Type I and II PRMTs inversely regulate post-transcriptional intron detention through Sm and CHTOP methylation.

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; Type II enzymes catalyze mono- and symmetric dimethylation. To understand the specific mechanisms of PRMT activity in splicing regulation, we inhibited Type I and II PRMTs and probed their transcriptomic consequences. Using the newly developed Splicing Kinetics and Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis of co-transcriptional splicing demonstrated that PRMT inhibition resulted in altered splicing rates. Surprisingly, co-transcriptional splicing kinetics did not correlate with final changes in splicing of polyadenylated RNA. This was particularly true for retained introns (RI). By using actinomycin D to inhibit ongoing transcription, we determined that PRMTs post-transcriptionally regulate RI. Subsequent proteomic analysis of both PRMT-inhibited chromatin and chromatin-associated polyadenylated RNA identified altered binding of many proteins, including the Type I substrate, CHTOP, and the Type II substrate, SmB. Targeted mutagenesis of all methylarginine sites in SmD3, SmB, and SmD1 recapitulated splicing changes seen with Type II PRMT inhibition, without disrupting snRNP assembly. Similarly, mutagenesis of all methylarginine sites in CHTOP recapitulated the splicing changes seen with Type I PRMT inhibition. Examination of subcellular fractions further revealed that RI were enriched in the nucleoplasm and chromatin. Taken together, these data demonstrate that, through Sm and CHTOP arginine methylation, PRMTs regulate the post-transcriptional processing of nuclear, detained introns.

Independent transcriptomic and proteomic regulation by type I and II protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) catalyze the post-translational monomethylation (Rme1), asymmetric (Rme2a), or symmetric (Rme2s) dimethylation of arginine. To determine the cellular consequences of type I (Rme2a) and II (Rme2s) PRMTs, we developed and integrated multiple approaches. First, we determined total cellular dimethylarginine levels, revealing that Rme2s was ∼3% of total Rme2 and that this percentage was dependent upon cell type and PRMT inhibition status. Second, we quantitatively characterized in vitro substrates of the major enzymes and expanded upon PRMT substrate recognition motifs. We also compiled our data with publicly available methylarginine-modified residues into a comprehensive database. Third, we inhibited type I and II PRMTs and performed proteomic and transcriptomic analyses to reveal their phenotypic consequences. These experiments revealed both overlapping and independent PRMT substrates and cellular functions. Overall, this study expands upon PRMT substrate diversity, the arginine methylome, and the complex interplay of type I and II PRMTs.

H1 histones control the epigenetic landscape by local chromatin compaction.

H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.

Association of Race and Ethnicity With Comorbidities and Survival Among Patients With COVID-19 at an Urban Medical Center in New York.

Importance: As of May 11, 2020, there have been more than 290 000 deaths worldwide from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). Risk-adjusted differences in outcomes among patients of differing ethnicity and race categories are not well characterized. Objectives: To investigate whether presenting comorbidities in patients with COVID-19 in New York City differed by race/ethnicity and whether case fatality rates varied among different ethnic and racial groups, controlling for presenting comorbidities and other risk factors. Design, Setting, and Participants: This cohort study included 5902 patients who presented for care to the Montefiore Medical Center, a large urban academic medical center in the Bronx, New York, between March 14 and April 15, 2020, and tested positive for SARS-CoV-2 on reverse transcription quantitative polymerase chain reaction assay. Final data collection was April 27, 2020. Exposures: Patient characteristics, including self-identified ethnicity/race, age, sex, socioeconomic status, and medical comorbidities, were tabulated. Main Outcomes and Measures: Overall survival. Associations between patient demographic characteristics, comorbidities, and race/ethnicity were examined using χ2 tests, and the association with survival was assessed using univariable and multivariable Cox proportional hazards regression, based on time from positive COVID-19 test. Results: Of 9268 patients who were tested, 5902 ethnically diverse patients (63.7%) had SARS-CoV-2. Of these, 3129 patients (53.0%) were women, and the median (interquartile range) age was 58 (44-71) years. A total of 918 patients (15.5%) died within the study time frame. Overall, 1905 patients (32.3%) identified as Hispanic; 1935 (32.8%), non-Hispanic Black; 509 (8.6%), non-Hispanic White; and 171 (2.9%), Asian; the death rates were 16.2% (309), 17.2% (333), 20.0% (102), and 17.0% (29), respectively (P = .25). Hispanic and non-Hispanic Black patients had a higher proportion of more than 2 medical comorbidities with 654 (34.3%) and 764 (39.5%), respectively, compared with 147 (28.9%) among non-Hispanic White patients (P < .001). Hispanic and non-Hispanic Black patients were also more likely to test positive for COVID-19 than White patients, with 1905 of 2919 Hispanic patients (65.3%), 1935 of 2823 non-Hispanic Black patients (68.5%), and 509 of 960 non-Hispanic White patients (53.0%) having positive test results for SARS-CoV-2 (P < .001). While controlling for age, sex, socioeconomic status and comorbidities, patients identifying as Hispanic (hazard ratio, 0.77; 95% CI, 0.61-0.98; P = .03) or non-Hispanic Black (hazard ratio, 0.69; 95% CI, 0.55-0.87; P = .002) had slightly improved survival compared with non-Hispanic White patients. Conclusions and Relevance: In this cohort study of patients with COVID-19 who presented for care at the same urban medical center, non-Hispanic Black and Hispanic patients did not experience worse risk-adjusted outcomes compared with their White counterparts. This finding is important for understanding the observed population differences in mortality by race/ethnicity reported elsewhere.

