Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

PPM1D modulates hematopoietic cell fitness and response to DNA damage and is a therapeutic target in myeloid malignancy.

PPM1D encodes a phosphatase that is recurrently activated across cancer, most notably in therapy-related myeloid neoplasms. However, the function of PPM1D in hematopoiesis and its contribution to tumor cell growth remain incompletely understood. Using conditional mouse models, we uncover a central role for Ppm1d in hematopoiesis and validate its potential as a therapeutic target. We find that Ppm1d regulates the competitive fitness and self-renewal of hematopoietic stem cells (HSCs) with and without exogenous genotoxic stresses. We also show that although Ppm1d activation confers cellular resistance to cytotoxic therapy, it does so to a lesser degree than p53 loss, informing the clonal competition phenotypes often observed in human studies. Notably, loss of Ppm1d sensitizes leukemias to cytotoxic therapies in vitro and in vivo, even in the absence of a Ppm1d mutation. Vulnerability to PPM1D inhibition is observed across many cancer types and dependent on p53 activity. Importantly, organism-wide loss of Ppm1d in adult mice is well tolerated, supporting the tolerability of pharmacologically targeting PPM1D. Our data link PPM1D gain-of-function mutations to the clonal expansion of HSCs, inform human genetic observations, and support the therapeutic targeting of PPM1D in cancer.

An improved index for diagnosis and mortality prediction in malignancy-associated hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening inflammatory syndrome that may complicate hematologic malignancies (HMs). The appropriateness of current criteria for diagnosing HLH in the context of HMs is unknown because they were developed for children with familial HLH (HLH-2004) or derived from adult patient cohorts in which HMs were underrepresented (HScore). Moreover, many features of these criteria may directly reflect the underlying HM rather than an abnormal inflammatory state. To improve and potentially simplify HLH diagnosis in patients with HMs, we studied an international cohort of 225 adult patients with various HMs both with and without HLH and for whom HLH-2004 criteria were available. Classification and regression tree and receiver-operating curve analyses were used to identify the most useful diagnostic and prognostic parameters and to optimize laboratory cutoff values. Combined elevation of soluble CD25 (>3900 U/mL) and ferritin (>1000 ng/mL) best identified HLH-2004-defining features (sensitivity, 84%; specificity, 81%). Moreover, this combination, which we term the optimized HLH inflammatory (OHI) index, was highly predictive of mortality (hazard ratio, 4.3; 95% confidence interval, 3.0-6.2) across diverse HMs. Furthermore, the OHI index identified a large group of patients with high mortality risk who were not defined as having HLH according to HLH-2004/HScore. Finally, the OHI index shows diagnostic and prognostic value when used for routine surveillance of patients with newly diagnosed HMs as well as those with clinically suspected HLH. Thus, we conclude that the OHI index identifies patients with HM and an inflammatory state associated with a high mortality risk and warrants further prospective validation.

Lenalidomide promotes the development of TP53-mutated therapy-related myeloid neoplasms.

There is a growing body of evidence that therapy-related myeloid neoplasms (t-MNs) with driver gene mutations arise in the background of clonal hematopoiesis (CH) under the positive selective pressure of chemo- and radiation therapies. Uncovering the exposure relationships that provide selective advantage to specific CH mutations is critical to understanding the pathogenesis and etiology of t-MNs. In a systematic analysis of 416 patients with t-MN and detailed prior exposure history, we found that TP53 mutations were significantly associated with prior treatment with thalidomide analogs, specifically lenalidomide. We demonstrated experimentally that lenalidomide treatment provides a selective advantage to Trp53-mutant hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, the effect of which was specific to Trp53-mutant HSPCs and was not observed in HSPCs with other CH mutations. Because of the differences in CK1α degradation, pomalidomide treatment did not provide an equivalent level of selective advantage to Trp53-mutant HSPCs, providing a biological rationale for its use in patients at high risk for t-MN. These findings highlight the role of lenalidomide treatment in promoting TP53-mutated t-MNs and offer a potential alternative strategy to mitigate the risk of t-MN development.

TET2-mutant clonal hematopoiesis and risk of gout.

Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1ß, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction [VAF] ≥2%: odds ratio [OR], 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio [HR], 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1ß secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1ß in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1ß levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.

Association of clonal hematopoiesis with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, -5.7%; adjusted 95% CI, -8.8% to -2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

Preventing Sexual Harm in Nightlife Settings: A Scoping Review.

Sexual harm within nightlife settings is a pervasive global concern; however, little is known about the nature of available interventions. The current study aims to review the literature on the nature and effectiveness of nightlife-related sexual harm interventions. A systematic literature search of six databases was conducted to identify records that were published between 1970 and June 2023 and reported approaches that aimed to reduce or prevent nightlife-related sexual harm. Records were included if they theorized, discussed, or evaluated an intervention, prevention or response strategy; however, individual safety strategies were excluded. Results were categorized according to intervention type. Thirty-five peer-reviewed journal articles and 16 gray literature records were identified. The most common nightlife-related sexual harm intervention strategies covered by the literature included targeted policies, laws, and regulations, bystander interventions, and awareness-raising campaigns. Literature in the area is increasing, with the majority of the articles (77.1%) being published in the previous six years; however, there are very few interventions that have been critically evaluated (22.9%). Promising areas for intervention include targeted alcohol regulations (e.g., lockout policies), venue-level policies, and environmental interventions. However, an increase in rigorous evaluative practices is urgently required to ensure future interventions are based on sound theoretical work and empirical evidence.

Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

Effect of Thromboprophylaxis on Clinical Outcomes After COVID-19 Hospitalization.

BACKGROUND: Patients hospitalized with COVID-19 have an increased incidence of thromboembolism. The role of extended thromboprophylaxis after hospital discharge is unclear. OBJECTIVE: To determine whether anticoagulation is superior to placebo in reducing death and thromboembolic complications among patients discharged after COVID-19 hospitalization. DESIGN: Prospective, randomized, double-blind, placebo-controlled clinical trial. (ClinicalTrials.gov: NCT04650087). SETTING: Done during 2021 to 2022 among 127 U.S. hospitals. PARTICIPANTS: Adults aged 18 years or older hospitalized with COVID-19 for 48 hours or more and ready for discharge, excluding those with a requirement for, or contraindication to, anticoagulation. INTERVENTION: 2.5 mg of apixaban versus placebo twice daily for 30 days. MEASUREMENTS: The primary efficacy end point was a 30-day composite of death, arterial thromboembolism, and venous thromboembolism. The primary safety end points were 30-day major bleeding and clinically relevant nonmajor bleeding. RESULTS: Enrollment was terminated early, after 1217 participants were randomly assigned, because of a lower than anticipated event rate and a declining rate of COVID-19 hospitalizations. Median age was 54 years, 50.4% were women, 26.5% were Black, and 16.7% were Hispanic; 30.7% had a World Health Organization severity score of 5 or greater, and 11.0% had an International Medical Prevention Registry on Venous Thromboembolism risk prediction score of greater than 4. Incidence of the primary end point was 2.13% (95% CI, 1.14 to 3.62) in the apixaban group and 2.31% (CI, 1.27 to 3.84) in the placebo group. Major bleeding occurred in 2 (0.4%) and 1 (0.2%) and clinically relevant nonmajor bleeding occurred in 3 (0.6%) and 6 (1.1%) apixaban-treated and placebo-treated participants, respectively. By day 30, thirty-six (3.0%) participants were lost to follow-up, and 8.5% of apixaban and 11.9% of placebo participants permanently discontinued the study drug treatment. LIMITATIONS: The introduction of SARS-CoV-2 vaccines decreased the risk for hospitalization and death. Study enrollment spanned the peaks of the Delta and Omicron variants in the United States, which influenced illness severity. CONCLUSION: The incidence of death or thromboembolism was low in this cohort of patients discharged after hospitalization with COVID-19. Because of early enrollment termination, the results were imprecise and the study was inconclusive. PRIMARY FUNDING SOURCE: National Institutes of Health.

Specialist-led urgent cholecystectomy for acute gallstone disease.

BACKGROUND: Despite overwhelming evidence of the clinical and financial benefit of urgent cholecystectomy, there is variable enthusiasm and uptake across the UK. In 2014, following the First National Emergency Laparotomy Audit Organisational Report, we implemented a specialist-led urgent surgery service, whereby all patients with gallstone-related pathologies were admitted under the direct care of specialist upper gastrointestinal surgeons. We have analysed 5 years of data to investigate the results of this service model. METHODS: Computerised operating theatre records were interrogated to identify all patients within a 5-year period undergoing cholecystectomy. Patient demographics, admission details, length of stay, duration of surgery, and complications were analysed. RESULTS: Between 01/01/2016 and 31/12/2020, a total of 4870 cholecystectomies were performed; 1793 (36.8%) were urgent cases and 3077 (63.2%) were elective cases. All cases were started laparoscopically; 25 (0.5%) were converted to open surgery-14 of 1793 (0.78%) urgent cases and 11 of 3077 (0.36%) elective cases. Urgent cholecystectomy took 20 min longer than elective surgery (median 74 versus 52 min). No relevant difference in conversion rate was observed when urgent cholecystectomy was performed within 2 days, between 2 and 4 days, or greater than 4 days from admission (P = 0.197). Median total hospital stay was 4 days. CONCLUSION: Urgent laparoscopic cholecystectomy is safe and feasible in most patients with acute gall bladder disease. Surgery under the direct care of upper gastrointestinal specialist surgeons is associated with a low conversion rate, low complication rate, and short hospital stay. Timing of surgery has no effect on conversion rate or complication rate.

