Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing.

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif.

mRNA structure determines modification by pseudouridine synthase 1.

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. Messenger RNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets that were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the probable mechanism to regulate mRNA pseudouridylation.

Comparing Changes in Controlled Substance Prescribing Trends by Provider Type.

BACKGROUND AND OBJECTIVES: Forty-nine out of 50 states have implemented Prescription Drug Monitoring Programs (PDMPs) to monitor controlled substance (CS) prescribing. PDMPs change health care provider behavior, but few studies have examined changes in CS prescription by health care provider type. METHODS: Aggregated yearly data, including number of CS prescriptions, doses, and doses per prescription by health care provider type (physician, advanced practice registered nurse [APRN], and dentist) for each year from 2011 to 2017 was provided by the state PDMP, Kentucky All Schedule Prescription Electronic Reporting System (KASPER). In aggregate, this data set included 64,578,307 total prescriptions and 3,982,130,994 total doses of Schedule II-V medications. RESULTS: Physicians and dentists showed a trend of decreasing prescriptions and doses for Schedule II opioids from 2012 to 2017 (27-32% reduction in 2017 compared to 2011). APRNs showed a substantive increase in the number of doses and prescriptions (121-204% increase in 2017 compared to 2011), with increases remaining when controlling for number of providers. Physicians increased doses and prescriptions of Schedule II stimulants (37% increase for both doses and prescriptions), but by a smaller magnitude than APRN increases in stimulants (334-360% increase). Dentists showed decreases in Schedule II stimulants prescribed (69-80% reduction). Similar trends, but more modest in magnitude, were observed for Schedule III-IV. DISCUSSION AND CONCLUSIONS: Although monitoring and continuing education requirements are similar across all providers in Kentucky, differences in prescription trends for Schedule II opioids and stimulants were noted for physicians, APRNs, and dentists. SCIENTIFIC SIGNIFICANCE: Changes in prescribing following introduction of mandatory use of KASPER markedly differed based on provider type, with increases observed for APRNs compared with physicians and dentists. These findings advance prior research by providing a detailed examination of prescribing trends by provider type subsequent to a PDMPs mandatory use law. (Am J Addict 2019;00:00-00).

Developmental Dynamics of X-Chromosome Dosage Compensation by the DCC and H4K20me1 in C. elegans.

In Caenorhabditis elegans, the dosage compensation complex (DCC) specifically binds to and represses transcription from both X chromosomes in hermaphrodites. The DCC is composed of an X-specific condensin complex that interacts with several proteins. During embryogenesis, DCC starts localizing to the X chromosomes around the 40-cell stage, and is followed by X-enrichment of H4K20me1 between 100-cell to comma stage. Here, we analyzed dosage compensation of the X chromosome between sexes, and the roles of dpy-27 (condensin subunit), dpy-21 (non-condensin DCC member), set-1 (H4K20 monomethylase) and set-4 (H4K20 di-/tri-methylase) in X chromosome repression using mRNA-seq and ChIP-seq analyses across several developmental time points. We found that the DCC starts repressing the X chromosomes by the 40-cell stage, but X-linked transcript levels remain significantly higher in hermaphrodites compared to males through the comma stage of embryogenesis. Dpy-27 and dpy-21 are required for X chromosome repression throughout development, but particularly in early embryos dpy-27 and dpy-21 mutations produced distinct expression changes, suggesting a DCC independent role for dpy-21. We previously hypothesized that the DCC increases H4K20me1 by reducing set-4 activity on the X chromosomes. Accordingly, in the set-4 mutant, H4K20me1 increased more from the autosomes compared to the X, equalizing H4K20me1 level between X and autosomes. H4K20me1 increase on the autosomes led to a slight repression, resulting in a relative effect of X derepression. H4K20me1 depletion in the set-1 mutant showed greater X derepression compared to equalization of H4K20me1 levels between X and autosomes in the set-4 mutant, indicating that H4K20me1 level is important, but X to autosomal balance of H4K20me1 contributes slightly to X-repression. Thus H4K20me1 is not only a downstream effector of the DCC [corrected].In summary, X chromosome dosage compensation starts in early embryos as the DCC localizes to the X, and is strengthened in later embryogenesis by H4K20me1.

The lifetime medical cost savings from preventing HIV in the United States.

