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Introduction: Overdose deaths from high-potency synthetic opioids, including fentanyl and its analogs, continue to rise along with emergency department (ED) visits for complications of opioid use disorder (OUD). Fentanyl accumulates in adipose tissue; although rare, this increases the risk of precipitated withdrawal in patients upon buprenorphine initiation. Many EDs have implemented medication for opioid use disorder (MOUD) programs using buprenorphine. However, few offer methadone, a proven therapy without the risk of precipitated withdrawal associated with buprenorphine initiation. We describe the addition of an ED-initiated methadone treatment pathway and compared its 72-hour follow-up outpatient treatment engagement rates to our existing ED-initiated buprenorphine MOUD program., Methods: We expanded our ED MOUD program with a methadone treatment pathway. From February 20-September 19, 2023, we screened 20,504 ED arrivals; 5.1% had signs of OUD. We enrolled 61 patients: 28 in the methadone; and 33 in the buprenorphine pathways. For patients who screened positive for opioid use, shared decision-making was employed to determine whether buprenorphine or methadone therapy was more appropriate. Patients in the methadone pathway received their first dose of up to 30 milligrams (mg) of methadone in the ED. Two additional methadone doses of up to 40 mg were dispensed at the time of the ED visit and held in the department, allowing patients to return each day for observed dosing until intake at an opioid treatment program (OTP). We compared 72-hour rates of outpatient follow-up treatment engagement at the OTP (for those on methadone) or at the addiction treatment center (ATC) (for those on buprenorphine) for the two treatment pathways., Results: Of the 28 patients enrolled in the methadone pathway, 12 (43%) successfully engaged in follow-up treatment at the OTP. Of the 33 patients enrolled in the buprenorphine pathway, 15 (45%) successfully engaged in follow-up treatment at the ATC (relative risk 1.06; 95% confidence interval 0.60-1.87)., Conclusion: Methadone initiation in the ED to treat patients with OUD resulted in similar 72-hour follow-up outpatient treatment engagement rates compared to ED-buprenorphine initiation, providing another viable option for MOUD., Competing Interests: Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. This study was funded by the Substance Abuse and Mental Health Services Administration (SAMHSA) 1H79TI085330 and the 2019 University of Vermont Health Network Innovation Grant. There are no other conflicts of interest or sources of funding to declare.

Objective: to identify factors associated with COVID19 vaccine hesitancy, including sources of information among residents of Maine. Methods: 148 study participants, recruited through community partners and primary care offices in Maine, completed an anonymous 15 item online survey. Recruitment and data collection occurred from May to September, 2021. Hesitancy was determined through a single question, “Will you get one of the COVID vaccines when it is offered to you?” Results: vaccine hesitant respondents were younger than not hesitant respondents (p = 0.01). Hesitant individuals were significantly more likely to report concerns regarding the speed of COVID-19 vaccine production, vaccine efficacy, and potential vaccine side effects (p < 0.05 for each). Hesitant individuals were also significantly more likely to have discussed vaccination with their primary physician (p = 0.04). Conclusions: overall, hesitant individuals are more likely to be younger and had less trust in information from government sources, but they sought input from primary care. They were also more concerned about efficacy, side effects, and the rapid development of COVID-19 vaccines. Primary care physicians are in key positions to address these concerns due to contact with individuals who need accurate information.

Understanding the contributions of transcription factor DNA binding sites to transcriptional enhancers is a significant challenge. We developed Quantitative enhancer-FACS-Seq for highly parallel quantification of enhancer activities from a genomically integrated reporter in Drosophila melanogaster embryos. We investigate the contributions of the DNA binding motifs of four poorly characterized TFs to the activities of twelve embryonic mesodermal enhancers. We measure quantitative changes in enhancer activity and discover a range of epistatic interactions among the motifs, both synergistic and alleviating. We find that understanding the regulatory consequences of TF binding motifs requires that they be investigated in combination across enhancer contexts., (© 2021. The Author(s).)