Extent of Prior Lung Irradiation and Mortality in COVID-19 Patients With a Cancer History.

PURPOSE: There have been nearly 200,000 deaths worldwide so far from coronavirus disease 2019 (COVID-19), which is caused by a coronavirus called SARS-CoV-2. Cancer history appears to be a poor prognostic factor for COVID-19 patients, although the reasons for this are unclear. In this report, we assess whether extent of prior lung irradiation is a risk factor for death as a result of COVID-19 infection. METHODS AND MATERIALS: Patients who tested positive for COVID-19 between March 14 and April 15, 2020, at our institution and who previously received radiation therapy for cancer in our department were included in this analysis. Patient characteristics and metrics describing the extent of lung irradiation were tabulated. Cox regression models were used to identify predictors of death after COVID-19 diagnosis. A logistic model was used to characterize the association between mean lung radiation therapy dose and 14-day mortality risk after COVID-19 diagnosis. RESULTS: For the study, 107 patients met the inclusion criteria. With a median follow-up of 7 days from COVID-19 diagnosis for surviving patients, 24 deaths have been observed. The actuarial survival rate 14 days after COVID-19 testing is 66%. Increasing mean lung dose (hazard ratio [HR] per Gy = 1.1, P = .002), lung cancer diagnosis (HR = 3.0, P = .034), and receiving radiation therapy between 1 month and 1 year before COVID-19 testing (HR = 3.4, P = .013) were associated with increased risk of death. Our survival model demonstrates a near linear relationship between mortality risk after COVID-19 diagnosis and mean lung radiation therapy dose. CONCLUSIONS: COVID-19 patients with a history of radiation therapy for cancer have a poor prognosis, and mortality risk appears to be associated with extent of lung irradiation. Validation of these findings will be critical as the COVID-19 pandemic continues.

Implementation of Electronic Decision Support for Diabetic Care in a Student-Run Clinic.

Background and objectives Type 2 diabetes mellitus (T2DM) is a complex disease that can lead to complications. Electronic decision support in the electronic medical record (EMR) aids management. There is no study demonstrating the effectiveness of electronic decision support in assisting medical student providers in student-run free clinics. Methods There were 71 T2DM patients seen by medical students. Twenty-three encounters used a Diabetes Progress Note (DPN) that was created from consensus, opinion-based guidelines. Each note received a total composite score based on an eight-point scale for adherence to guidelines. Statistical comparisons between mean composite scores were performed using independent t-tests. Results The mean total composite score of DPN users was significantly greater than DPN non-users (5.35 vs. 4.23, p = 0.008), with a significant difference in the physical exam component (1.70 vs. 1.31, p = 0.002). Conclusions In this exploratory study, medical student providers at an attending-supervised, student-run free clinic that used electronic decision support during T2DM patient visits improved adherence to screening for diabetic complications and standard of care.

Enhancing Medical Student-Interpreter Collaboration in an Urban Free Clinic.

BACKGROUND AND OBJECTIVES: Cultural barriers and patient-provider language discordance exert deleterious effects on patient care. One solution has been the integration of medical interpreters into the care of patients with limited English proficiency. While medical schools and residency programs have started developing training programs on how to work with medical interpreters, no similar endeavor has been reported by student-run free clinics. METHODS: Over 1 year, 76 third-year medical students (MS3s) were enrolled in control and intervention groups, and evaluated by in-person interpreters during interpreted real-patient encounters. MS3s in the intervention group received a lesson- and reminder-based training program on how to work with in-person interpreters. RESULTS: MS3s who received the intervention were more likely to ask the patient one question at a time (odds ratio [OR] 3.54, P=.0079), listen to the interpreter without unnecessary interruption (OR 3.30, P=.022), and speak in short, simple sentences with pauses for interpretation (OR 3.08, P=.017). CONCLUSIONS: Our lesson- and reminder-based training program on provider-interpreter collaboration can improve the performance of MS3s within a select skill set with minimal cost and time investment.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis.

The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050.

New strategies targeting Plasmodium falciparum gametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective against P. falciparum gametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations against P. falciparum sexual stages. In this study, we showed that maduramicin has an EC50 (effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocks in vivo transmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidly in vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na(+) concentration that is proposed to be due to inhibition of PfATP4, a putative Na(+) pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stage P. falciparum parasites and the potential of targeting this pathway for drug development.

Potent Plasmodium falciparum gametocytocidal activity of diaminonaphthoquinones, lead antimalarial chemotypes identified in an antimalarial compound screen.

Forty percent of the world's population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria.