Community-Based Medications First for Opioid Use Disorder - Care Utilization and Mortality Outcomes.

Purpose: A large treatment gap exists for people who could benefit from medications for opioid use disorder (MOUD). People OUD accessing services in harm reduction and community-based organizations often have difficulty engaging in MOUD at opioid treatment programs and traditional health care settings. We conducted a study to test the impacts of a community-based medications first model of care in six Washington (WA) State communities that provided drop-in MOUD access. Participants and Methods: Participants included people newly prescribed MOUD. Settings included harm reduction and homeless services programs. A prospective cohort analysis tested the impacts of the intervention on MOUD and care utilization. Intervention impacts on mortality were tested via a synthetic comparison group analysis matching on demographics, MOUD history, and geography using WA State agency administrative data. Results: 825 people were enrolled in the study of whom 813 were matched to state records for care utilization and outcomes. Cohort analyses indicated significant increases for days' supply of buprenorphine, months with any MOUD, and months with any buprenorphine for people previously on buprenorphine (all results p<0.05). Months with an emergency department overdose did not change. Months with an inpatient hospital stay increased (p<0.05). The annual death rate in the first year for the intervention group was 0.45% (3 out of 664) versus 2.2% (222 out of 9893) in the comparison group in the 12 months; a relative risk of 0.323 (95% CI 0.11-0.94). Conclusion: Findings indicated a significant increase in MOUD for the intervention group and a lower mortality rate relative to the comparison group. The COVID-19 epidemic and rapid increase in non-pharmaceutical-fentanyl may have lessened the intervention impact as measured in the cohort analysis. Study findings support expanding access to a third model of low barrier MOUD care alongside opioid treatment programs and traditional health care settings.

RluA is the major mRNA pseudouridine synthase in Escherichia coli.

Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life. Conserved pseudouridine synthases modify the mRNAs of diverse eukaryotes, but the modification has yet to be identified in bacterial mRNAs. Here, we report the discovery of pseudouridines in mRNA from E. coli. By testing the mRNA modification capacity of all 11 known pseudouridine synthases, we identify RluA as the predominant mRNA-modifying enzyme. RluA, a known tRNA and 23S rRNA pseudouridine synthase, modifies at least 31 of the 44 high-confidence sites we identified in E. coli mRNAs. Using RNA structure probing data to inform secondary structures, we show that the target sites of RluA occur in a common sequence and structural motif comprised of a ΨURAA sequence located in the loop of a short hairpin. This recognition element is shared with previously identified target sites of RluA in tRNAs and rRNA. Overall, our work identifies pseudouridine in key mRNAs and suggests the capacity of Ψ to regulate the transcripts that contain it.

Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs.

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs. Highlights: Measurement of >30,000 human 5' UTRs reveals a 250-fold range of translation outputSystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elementsN1-methylpseudouridine alters translation initiation in a sequence-specific mannerOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines.

Recent developments, opportunities, and challenges in the study of mRNA pseudouridylation.

Pseudouridine is an abundant mRNA modification found in diverse organisms ranging from bacteria and viruses to multicellular plants and humans. New developments in pseudouridine profiling provide quantitative tools to map mRNA pseudouridylation sites. Sparse biochemical studies establish the potential for mRNA pseudouridylation to affect most stages of the mRNA life cycle from birth to death. This recent progress sets the stage for deeper investigations into the molecular and cellular functions of specific mRNA pseudouridines, including in disease.

Beyond reader proteins: RNA binding proteins and RNA modifications in conversation to regulate gene expression.