OBJECTIVE: Enhanced HIV prevention interventions, such as preexposure prophylaxis for high-risk individuals, require substantial investments. We sought to estimate the medical cost saved by averting 1 HIV infection in the United States. METHODS: We estimated lifetime medical costs in persons with and without HIV to determine the cost saved by preventing 1 HIV infection. We used a computer simulation model of HIV disease and treatment (CEPAC) to project CD4 cell count, antiretroviral treatment status, and mortality after HIV infection. Annual medical cost estimates for HIV-infected persons, adjusted for age, sex, race/ethnicity, and transmission risk group, were from the HIV Research Network (range, $1854-$4545/mo) and for HIV-uninfected persons were from the Medical Expenditure Panel Survey (range, $73-$628/mo). Results are reported as lifetime medical costs from the US health system perspective discounted at 3% (2012 USD). RESULTS: The estimated discounted lifetime cost for persons who become HIV infected at age 35 is $326,500 (60% for antiretroviral medications, 15% for other medications, 25% nondrug costs). For individuals who remain uninfected but at high risk for infection, the discounted lifetime cost estimate is $96,700. The medical cost saved by avoiding 1 HIV infection is $229,800. The cost saved would reach $338,400 if all HIV-infected individuals presented early and remained in care. Cost savings are higher taking into account secondary infections avoided and lower if HIV infections are temporarily delayed rather than permanently avoided. CONCLUSIONS: The economic value of HIV prevention in the United States is substantial given the high cost of HIV disease treatment.

The clinical and economic impact of point-of-care CD4 testing in mozambique and other resource-limited settings: a cost-effectiveness analysis.

BACKGROUND: Point-of-care CD4 tests at HIV diagnosis could improve linkage to care in resource-limited settings. Our objective is to evaluate the clinical and economic impact of point-of-care CD4 tests compared to laboratory-based tests in Mozambique. METHODS AND FINDINGS: We use a validated model of HIV testing, linkage, and treatment (CEPAC-International) to examine two strategies of immunological staging in Mozambique: (1) laboratory-based CD4 testing (LAB-CD4) and (2) point-of-care CD4 testing (POC-CD4). Model outcomes include 5-y survival, life expectancy, lifetime costs, and incremental cost-effectiveness ratios (ICERs). Input parameters include linkage to care (LAB-CD4, 34%; POC-CD4, 61%), probability of correctly detecting antiretroviral therapy (ART) eligibility (sensitivity: LAB-CD4, 100%; POC-CD4, 90%) or ART ineligibility (specificity: LAB-CD4, 100%; POC-CD4, 85%), and test cost (LAB-CD4, US$10; POC-CD4, US$24). In sensitivity analyses, we vary POC-CD4-specific parameters, as well as cohort and setting parameters to reflect a range of scenarios in sub-Saharan Africa. We consider ICERs less than three times the per capita gross domestic product in Mozambique (US$570) to be cost-effective, and ICERs less than one times the per capita gross domestic product in Mozambique to be very cost-effective. Projected 5-y survival in HIV-infected persons with LAB-CD4 is 60.9% (95% CI, 60.9%-61.0%), increasing to 65.0% (95% CI, 64.9%-65.1%) with POC-CD4. Discounted life expectancy and per person lifetime costs with LAB-CD4 are 9.6 y (95% CI, 9.6-9.6 y) and US$2,440 (95% CI, US$2,440-US$2,450) and increase with POC-CD4 to 10.3 y (95% CI, 10.3-10.3 y) and US$2,800 (95% CI, US$2,790-US$2,800); the ICER of POC-CD4 compared to LAB-CD4 is US$500/year of life saved (YLS) (95% CI, US$480-US$520/YLS). POC-CD4 improves clinical outcomes and remains near the very cost-effective threshold in sensitivity analyses, even if point-of-care CD4 tests have lower sensitivity/specificity and higher cost than published values. In other resource-limited settings with fewer opportunities to access care, POC-CD4 has a greater impact on clinical outcomes and remains cost-effective compared to LAB-CD4. Limitations of the analysis include the uncertainty around input parameters, which is examined in sensitivity analyses. The potential added benefits due to decreased transmission are excluded; their inclusion would likely further increase the value of POC-CD4 compared to LAB-CD4. CONCLUSIONS: POC-CD4 at the time of HIV diagnosis could improve survival and be cost-effective compared to LAB-CD4 in Mozambique, if it improves linkage to care. POC-CD4 could have the greatest impact on mortality in settings where resources for HIV testing and linkage are most limited. Please see later in the article for the Editors' Summary.

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

Intermittent theta burst stimulation and functional connectivity in people living with HIV/AIDS who smoke tobacco cigarettes: a preliminary pilot study.