Facility-Based Maternal Quality of Care Frameworks: A Systematic Review and Best Fit Framework Analysis.

OBJECTIVES: The World Health Organization has adopted two main strategies to improve the quality of maternal health: increasing the number of deliveries by skilled birth attendants and increasing access to emergency obstetric care. Despite increased access to care, there continue to be high rates of maternal morbidity and mortality in part due to quality of care. This study aims to identify and summarize existing frameworks for measuring quality of maternal care at a facility-level. METHODS: PubMed, Health Systems Evidence, Embase, Global Health, OVID Healthstar, OVID Medline, PsycINFO, and Web of Science were searched for frameworks, tools, theories, or components of frameworks relevant to maternal quality of care in facility-level settings. Title/abstract and full-text screening were completed by two independent reviewers and conflicts resolved through consensus or a third reviewer. RESULTS: An initial search resulted in 3182 studies. Fifty-four studies were included in the qualitative analysis. A best fit framework analysis was done using the updated Hulton framework as the conceptual framework. A facility-based maternal quality of care framework is proposed including the following components, separated into provision and experience of care: (1) human resources; (2) infrastructure; (3) equipment, supplies and medicine; (4) evidence and information; (5) referral and networks of care; (6) cultural competence; (7) clinical practice; (8) financing; (9) leadership and governance; (10) cognition; and 11) respect, dignity, equity, and emotional support.

Despite increased institutional access to care, there continue to be high rates of maternal morbidity and mortality in some low- and middle-income countries. This is in part due to the quality of maternal care once care has been accessed. Several frameworks have been proposed for understanding the complexities surrounding quality of care in maternal health but there has not been a single framework consistently used in the literature for facility-based quality of maternal care. This paper identifies and summarizes existing frameworks and tools, and uses a best fit framework analysis to propose a comprehensive framework for assessing facility-based maternal quality of care.

Using the Cardiff model to reduce late-night alcohol-related presentations in regional Australia.

INTRODUCTION: The Cardiff model is a data sharing approach that aims to reduce the volume of intoxicated patients in emergency departments (EDs). This approach has not been tested in a rural setting. OBJECTIVE: This study assessed whether this approach would reduce the number of alcohol-associated presentations during high-alcohol hours (HAH) in a regional ED. DESIGN: From July 2017, people over the age of 18 attending the ED were asked by the triage nurse (1) whether they had consumed alcohol in the past 12 h, (2) their typical alcohol consumption level, (3) the location where most alcohol was purchased and (4) the location of the last drink. From April 2018, quarterly letters were sent to the top five venues reported within the ED. Deidentified, aggregated data were shared with local police, licensing authorities and local government, identifying the top five venues reported in the ED and providing a summary of alcohol-related attendances to the ED. Interrupted time series analyses were used to estimate the influence of the intervention on monthly injury and alcohol-related ED presentations. FINDINGS: ITS models found that there was a significant gradual decrease in the monthly rate of injury attendances during HAH (Coefficient = -0.004, p = 0.044). No other significant results were found. DISCUSSION: Our study found that sharing last drinks data collected in the ED with a local violence prevention committee was associated with a small, but significant reduction in the rate of injury presentations compared with all ED presentations. CONCLUSION: This intervention continues to have promise for reducing alcohol-related harm.

Sarcopenia and Myosteatosis Predict Adverse Outcomes After Emergency Laparotomy: A Multi-center Observational Cohort Study.