Post-transcriptional mRNA modifications play diverse roles in gene expression and RNA function. In many cases, RNA modifications function by altering how cellular machinery such as RNA binding proteins (RBPs) interact with RNA substrates. For instance, N6-methyladenosine (m6A) is recognized by the well-characterized YTH domain-containing family of "reader" proteins. For other mRNA modifications, similar global readers of modification status have not been clearly defined. Rather, most interactions between RBPs and RNA modifications have a more complicated dependence on sequence context and binding modality. The current handful of studies that demonstrate modifications impacting protein binding likely represent only a fraction of the full landscape. In this review, we dissect the known instances of RNA modifications altering RBP binding, specifically m6A, N1-methyladenosine (m1A), 5-methylcytosine (m5C), pseudouridine (Ψ), and internal N7-methylguanosine. We then review the biochemical properties of these and other identified mRNA modifications including dihydrouridine (D), N4-acetylcytosine (ac4C), and 2'-O-Methylation (Nme). We focus on how these properties would be likely to impact RNA:RBP interactions, including by changes to hydrogen bond potential, base-stacking efficiency, and RNA conformational preferences. The effects of RNA modifications on secondary structure have been well-studied, and we briefly discuss how structural effects imparted by modifications can lead to protein binding changes. Finally, we discuss strategies for uncovering as-yet-to-be identified modification-sensitive RBP:RNA Interactions. Coordinating future efforts to intersect the epitranscriptome and the RNA-protein interactome will illuminate the rules governing RNA modification recognition and the mechanisms responsible for the biological consequences of mRNA modification. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > RNA Editing and Modification.

D-Seq: Genome-wide detection of dihydrouridine modifications in RNA.

In addition to A, C, G and U, RNA contains over 100 additional chemically distinct residues. An abundant modified base frequently found in tRNAs, dihydrouridine (D) has recently been mapped to over 100 positions in mRNAs in yeast and human cells. Multiple highly conserved dihydrouridine synthases associate with and modify mRNA, suggesting there are many D sites yet to be found. Because D alters RNA structure, installation of D in mRNA is likely to effect multiple steps in mRNA metabolism including processing, trafficking, translation, and degradation. Here, we introduce D-seq, a method to chart the D landscape at single nucleotide resolution. The included protocols start with RNA isolation and carry through D-seq library preparation and data analysis. While the protocols below are tailored to map Ds in mRNA, the D-seq method is generalizable to any RNA type of interest, including non-coding RNAs, which have also recently been identified as dihydrouridine synthase targets.

Structural basis for translation inhibition by MERS-CoV Nsp1 reveals a conserved mechanism for betacoronaviruses.

All betacoronaviruses (ß-CoVs) encode non-structural protein 1 (Nsp1), an essential pathogenicity factor that potently restricts host gene expression. Among the ß-CoV family, MERS-CoV is the most distantly related member to SARS-CoV-2, and the mechanism for host translation inhibition by MERS-CoV Nsp1 remains controversial. Herein, we show that MERS-CoV Nsp1 directly interacts with the 40S ribosomal subunit. Using cryogenic electron microscopy (cryo-EM), we report a 2.6-Å structure of the MERS-CoV Nsp1 bound to the human 40S ribosomal subunit. The extensive interactions between C-terminal domain of MERS-CoV Nsp1 and the mRNA entry channel of the 40S ribosomal subunit are critical for its translation inhibition function. This mechanism of MERS-CoV Nsp1 is strikingly similar to SARS-CoV and SARS-CoV-2 Nsp1, despite modest sequence conservation. Our results reveal that the mechanism of host translation inhibition is conserved across ß-CoVs and highlight a potential therapeutic target for the development of antivirals that broadly restrict ß-CoVs.

mRNA Regulation by RNA Modifications.

Chemical modifications on mRNA represent a critical layer of gene expression regulation. Research in this area has continued to accelerate over the last decade, as more modifications are being characterized with increasing depth and breadth. mRNA modifications have been demonstrated to influence nearly every step from the early phases of transcript synthesis in the nucleus through to their decay in the cytoplasm, but in many cases, the molecular mechanisms involved in these processes remain mysterious. Here, we highlight recent work that has elucidated the roles of mRNA modifications throughout the mRNA life cycle, describe gaps in our understanding and remaining open questions, and offer some forward-looking perspective on future directions in the field.

Internally controlled RNA sequencing comparisons using nucleoside recoding chemistry.

Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.