A healthy mitochondrial network is essential for the maintenance of neuronal synaptic integrity. Mitochondrial and metabolic dysfunction contributes to the pathogenesis of many neurodegenerative diseases including dementia. OPA1 is the master regulator of mitochondrial fusion and fission and is likely to play an important role during neurodegenerative events. To explore this, we quantified hippocampal dendritic and synaptic integrity and the learning and memory performance of aged Opa1 haploinsufficient mice carrying the Opa1 Q285X mutation (B6; C3- Opa1 Q285STOP ; Opa1 +/- ). We demonstrate that heterozygous loss of Opa1 results in premature age-related loss of spines in hippocampal pyramidal CA1 neurons and a reduction in synaptic density in the hippocampus. This loss is associated with subtle memory deficits in both spatial novelty and object recognition. We hypothesize that metabolic failure to maintain normal neuronal activity at the level of a single spine leads to premature age-related memory deficits. These results highlight the importance of mitochondrial homeostasis for maintenance of neuronal function during ageing., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Transcription factors (TFs) control gene expression by binding DNA recognition sites in genomic regulatory regions. Although most forkhead TFs recognize a canonical forkhead (FKH) motif, RYAAAYA, some forkheads recognize a completely different (FHL) motif, GACGC. Bispecific forkhead proteins recognize both motifs, but the molecular basis for bispecific DNA recognition is not understood. We present co-crystal structures of the FoxN3 DNA binding domain bound to the FKH and FHL sites, respectively. FoxN3 adopts a similar conformation to recognize both motifs, making contacts with different DNA bases using the same amino acids. However, the DNA structure is different in the two complexes. These structures reveal how a single TF binds two unrelated DNA sequences and the importance of DNA shape in the mechanism of bispecific recognition., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40-60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent., (© 2017 Dukler et al.; Published by Cold Spring Harbor Laboratory Press.)

We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activation—including expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor binding—than those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.

Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.

Fluctuations in nutrient availability profoundly impact gene expression. Previous work revealed postrecruitment regulation of RNA polymerase II (Pol II) during starvation and recovery in Caenorhabditis elegans, suggesting that promoter-proximal pausing promotes rapid response to feeding. To test this hypothesis, we measured Pol II elongation genome wide by two complementary approaches and analyzed elongation in conjunction with Pol II binding and expression. We confirmed bona fide pausing during starvation and also discovered Pol II docking. Pausing occurs at active stress-response genes that become downregulated in response to feeding. In contrast, "docked" Pol II accumulates without initiating upstream of inactive growth genes that become rapidly upregulated upon feeding. Beyond differences in function and expression, these two sets of genes have different core promoter motifs, suggesting alternative transcriptional machinery. Our work suggests that growth and stress genes are both regulated postrecruitment during starvation but at initiation and elongation, respectively, coordinating gene expression with nutrient availability., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5' caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5' ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex. DOI:http://dx.doi.org/10.7554/eLife.00808.001.

Fcp1 dephosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II) to recycle it into a form that can initiate a new round of transcription. Previously, we identified Drosophila Fcp1 as an important factor in optimal Hsp70 mRNA accumulation after heat shock. Here, we examine the role of Fcp1 in transcription of heat shock genes in vivo. We demonstrate that Fcp1 localizes to active sites of transcription including the induced Hsp70 gene. The reduced Hsp70 mRNA accumulation seen by RNA interference (RNAi) depletion of Fcp1 in S2 cells is a result of a loss of Pol II in the coding region of highly transcribed heat shock-induced genes: Hsp70, Hsp26, and Hsp83. Moreover, Fcp1 depletion dramatically increases phosphorylation of the non-chromatin-bound Pol II. Reexpression of either wild-type or catalytically dead versions of Fcp1 demonstrates that both the reduced Pol II levels on heat shock genes and the increased levels of phosphorylated free Pol II are dependent on the catalytic activity of Fcp1. Our results indicate that Fcp1 is required to maintain the pool of initiation-competent unphosphorylated Pol II, and this function is particularly important for the highly transcribed heat shock genes.

