Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Multi-task transfer learning for the prediction of entity modifiers in clinical text: application to opioid use disorder case detection.

BACKGROUND: The semantics of entities extracted from a clinical text can be dramatically altered by modifiers, including entity negation, uncertainty, conditionality, severity, and subject. Existing models for determining modifiers of clinical entities involve regular expression or features weights that are trained independently for each modifier. METHODS: We develop and evaluate a multi-task transformer architecture design where modifiers are learned and predicted jointly using the publicly available SemEval 2015 Task 14 corpus and a new Opioid Use Disorder (OUD) data set that contains modifiers shared with SemEval as well as novel modifiers specific for OUD. We evaluate the effectiveness of our multi-task learning approach versus previously published systems and assess the feasibility of transfer learning for clinical entity modifiers when only a portion of clinical modifiers are shared. RESULTS: Our approach achieved state-of-the-art results on the ShARe corpus from SemEval 2015 Task 14, showing an increase of 1.1% on weighted accuracy, 1.7% on unweighted accuracy, and 10% on micro F1 scores. CONCLUSIONS: We show that learned weights from our shared model can be effectively transferred to a new partially matched data set, validating the use of transfer learning for clinical text modifiers.

Community-Based Services for Hospitalized Patients With Serious Injection-Related Infections in Alabama: A Brief Report.

Injection-related infections continue to rise, particularly in the South. People who inject drugs are increasingly utilizing hospital services for serious injection-related infections but may be discharged to areas without harm reduction services. We explored the availability and travel time to services for HIV and substance use in Alabama.

National HIV and HCV Screening Rates for Hospitalized People who Use Drugs Are Suboptimal and Heterogeneous Across 11 US Hospitals.

Background: To end the HIV and hepatitis C virus (HCV) epidemics, people who use drugs (PWUD) need more opportunities for testing. While inpatient hospitalizations are an essential opportunity to test people who use drugs (PWUD) for HIV and HCV, there is limited research on rates of inpatient testing for HIV and HCV among PWUD. Methods: Eleven hospital sites were included in the study. Each site created a cohort of inpatient encounters associated with injection drug use. From these cohorts, we collected data on HCV and HIV testing rates and HIV testing consent policies from 65 276 PWUD hospitalizations. Results: Hospitals had average screening rates of 40% for HIV and 32% for HCV, with widespread heterogeneity in screening rates across facilities. State consent laws and opt-out testing policies were not associated with statistically significant differences in HIV screening rates. On average, hospitals that reflexed HCV viral load testing on HCV antibody testing did not have statistically significant differences in HCV viral load testing rates. We found suboptimal testing rates during inpatient encounters for PWUD. As treatment (HIV) and cure (HCV) are necessary to end these epidemics, we need to prioritize understanding and overcoming barriers to testing.

Hospitalization is a missed opportunity for HIV screening, pre-exposure prophylaxis, and treatment.

BACKGROUND: Hospitalization is a "reachable moment" for people who inject drugs (PWID), but preventive care including HIV testing, prevention and treatment is rarely offered within inpatient settings. METHODS: We conducted a multisite, retrospective cohort study of patients with opioid use disorder with infectious complications of injection drug use hospitalized between 1/1/2018-12/31/2018. We evaluated HIV care continuum outcomes using descriptive statistics and hypothesis tests for intergroup differences. RESULTS: 322 patients were included. Of 300 patients without known HIV, only 2 had a documented discussion of PrEP, while only 1 was prescribed PrEP on discharge. Among the 22 people with HIV (PWH), only 13 (59%) had a viral load collected during admission of whom all were viremic and 10 (45%) were successfully linked to care post-discharge. Rates of readmission, Medicaid or uninsured status, and unstable housing were high in both groups. DISCUSSION: We observed poor provision of HIV testing, PrEP and other HIV services for hospitalized PWID across multiple U.S. medical centers. Future initiatives should focus on providing this group with comprehensive HIV testing and treatment services through a status neutral approach.

Xylazine co-occurrence with illicit fentanyl is a growing threat in the Deep South: a retrospective study of decedent data.

BACKGROUND: Xylazine is a dangerous veterinary sedative found mainly in illicit fentanyl in the Northeast and Midwest. Its role in the Deep South overdose crisis is not well-characterized. METHODS: We conducted a retrospective review of autopsy data in Jefferson County, Alabama to identify trends in xylazine prevalence among people who fatally overdosed from June 2019 through June 2023. RESULTS: 165 decedents met inclusion criteria. While the first identified xylazine-associated overdose was in June 2019, xylazine has become consistently prevalent since January 2021. All cases of xylazine-associated fatal overdoses were accompanied by fentanyl, and most (75.4%) involved poly-drug stimulant use. The average age was 42.2, and most decedents were white (58.8%) and male (68.5%). Overall, 18.2% of people were unhoused at the time of death. DISCUSSION: Xylazine is prevalent in the Deep South. Efforts to promote harm reduction, publicly viewable drug supply trends, and legalization of drug checking and syringe service programs should be prioritized.