The Community-Based Medication-First program for opioid use disorder: a hybrid implementation study protocol of a rapid access to buprenorphine program in Washington State.

BACKGROUND: Opioid use disorder (OUD) is a serious health condition that is effectively treated with buprenorphine. However, only a minority of people with OUD are able to access buprenorphine. Many access points for buprenorphine have high barriers for initiation and retention. Health care and drug treatment systems have not been able to provide services to all-let alone the majority-who need it, and many with OUD report extreme challenges starting and staying on buprenorphine in those care settings. We describe the design and protocol for a study of a rapid access buprenorphine program model in six Washington State communities at existing sites serving people who are unhoused and/or using syringe services programs. This study aimed to test the effectiveness of a Community-Based Medication-First Program model. METHODS: We are conducting a hybrid effectiveness-implementation study of a rapid access buprenorphine model of care staffed by prescribers, nurse care managers, and care navigators. The Community-Based Medication-First model of care was designed as a 6-month, induction-stabilization-transition model to be delivered between 2019 and 2022. Effectiveness outcomes will be tested by comparing the intervention group with a comparison group derived from state records of people who had OUD. Construction of the comparison group will align characteristics such as geography, demographics, historical rates of arrests, OUD medication, and health care utilization, using restriction and propensity score techniques. Outcomes will include arrests, emergency and inpatient health care utilization, and mortality rates. Descriptive statistics for buprenorphine utilization patterns during the intervention period will be documented with the prescription drug monitoring program. DISCUSSION: Results of this study will help determine the effectiveness of the intervention. Given the serious population-level and individual-level impacts of OUD, it is essential that services be readily available to all people with OUD, including those who cannot readily access care due to their circumstances, capacity, preferences, and related systems barriers.

Transcriptome-wide mapping reveals a diverse dihydrouridine landscape including mRNA.

Dihydrouridine is a modified nucleotide universally present in tRNAs, but the complete dihydrouridine landscape is unknown in any organism. We introduce dihydrouridine sequencing (D-seq) for transcriptome-wide mapping of D with single-nucleotide resolution and use it to uncover novel classes of dihydrouridine-containing RNA in yeast which include mRNA and small nucleolar RNA (snoRNA). The novel D sites are concentrated in conserved stem-loop regions consistent with a role for D in folding many functional RNA structures. We demonstrate dihydrouridine synthase (DUS)-dependent changes in splicing of a D-containing pre-mRNA in cells and show that D-modified mRNAs can be efficiently translated by eukaryotic ribosomes in vitro. This work establishes D as a new functional component of the mRNA epitranscriptome and paves the way for identifying the RNA targets of multiple DUS enzymes that are dysregulated in human disease.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

Sexual Assault Nurse Examiner Training: A Review of Literature and Implication for Nursing Education and Service to Rural Communities.

ABSTRACT: Sexual assault nurse examiners (SANEs) play a critical role in providing quality care to patients who have experienced sexual violence. Recent national legislative initiatives have been launched both to better explore state-specific needs in regard to training SANE nurses as well as pilot programs that will establish evidence-based methods of expanding SANE training to remote, rural, and underserved communities.A scoping literature review was conducted to identify best practices in SANE training as well as implications for delivering this education to rural and underserved communities. This review analyzed the scientific strengths and weaknesses of existing research, provided an overview of findings within the literature, and identified areas where further research could be directed to inform local, state, and national strategy on SANE education.Blended learning programs that pair online education with simulated clinical experiences are promising alternatives to the traditional classroom experience; however, more research is needed to know how to enhance retention and engagement of nurses utilizing these methods to obtain preliminary SANE training.

Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation.

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5' untranslated regions that confer 1,000-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we determined a functional role for most alternative 5' UTR isoforms expressed in yeast, revealed a general mode of increased translation via direct binding to a core translation factor, and identified numerous translational control elements including C-rich silencers that are sufficient to repress translation both in vitro and in vivo. DART enables systematic assessment of the translational regulatory potential of 5' UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

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.