Background: In Xenopus retinogenesis, p27Xic1, a Xenopus cyclin dependent kinase inhibitor, functions as a cell fate determinant in both gliogenesis and neurogenesis in a context dependent manner. This activity is essential for co-ordination of determination and cell cycle regulation. However, very little is known about the mechanism regulating the context dependent choice between gliogenesis versus neurogenesis., Results: We have identified NM23-X4, a NM23 family member, as a binding partner of p27Xic1. NM23-X4 is expressed at the periphery of the ciliary marginal zone of the Xenopus retina and the expression overlaps with p27Xic1 at the central side. Our in vivo functional analysis in Xenopus retina has shown that knockdown of NM23-X4 activates gliogenesis. Furthermore, co-overexpression of NM23-X4 with p27Xic1 results in the inhibition of p27Xic1-mediated gliogenesis, through direct interaction of NM23-X4 with the amino-terminal side of p27Xic1. This inhibitory effect on gliogenesis requires serine-150 and histidine-148, which correspond to the important residues for the kinase activities of NM23 family members., Conclusion: This study demonstrates that NM23-X4 functions as an inhibitor of p27Xic1-mediated gliogenesis in Xenopus retina and suggests that this activity contributes to the proper spatio-temporal regulation of gliogenesis.

Background/aim: X linked retinoschisis (XLRS) is caused by mutations in RS1 which encodes the discoidin domain protein retinoschisin, secreted by photoreceptors and bipolar cells. Missense mutations occur throughout the gene and some of these are known to interfere with protein secretion. This study was designed to investigate the functional consequences of missense mutations at different locations in retinoschisin., Methods and Results: The authors developed a structural model of the retinoschisin discoidin domain and used this to predict the effects of missense mutations. They expressed disease associated mutations and found that those affecting conserved residues prevented retinoschisin secretion. Most of the remaining mutations cluster within a series of loops on the surface of the beta barrel structure and do not interfere with secretion, suggesting this region may be a ligand binding site. They also demonstrated that wild type retinoschisin octamerises and associates with the cell surface. A subgroup of secreted mutations reduce oligomerisation (C59S, C219G, C223R)., Conclusions: It is suggested that there are three different molecular mechanisms which lead to XLRS: mutations interfering with secretion, mutations interfering with oligomerisation, and mutations that allow secretion and oligomerisation but interfere with retinoschisin function. The authors conclude that binding of oligomerised retinoschisin at the cell surface is important in its presumed role in cell adhesion.

X-linked retinoschisis results in visual loss in early life with splitting within the inner retinal layers. Many missense and protein truncating mutations of the causative gene RS1 (encoding retinoschisin) have been identified but disease severity is not mutation-dependent. Retinoschisin is a soluble secretory protein predicted to have a globular conformation. Missense mutations would be expected to interfere with protein folding leading to an abnormal conformation and intracellular retention and elimination. To test this hypothesis we have expressed seven pathological RS1 mutations (L12H, C59S, G70S, R102W, G109R, R141G and R213W) in COS-7 cells and investigated their intracellular processing and transport. Using immunoblotting and confocal fluorescent immunocytochemistry we show normal secretion of WT RS1, but either reduced (C59S and R141G) or absent (L12H, G70S, R102W, G109R and R213W) secretion of mutant RS1 and intracellular retention. In addition, we show that L12H RS1 is degraded by proteasomes and in vitro transcription/translation revealed the defects in both cleavage of its signal peptide and translocation into the endoplasmic reticulum. Our results indicate the pathological basis of RS1 is intracellular retention of the majority of mutant proteins, which may explain why disease severity is not mutation-specific. Furthermore, we have shown that in vitro expression of RS1 may be a useful functional assay to investigate the pathogenicity of sequence changes within the RS1 gene.