Background: People living with HIV (PLWHA) smoke at three times the rate of the general population and respond poorly to cessation strategies. Previous studies examined repetitive transcranial magnetic stimulation (rTMS) over left dorsolateral prefrontal cortex (L. dlPFC) to reduce craving, but no studies have explored rTMS among PLWHA who smoke. The current pilot study compared the effects of active and sham intermittent theta-burst stimulation (iTBS) on resting state functional connectivity (rsFC), cigarette cue attentional bias, and cigarette craving in PLWHA who smoke. Methods: Eight PLWHA were recruited (single-blind, within-subject design) to receive one session of iTBS (n=8) over the L. dlPFC using neuronavigation and, four weeks later, sham iTBS (n=5). Cigarette craving and attentional bias assessments were completed before and after both iTBS and sham iTBS. rsFC was assessed before iTBS (baseline) and after iTBS and sham iTBS. Results: Compared to sham iTBS, iTBS enhanced rsFC between the L. dlPFC and bilateral medial prefrontal cortex and pons. iTBS also enhanced rsFC between the right insula and right occipital cortex compared to sham iTBS. iTBS also decreased cigarette craving and cigarette cue attentional bias. Conclusion: iTBS could potentially offer a therapeutic option for smoking cessation in PLWHA.

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

High Coronary Artery Calcium Score Is Associated With Increased Major Adverse Cardiac Events After Liver Transplantation.

Liver transplantation (LT) candidates frequently have multiple cardiovascular risk factors, and cardiovascular disease is a major cause of morbidity and mortality after LT. Coronary artery calcium (CAC) scores are a noninvasive assessment of coronary artery disease using computed tomography. This study examines CAC scores and cardiac risk factors and their association with outcomes after LT. Methods: Patients who underwent LT between January 2010 and June 2019 with a pretransplant CAC score were included in this study. Patients were divided by CAC score into 4 groups (CAC score 0, CAC score 1-100, CAC score 101-400, CAC score >400). Major adverse cardiovascular events (MACEs) were defined as myocardial infarction, stroke, revascularization, heart failure, atrial fibrillation, and cardiovascular death. Associations between CAC score and MACE or all-cause mortality within the 5-y post-LT follow-up period were analyzed using Cox regression. Statistical significance was defined as P < 0.05. Results: During the study period, 773 adult patients underwent their first LT, and 227 patients met our study criteria. The median follow-up time was 3.4 (interquartile range 1.9, 5.3) y. After 5 y, death occurred in 47 patients (20.7%) and MACE in 47 patients (20.7%). In multivariable analysis, there was no difference in death between CAC score groups. There was significantly higher risk of MACE in the CAC score >400 group, with a hazard ratio 2.58 (95% confidence interval 1.05, 6.29). Conclusions: CAC score was not associated with all-cause mortality. Patients with CAC score >400 had an increase in MACEs within the 5-y follow-up period compared with patients with a CAC score = 0. Further research with larger cohorts is needed to examine cardiac risk stratification in this vulnerable patient population.

Two Acute Liver Injuries in a Patient With Malnutrition.

Chronic starvation and refeeding have been associated with liver injury (LI). We present a patient with anorexia nervosa who exhibited both phenomena of malnutrition-related LI. At presentation, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were elevated at 154 and 136 U/L, respectively, and rose rapidly to peaks of 750 and 638 U/L, respectively, as nutrition was introduced. Mechanisms of starvation-related LI include impaired degradation and secretion of lipids, as well as starvation-induced autophagy. LI during refeeding may be related to rapid increase in glucose availability. These phenomena are crucial to consider in patients with chronic starvation undergoing refeeding.

Timing is everything: Early do-not-resuscitate orders in the intensive care unit and patient outcomes.

BACKGROUND: The use of Do-Not-Resuscitate (DNR) orders has increased but many are placed late in the dying process. This study is to determine the association between the timing of DNR order placement in the intensive care unit (ICU) and nurses' perceptions of patients' distress and quality of death. METHODS: 200 ICU patients and the nurses (n = 83) who took care of them during their last week of life were enrolled from the medical ICU and cardiac care unit of New York Presbyterian Hospital/Weill Cornell Medicine in Manhattan and the surgical ICU at the Brigham and Women's Hospital in Boston. Nurses were interviewed about their perceptions of the patients' quality of death using validated measures. Patients were divided into 3 groups-no DNR, early DNR, late DNR placement during the patient's final ICU stay. Logistic regression analyses modeled perceived patient quality of life as a function of timing of DNR order placement. Patient's comorbidities, length of ICU stay, and procedures were also included in the model. RESULTS: 59 patients (29.5%) had a DNR placed within 48 hours of ICU admission (early DNR), 110 (55%) placed after 48 hours of ICU admission (late DNR), and 31 (15.5%) had no DNR order placed. Compared to patients without DNR orders, those with an early but not late DNR order placement had significantly fewer non-beneficial procedures and lower odds of being rated by nurses as not being at peace (Adjusted Odds Ratio namely AOR = 0.30; [CI = 0.09-0.94]), and experiencing worst possible death (AOR = 0.31; [CI = 0.1-0.94]) before controlling for procedures; and consistent significance in severe suffering (AOR = 0.34; [CI = 0.12-0.96]), and experiencing a severe loss of dignity (AOR = 0.33; [CI = 0.12-0.94]), controlling for non-beneficial procedures. CONCLUSIONS: Placement of DNR orders within the first 48 hours of the terminal ICU admission was associated with fewer non-beneficial procedures and less perceived suffering and loss of dignity, lower odds of being not at peace and of having the worst possible death.