Differences in Loneliness Across the Rural-Urban Continuum Among Adults Living in Washington State.

PURPOSE: Rural residents may be at higher risk for loneliness than urban residents due to factors such as social isolation, poorer health, and socioeconomic disadvantage. To date, there have been few studies examining rural-urban differences in loneliness among adults in the United States. We examined differences in loneliness across the rural-urban continuum among adult residents living in Washington State. METHODS: Stratified random sampling was used to select 2,575 adults from small rural, large rural, suburban, and urban areas who were invited to complete a survey on factors affecting health. Data were obtained from 616 adults (278 from small rural, 100 from large rural, 98 from suburban, and 140 from urban areas) from June 2018 through October 2019. Loneliness was measured using the UCLA Loneliness Scale (3rd version). Multivariable linear and logistic regressions were used to examine geographic differences in loneliness (measured continuously and dichotomously). FINDINGS: Mean unadjusted loneliness scores were lower in suburban compared to urban areas (35.06 vs 38.57, P = .03). The prevalence of loneliness was 50.7%, 59.0%, 40.8%, and 54.3% in small rural, large rural, suburban, and urban areas, respectively. Suburban living was associated with lower odds for being lonely compared to urban living (unadjusted OR = 0.58; 95% CI = 0.34-0.98), but this association was not statistically significant in the adjusted model (OR = 0.63; 95% CI = 0.33-1.19). CONCLUSION: Loneliness is a prevalent health issue across the rural-urban continuum among Washington State adults.

Optimized protocol for quantifying 5' UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting.

Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1.

Pseudouridine site assignment by high-throughput in vitro RNA pseudouridylation and sequencing.

Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNAs. High-throughput pseudouridine profiling of eukaryotic mRNAs from cells has revealed novel sites of modification across the transcriptome. Pseudouridine affects RNA structure and RNA-protein interactions with the potential to influence many steps of mRNA metabolism and thereby affect gene expression. Identifying the mechanisms by which individual pseudouridines sites are modified by pseudouridine synthases (PUS) will facilitate studies on the molecular functions of Ψ. Multiple pseudouridine synthases are expressed in all organisms and might direct pseudouridylation of diverse cellular RNAs, but the RNA targets of many enzymes and their specificity determinants remain to be defined. We developed a high-throughput in vitro pseudouridylation assay followed by sequencing that allows validation of candidate sites identified in cells, assignment of sites as direct targets of PUS and interrogation of the RNA sequence and structural features that direct modification. We also implemented an analysis pipeline to assign Ψ sites from these data, including an updated approach to peak-calling that accounts for noisy signal from low-abundance transcripts.

Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing.

Pseudouridine profiling has revealed many previously unknown sites of the RNA modification pseudouridine (Ψ) in cellular RNAs. All organisms express multiple pseudouridine synthases (PUS) whose RNA targets and mechanisms of targeting remain to be elucidated. Here, we describe a high-throughput in vitro pseudouridylation assay to interrogate pseudouridine status upon incubation with recombinant pseudouridine synthases (PUS) at thousands of RNA sequences of interest in parallel. This approach allows validation of sites provisionally identified in cells, identification of the direct targets of individual PUS, and interrogation of the determinants of target recognition including primary sequence and RNA secondary structure.

Restriction of SARS-CoV-2 replication by targeting programmed -1 ribosomal frameshifting.

Translation of open reading frame 1b (ORF1b) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a programmed -1 ribosomal frameshift (-1 PRF) promoted by an RNA pseudoknot. The extent to which SARS-CoV-2 replication may be sensitive to changes in -1 PRF efficiency is currently unknown. Through an unbiased, reporter-based high-throughput compound screen, we identified merafloxacin, a fluoroquinolone antibacterial, as a -1 PRF inhibitor for SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on -1 PRF of other betacoronaviruses. Consistent with the essential role of -1 PRF in viral gene expression, merafloxacin impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing proof-of-principle for targeting -1 PRF as a plausible and effective antiviral strategy for SARS-CoV-2 and other coronaviruses.