use thiopurine methyltransferase (TPMT) testing to predict leukopenia in patients taking azathioprine. We hypothesized that a risk score incorporating additional clinical and genetic variables would improve the prediction of azathioprine-associated leukopenia. In the discovery phase, we developed four risk score models: (1) age, sex, and TPMT metabolizer status; (2) model 1 plus additional clinical variables; (3) sixty candidate single nucleotide polymorphisms; and (4) model 2 plus model 3. The area under the receiver-operating-characteristic curve (AUC) of the risk scores was 0.59 (95% CI: 0.54-0.64), 0.75 (0.71-0.80), 0.66 (0.61-0.71), and 0.78 (0.74-0.82) for models 1, 2, 3, and 4, respectively. During the replication phase, models 2 and 4 (AUC = 0.64, 95% CI: 0.59-0.70 and AUC = 0.63, 95% CI: 0.58-0.69, respectively) were significant in an independent group. Compared with TPMT testing alone, additional genetic and clinical variables improve the prediction of azathioprine-associated leukopenia.

Background: Blood pressure visit-to-visit variability is a novel risk factor for deleterious long-term cardiac and renal outcomes in the general population. We hypothesized that patients with systemic lupus erythematosus (SLE) have greater blood pressure visit-to-visit variability than control subjects and that blood pressure visit-to-visit variability is associated with a higher comorbidity burden., Methods: We studied 899 patients with SLE and 4172 matched controls using de-identified electronic health records from an academic medical center. We compared blood pressure visit-to-visit variability measures in patients with SLE and control subjects and examined the association between blood pressure visit-to-visit variability and patients' characteristics., Results: Patients with SLE had higher systolic blood pressure visit-to-visit variability 9.7% (7.8-11.8%) than the control group 9.2% (7.4-11.2%), P  < 0.001 by coefficient of variation. Additional measures of systolic blood pressure visit-to-visit variability (i.e. standard deviation, average real variation, successive variation and maximum measure-to-measure change) were also significantly higher in patients with SLE than in control subjects. In patients with SLE, blood pressure visit-to-visit variability correlated significantly with age, creatinine, CRP, triglyceride concentrations and the Charlson comorbidity score (all P  < 0.05). Hydroxychloroquine use was associated with reduced blood pressure visit-to-visit variability ( P  < 0.001), whereas the use of antihypertensives, cyclophosphamide, mycophenolate mofetil and corticosteroids was associated with increased blood pressure visit-to-visit variability ( P  < 0.05)., Conclusion: Patients with SLE had higher blood pressure visit-to-visit variability than controls, and this increased blood pressure visit-to-visit variability was associated with greater Charlson comorbidity scores, several clinical characteristics and immunosuppressant medications. In particular, hydroxychloroquine prescription was associated with lower blood pressure visit-to-visit variability.

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tizanidine, a widely used muscle relaxant that can lower blood pressure, is metabolized by the cytochrome P450 1A2 (CYP1A2). We studied 1,626 patients prescribed tizanidine and 5,012 prescribed cyclobenzaprine concurrently with a strong CYP1A2 inhibitor. The primary outcome was severe hypotension, defined as systolic blood pressure (SBP) ≤ 70 mmHg during periods of drug co-exposure. Severe hypotension occurred more often in the tizanidine group (2.03%; n = 33) than the cyclobenzaprine group (1.28%; n = 64); odds ratio (OR) = 1.60; P = 0.029. This difference remained statistically significant after adjustment for a log-transformed propensity score that included age, sex, race, Charlson's comorbidity index, and concurrent use of antihypertensive medications (OR = 1.57; P = 0.049). A sensitivity analysis that defined hypotension as SBP < 90 mmHg also yielded higher rates of hypotension among patients prescribed tizanidine. In conclusion, CYP1A2 inhibition increases the risk of hypotensive episodes associated with the use of tizanidine in routine clinical practice., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

The spine is a segmented axial structure made of alternating vertebral bodies (centra) and intervertebral discs (IVDs) assembled around the notochord. Here, we show that, prior to centra formation, the outer epithelial cell layer of the zebrafish notochord, the sheath, segments into alternating domains corresponding to the prospective centra and IVD areas. This process occurs sequentially in an anteroposterior direction via the activation of Notch signaling in alternating segments of the sheath, which transition from cartilaginous to mineralizing domains. Subsequently, osteoblasts are recruited to the mineralized domains of the notochord sheath to form mature centra. Tissue-specific manipulation of Notch signaling in sheath cells produces notochord segmentation defects that are mirrored in the spine. Together, our findings demonstrate that notochord sheath segmentation provides a template for vertebral patterning in the zebrafish spine., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chromatin regulation is a principal mechanism governing animal development, yet it is unclear to what extent structural changes in chromatin underlie tissue regeneration. Non-mammalian vertebrates such as zebrafish activate cardiomyocyte (CM) division after tissue damage to regenerate lost heart muscle. Here, we generated transgenic zebrafish expressing a biotinylatable H3.3 histone variant in CMs and derived cell-type-specific profiles of histone replacement. We identified an emerging program of putative enhancers that revise H3.3 occupancy during regeneration, overlaid upon a genome-wide reduction of H3.3 from promoters. In transgenic reporter lines, H3.3-enriched elements directed gene expression in subpopulations of CMs. Other elements increased H3.3 enrichment and displayed enhancer activity in settings of injury- and/or Neuregulin1-elicited CM proliferation. Dozens of consensus sequence motifs containing predicted transcription factor binding sites were enriched in genomic regions with regeneration-responsive H3.3 occupancy. Thus, cell-type-specific regulatory programs of tissue regeneration can be revealed by genome-wide H3.3 profiling., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration. We performed a genome-wide profiling screen for secreted factors that are up-regulated during zebrafish spinal cord regeneration. We found that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupted spinal cord repair, and transgenic ctgfa overexpression or local delivery of human CTGF recombinant protein accelerated bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration., (Copyright © 2016, American Association for the Advancement of Science.)