Clinical and Socioeconomic Predictors of Heart Failure Readmissions: A Review of Contemporary Literature.

Heart failure represents a clinical syndrome that results from a constellation of disease processes affecting myocardial function. Although recent studies have suggested a declining or stable incidence of heart failure, patients with heart failure continue to have high hospitalization and readmission rates, resulting in a substantial economic and public health burden. We searched PubMed and Google Scholar to identify published literature from 1998 through 2018 using the following keywords: heart failure, readmissions, predictors, prediction models, and interventions. Cited references were also used to identify relevant literature. Developments in the diagnosis and management of patients with heart failure have improved hospitalization and readmission rates in the past few decades. However, heart failure remains the most common cause of hospitalization in persons older than 65 years. As a result, given the enormous clinical and financial burden associated with heart failure readmissions on health care, there has been growing interest in the investigation of mechanisms aimed at improving outcomes and curtailing associated costs of care. Herein, we review the current literature on clinical and socioeconomic predictors of heart failure readmissions, briefly discussing limitations of existing strategies and providing an overview of current technology aimed at reducing hospitalizations.

A cross-sectional survey of coronary plaque composition in individuals on non-statin lipid lowering drug therapies and undergoing coronary computed tomography angiography.

INTRODUCTION: Non-statin therapy (NST) is used as second-line treatment when statin monotherapy is inadequate or poorly tolerated. OBJECTIVE: To determine the association of NST with plaque composition, alone or in combination with statins, in patients undergoing coronary computed tomography angiography (coronary CTA). METHODS: From the multicenter CONFIRM registry, we analyzed individuals who underwent coronary CTA with known lipid-lowering therapy status and without prior coronary artery disease at baseline. We created a propensity score for being on NST, followed by stepwise multivariate linear regression, adjusting for the propensity score as well as risk factors, to determine the association between NST and the number of coronary artery segments with each plaque type (non-calcified (NCP), partially calcified (PCP) or calcified (CP)) and segment stenosis score (SSS). RESULTS: Of the 27,125 subjects in CONFIRM, 4,945 met the inclusion criteria; 371 (7.5%) took NST. At baseline, patients on NST had more prevalent risk factors and were more likely to be on concomitant cardiac medications. After multivariate and propensity score adjustment, NST was not associated with plaque composition: NCP (0.07 increase, 95% CI: -0.05, 0.20; p = 0.26), PCP (0.10 increase, 95% CI: -0.10, 0.31; p = 0.33), CP (0.18 increase, 95% CI: -0.10, 0.46; p = 0.21) or SSS (0.45 increase, 95% CI: -0.02,0.93; p = 0.06). The absence of an effect of NST on plaque type was not modified by statin use (p for interaction > 0.05 for all). CONCLUSION: In this cross-sectional study, non-statin therapy was not associated with differences in plaque composition as assessed by coronary CTA.

Beyond Pain: Nurses' Assessment of Patient Suffering, Dignity, and Dying in the Intensive Care Unit.

CONTEXT: Deaths in the intensive care unit (ICU) are increasingly common in the U.S., yet little is known about patients' experiences at the end of life in the ICU. OBJECTIVES: The objective of this study was to determine nurse assessment of symptoms experienced, and care received by ICU patients in their final week, and their associations with nurse-perceived suffering and dignity. METHODS: From September 2015 to March 2017, nurses who cared for 200 ICU patients who died were interviewed about physical and psychosocial dimensions of patients' experiences. Medical chart abstraction was used to document baseline patient characteristics and care. RESULTS: The patient sample was 61% males, 70.2% whites, and on average 66.9 (SD 15.1) years old. Nurses reported that 40.9% of patients suffered severely and 33.1% experienced severe loss of dignity. The most common symptoms perceived to contribute to suffering and loss of dignity included trouble breathing (44.0%), edema (41.9%), and loss of control of limbs (36.1%). Most (n = 9) remained significantly (P < 0.05) associated with suffering, after adjusting for physical pain, including fever/chills, fatigue, and edema. Most patients received vasopressors and mechanical ventilation. Renal replacement therapy was significantly (<0.05) associated with severe suffering (adjusted odds ratio [AOR] 2.53) and loss of dignity (AOR 3.15). Use of feeding tube was associated with severe loss of dignity (AOR 3.12). CONCLUSION: Dying ICU patients are perceived by nurses to experience extreme indignities and suffer beyond physical pain. Attention to symptoms such as dyspnea and edema may improve the quality of death in the ICU.