Diagnostic accuracy of point-of-care ultrasound with artificial intelligence-assisted assessment of left ventricular ejection fraction.

Focused cardiac ultrasound (FoCUS) is becoming standard practice in a wide spectrum of clinical settings. There is limited data evaluating the real-world use of FoCUS with artificial intelligence (AI). Our objective was to determine the accuracy of FoCUS AI-assisted left ventricular ejection fraction (LVEF) assessment and compare its accuracy between novice and experienced users. In this prospective, multicentre study, participants requiring a transthoracic echocardiogram (TTE) were recruited to have a FoCUS done by a novice or experienced user. The AI-assisted device calculated LVEF at the bedside, which was subsequently compared to TTE. 449 participants were enrolled with 424 studies included in the final analysis. The overall intraclass coefficient was 0.904, and 0.921 in the novice (n = 208) and 0.845 in the experienced (n = 216) cohorts. There was a significant bias of 0.73% towards TTE (p = 0.005) with a level of agreement of 11.2%. Categorical grading of LVEF severity had excellent agreement to TTE (weighted kappa = 0.83). The area under the curve (AUC) was 0.98 for identifying an abnormal LVEF (<50%) with a sensitivity of 92.8%, specificity of 92.3%, negative predictive value (NPV) of 0.97 and a positive predictive value (PPV) of 0.83. In identifying severe dysfunction (<30%) the AUC was 0.99 with a sensitivity of 78.1%, specificity of 98.0%, NPV of 0.98 and PPV of 0.76. Here we report that FoCUS AI-assisted LVEF assessments provide highly reproducible LVEF estimations in comparison to formal TTE. This finding was consistent among senior and novice echocardiographers suggesting applicability in a variety of clinical settings.

Blood culture identification (BCID) performance in polymicrobial bacteremia.

The rapid multiplex PCR (rmPCR)-based FilmArray® blood culture identification (BCID) assay reduces time from positive blood culture to organism identification. Polymicrobial bacteremia (PMB) is a known area of reduced diagnostic fidelity for BCID and remains incompletely characterized. All cases of clinically confirmed PMB at a large academic single center from a 23-month period were evaluated in a retrospective cohort analysis. A total of 207 samples were identified and studied. Overall, 49.3% (N = 102) of polymicrobial cultures were incompletely identified by FilmArray® result. There were no significant between-group differences in comorbidity status, length of stay, mortality, or source between patients with PMB who had complete versus incomplete BCID identification. Our results suggest that rmPCR-based assays frequently miss organisms in PMB and should be interpreted accordingly.

Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

Desmosomes: emerging pathways and non-canonical functions in cardiac arrhythmias and disease.

Desmosomes are critical adhesion structures in cardiomyocytes, with mutation/loss linked to the heritable cardiac disease, arrhythmogenic right ventricular cardiomyopathy (ARVC). Early studies revealed the ability of desmosomal protein loss to trigger ARVC disease features including structural remodeling, arrhythmias, and inflammation; however, the precise mechanisms contributing to diverse disease presentations are not fully understood. Recent mechanistic studies demonstrated the protein degradation component CSN6 is a resident cardiac desmosomal protein which selectively restricts cardiomyocyte desmosomal degradation and disease. This suggests defects in protein degradation can trigger the structural remodeling underlying ARVC. Additionally, a subset of ARVC-related mutations show enhanced vulnerability to calpain-mediated degradation, further supporting the relevance of these mechanisms in disease. Desmosomal gene mutations/loss has been shown to impact arrhythmogenic pathways in the absence of structural disease within ARVC patients and model systems. Studies have shown the involvement of connexins, calcium handling machinery, and sodium channels as early drivers of arrhythmias, suggesting these may be distinct pathways regulating electrical function from the desmosome. Emerging evidence has suggested inflammation may be an early mechanism in disease pathogenesis, as clinical reports have shown an overlap between myocarditis and ARVC. Recent studies focus on the association between desmosomal mutations/loss and inflammatory processes including autoantibodies and signaling pathways as a way to understand the involvement of inflammation in ARVC pathogenesis. A specific focus will be to dissect ongoing fields of investigation to highlight diverse pathogenic pathways associated with desmosomal mutations/loss.

Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

Prenatal exposure to bisphenols affects pregnancy outcomes and offspring development in rats.

The objective of this study was to evaluate the effects of gestational exposure to low doses of bisphenol A (BPA), bisphenol S (BPS), and bisphenol F (BPF) on pregnancy outcomes and offspring development. Pregnant Sprague-Dawley rats were orally dosed with vehicle, 5 µg/kg body weight (BW)/day of BPA, BPS and BPF, or 1 µg/kg BW/day of BPF on gestational days 6-21. Pregnancy and gestational outcomes, including number of abortions and stillbirths, were monitored. Male and female offspring were subjected to morphometry at birth, followed by pre- and post-weaning body weights, post-weaning food and water intakes, and adult organ weights. Ovarian follicular counts were also obtained from adult female offspring. We observed spontaneous abortions in over 80% of dams exposed to 5 µg/kg of BPF. BPA exposure increased Graafian follicles in female offspring, while BPS and BPF exposure decreased the number of corpora lutea, suggesting reduced ovulation rates. Moreover, BPA exposure increased male kidney and prostate gland weights, BPF decreased epididymal adipose tissue weights, and BPS had modest effects on male abdominal adipose tissue weights. Prenatal BPS exposure reduced anogenital distance (AGD) in male offspring, suggesting possible feminization, whereas both BPS and BPA induced oxidative stress in the testes. These results indicate that prenatal exposure to BPF affects pregnancy outcomes, BPS alters male AGD, and all three bisphenols alter certain organ weights in male offspring and ovarian function in female offspring. Altogether, it appears that prenatal exposure to BPA or its analogues can induce reproductive toxicity even at low doses.

DNA replication, transcription, and H3K56 acetylation regulate copy number and stability at tandem repeats.

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

How to Start an Enhanced Recovery After Surgery Cardiac Program.

In this review the authors introduce a practical approach to guide the initiation of an enhanced recovery after surgery (ERAS) cardiac surgery program. The first step in implementation is organizing a dedicated multidisciplinary ERAS cardiac team composed of representatives from nursing, surgery, anesthesiology, and other relevant allied health groups. Identifying a program coordinator or navigator who will have responsibilities for developing and implementing educational initiatives, troubleshooting, monitoring progress and setbacks, and data collection is also vital for success. An institution-specific protocol is then developed by leveraging national guidelines and local expertise.

Translation of small downstream ORFs enhances translation of canonical main open reading frames.

In addition to canonical open reading frames (ORFs), thousands of translated small ORFs (containing less than 100 codons) have been identified in untranslated mRNA regions (UTRs) across eukaryotes. Small ORFs in 5' UTRs (upstream (u)ORFs) often repress translation of the canonical ORF within the same mRNA. However, the function of translated small ORFs in the 3' UTRs (downstream (d)ORFs) is unknown. Contrary to uORFs, we find that translation of dORFs enhances translation of their corresponding canonical ORFs. This translation stimulatory effect of dORFs depends on the number of dORFs, but not the length or peptide they encode. We propose that dORFs represent a new, strong, and universal translation regulatory mechanism in vertebrates.

Role of the Internet in Solving the Last Mile Problem in Medicine.

Internet-augmented medicine has a strong role to play in ensuring that all populations benefit equally from discoveries in the medical sciences. Yet, data from the Centers for Disease Control and Prevention collected from 1999 to 2014 suggested that during the first phase of internet diffusion, progress against mortality has stalled, and in some cases, receded in rural areas that are traditionally underserved by medical and broadband resources. This problem of failing to extend the benefits of extant medical knowledge equitably to all populations regardless of geography can be framed as the "last mile problem in health care." In theory, the internet should help solve the last mile problem by making the best knowledge in the world available to everyone worldwide at a low cost and no delay. In practice, the antiquated supply chains of industrial age medicine have been slow to yield to the accelerative forces of evolving internet capacity. This failure is exacerbated by the expanding digital divide, preventing residents of isolated, geographically distant communities from taking full advantage of the digital health revolution. The result, according to the Federal Communications Commission's (FCC's) Connect2Health Task Force, is the unanticipated emergence of "double burden counties," ie, counties for which the mortality burden is high while broadband access is low. The good news is that a convergence of trends in internet-enabled health care is putting medicine within striking distance of solving the last mile problem both in the United States and globally. Specific trends to monitor over the next 25 years include (1) using community-driven approaches to bridge the digital divide, (2) addressing structural disconnects in care through P4 Medicine, (3) meeting patients at "point-of-need," (4) ensuring that no one is left behind through population management, and (5) self-correcting cybernetically through the learning health care system.