OBJECTIVE: To determine the relationship between BC, specifically low skeletal muscle mass (sarcopenia) and poor muscle quality (myosteatosis) and outcomes in emergency laparotomy patients. BACKGROUND: Emergency laparotomy has one of the highest morbidity and mortality rates of all surgical interventions. BC objectively identifies patients at risk of adverse outcomes in elective cancer cohorts, however, evidence is lacking in emergency surgery. METHODS: An observational cohort study of patients undergoing emergency laparotomy at ten English hospitals was performed. BC analyses were performed at the third lumbar vertebrae level using preoperative computed tomography images to quantify skeletal muscle index (SMI) and skeletal muscle radiation attenuation (SM-RA). Sex-specific SMI and SM-RA were determined, with the lower tertile splits defining sarcopenia (low SMI) and myosteatosis (low SM-RA). Accuracy of mortality risk prediction, incorporating SMI and SM-RA variables into risk models was assessed with regression modeling. RESULTS: Six hundred ten patients were included. Sarcopenia and myosteatosis were both associated with increased risk of morbidity (52.1% vs 45.1%, P = 0.028; 57.5% vs 42.6%, P = 0.014), 30-day (9.5% vs 3.6%, P = 0.010; 14.9% vs 3.4%, P < 0.001), and 1-year mortality (27.4% vs 11.5%, P < 0.001; 29.7% vs 12.5%, P < 0.001). Risk-adjusted 30-day mortality was significantly increased by sarcopenia [OR 2.56 (95% CI 1.12-5.84), P = 0.026] and myosteatosis [OR 4.26 (2.01-9.06), P < 0.001], similarly at 1-year [OR 2.66 (95% CI 1.57-4.52), P < 0.001; OR2.08 (95%CI 1.26-3.41), P = 0.004]. BC data increased discrimination of an existing mortality risk-prediction model (AUC 0.838, 95% CI 0.835-0.84). CONCLUSION: Sarcopenia and myosteatosis are associated with increased adverse outcomes in emergency laparotomy patients.

Contribution of clonal hematopoiesis to adult-onset hemophagocytic lymphohistiocytosis.

Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease of immune hyperactivation. Unlike pediatric HLH, adult HLH is rarely driven by germline genetic variants. Although numerous precipitating etiologies have been identified, the reason that HLH occurs in only a subset of individuals and how other factors contribute to the disease remains unknown. We hypothesized that clonal hematopoiesis (CH), a state in which somatic mutations in blood cells cause an expanded population of mutant hematopoietic cells and drive an aberrant inflammatory state, could contribute to adult-onset HLH. In a highly annotated cohort of older adults with HLH we found that CH was more prevalent than in control cohorts. Using the adult-onset HLH mouse model in which repeated treatments of the TLR9 agonist, ODN1826, was delivered to the mouse, we observed that macrophages carrying mutations in Tet2, one of the most commonly mutated genes in CH, have an enhanced inflammatory response to TLR9 agonism. Finally, mice carrying Tet2 mutations in the hematopoietic compartment (a common model for CH) displayed an exaggerated response to TLR9 agonism, including worse splenomegaly and anemia. Our data suggest that CH is more common in individuals with adult-onset HLH and can contribute to the pathophysiology of this disease.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Voting in a Pandemic: COVID-19 and Primary Turnout in Milwaukee, Wisconsin.

We report the first study of the effect of the novel coronavirus SARS-CoV-2 (COVID-19) on polling place consolidation and voting behavior. We draw upon individual-level observations from Milwaukee matched to similar observations in the surrounding municipalities to assess whether fewer polling places in the April 2020 presidential primary election decreased turnout in the city. We find polling place consolidation reduced overall turnout by about 8.7 points and reduced turnout among the Black population in the city by about 10 points. We conclude, based on these data, that polling place consolidation even accompanied by widespread absentee voting in the face of an emergency may result in disenfranchisement, particularly among Black voters.

An Examination of Community Awareness and Understanding of Patron Banning Provisions in Western Australia: Implications for Policy Development and Success.

Individuals who engage in problematic behaviours within Australian night-time entertainment precincts can be banned from entering certain locations. Bans are expected to deter recipients and the wider community from further inappropriate behaviours. The collective effect is intended to reduce crime and increase safety within entertainment precincts. This study examined public awareness and understanding of two patron banning mechanisms (police barring notices and prohibition orders) used across Western Australia (WA). An anonymous survey was completed by 1018 respondents, and interviews were conducted with 54 stakeholders. Survey participants had limited awareness of patron banning: 75% had not heard of police barring notices; 87% had not heard of prohibition orders. Knowledge was higher for individuals directly associated with a ban recipient. Stakeholders also perceived a low level of community awareness and understanding of patron banning. Patron banning may have some merit as a specific deterrent for recipients but, in WA, the lack of public knowledge means that the banning provisions may currently have limited effect as a general deterrent. Public awareness should be increased in order to optimise the direct and consequential effects of patron banning policy.

Spontaneous Selection of Cryptosporidium Drug Resistance in a Calf Model of Infection.

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.