Differential dopamine modulation of spinal reflex amplitudes is associated with the presence or absence of the autonomic nervous system.

The spinal cord contains a highly collateralized network of descending dopamine (DA) fibers that stem from the dorso-posterior hypothalamic A11 region in the brain, however, the modulatory actions of DA have generally only been assessed in lumbar segments L2-L5. In contrast to these exclusively sensorimotor segments, spinal cords segments T1-L2 and, in mouse, L6-S2, additionally contain the intermediolateral (IML) nucleus, the origin of autonomic nervous system (ANS). Here, we tested if the different spinal circuits in sensorimotor and IML-containing segments react differently to the modulation of the monosynaptic reflex (MSR) by DA. Bath-application of DA (1 µM) led to a decrease of MSR amplitude in L3-L5 segments; however, in IML-containing segments (T10-L2, and S1/2) the MSR response was facilitated. We did not observe any difference in the response between thoracic (sympathetic) and lumbosacral (parasympathetic) segments. Application of the D2-receptor agonists bromocriptine or quinpirole mimicked the effects of DA, while blocking D2 receptor pathways with raclopride or application with the D1-receptor agonist SKF 38393 led to an increase of the MSR in L3-L5 segments and a decrease of the MSR in IML-containing segments. In contrast, in the presence of the gap-junction blockers, carbenoloxone and quinine, DA modulatory actions in IML-containing segments were similar to those of sensorimotor L3-L5 segments. We suggest that DA modulates MSR amplitudes in the spinal cord in a segment-specific manner, and that the differential outcome observed in ANS segments may be a result of gap junctions in the IML.

BTBD9 and dopaminergic dysfunction in the pathogenesis of restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an urge to move legs, usually accompanied by uncomfortable sensations. RLS symptoms generally happen at night and can be relieved by movements. Genetic studies have linked polymorphisms in BTBD9 to a higher risk of RLS. Knockout of BTBD9 homolog in mice (Btbd9) and fly results in RLS-like phenotypes. A dysfunctional dopaminergic system is associated with RLS. However, the function of BTBD9 in the dopaminergic system and RLS is not clear. Here, we made use of the simple Caenorhabditis elegans nervous system. Loss of hpo-9, the worm homolog of BTBD9, resulted in hyperactive egg-laying behavior. Analysis of genetic interactions between hpo-9 and genes for dopamine receptors (dop-1, dop-3) indicated that hpo-9 and dop-1 worked similarly. Reporter assays of dop-1 and dop-3 revealed that hpo-9 knockout led to a significant increase of DOP-3 expression. This appears to be evolutionarily conserved in mice with an increased D2 receptor (D2R) mRNA in the striatum of the Btbd9 knockout mice. Furthermore, the striatal D2R protein was significantly decreased and Dynamin I was increased. Overall, activities of DA neurons in the substantia nigra were not altered, but the peripheral D1R pathway was potentiated in the Btbd9 knockout mice. Finally, we generated and characterized the dopamine neuron-specific Btbd9 knockout mice and detected an active-phase sleepiness, suggesting that dopamine neuron-specific loss of Btbd9 is sufficient to disturb the sleep. Our results suggest that increased activities in the D1R pathway, decreased activities in the D2R pathway, or both may contribute to RLS.

H3K36 Methylation and the Chromodomain Protein Eaf3 Are Required for Proper Cotranscriptional Spliceosome Assembly.

In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an "adapter protein" to support efficient, constitutive splicing.

The Exon Junction Complex: A Multitasking Guardian of the Transcriptome.

In a recent issue of Molecular Cell, Boehm et al. (2018), Blazquez et al. (2018), and Gonatopoulos-Pournatzis et al. (2018) uncover novel mechanisms by which the cell regulates splicing of cryptic splice sites and microexons.

Mathematical modeling identifies potential gene structure determinants of co-transcriptional control of alternative pre-mRNA splicing.

The spliceosome catalyzes the removal of introns from pre-messenger RNA (mRNA) and subsequent pairing of exons with remarkable fidelity. Some exons are known to be skipped or included in the mature mRNA in a cell type- or context-dependent manner (cassette exons), thereby contributing to the diversification of the human proteome. Interestingly, splicing is initiated (and sometimes completed) co-transcriptionally. Here, we develop a kinetic mathematical modeling framework to investigate alternative co-transcriptional splicing (CTS) and, specifically, the control of cassette exons' inclusion. We show that when splicing is co-transcriptional, default splice patterns of exon inclusion are more likely than when splicing is post-transcriptional, and that certain exons are more likely to be regulatable (i.e. cassette exons) than others, based on the exon-intron structure context. For such regulatable exons, transcriptional elongation rates may affect splicing outcomes. Within the CTS paradigm, we examine previously described hypotheses of co-operativity between splice sites of short introns (i.e. 'intron definition') or across short exons (i.e. 'exon definition'), and find that models encoding these faithfully recapitulate observations in the fly and human genomes, respectively.

Biomedical findings from NASA's Project Mercury: a case series.

The United States first sent humans into space during six flights of Project Mercury from May 1961 to May 1963. These flights were brief, with durations ranging from about 15 min to just over 34 h. A primary purpose of the project was to determine if humans could perform meaningful tasks while in space. This was supported by a series of biomedical measurements on each astronaut before, during (when feasible), and after flight to document the effects of exposure to the spaceflight environment. While almost all of the data presented here have been published in technical reports, this is the first integrated summary of the main results. One unexpected finding emerges: the major physiological changes associated with these short-term spaceflights are correlated more strongly with time spent by the astronaut in a spacesuit than with time spent in space per se. Thus, exposure to the direct stressors of short-duration (up to 34 h) spaceflight was not the dominant factor influencing human health and performance. This is relevant to current spaceflight programs and especially to upcoming commercial flights in which time spent in space (as on a suborbital flight) will be minor compared to the time spent in associated preparation, ascent, and return.

Identifying Subgroups of Adult Superutilizers in an Urban Safety-Net System Using Latent Class Analysis: Implications for Clinical Practice.

BACKGROUND: Patients with repeated hospitalizations represent a group with potentially avoidable utilization. Recent publications have begun to highlight the heterogeneity of this group. Latent class analysis provides a novel methodological approach to utilizing administrative data to identify clinically meaningful subgroups of patients to inform tailored intervention efforts. OBJECTIVE: The objective of the study was to identify clinically distinct subgroups of adult superutilizers. RESEARCH DESIGN: Retrospective cohort analysis. SUBJECTS: Adult patients who had an admission at an urban safety-net hospital in 2014 and 2 or more admissions within the preceding 12 months. MEASURES: Patient-level medical, mental health (MH) and substance use diagnoses, social characteristics, demographics, utilization and charges were obtained from administrative data. Latent class analyses were used to determine the number and characteristics of latent subgroups that best represented these data. RESULTS: In this cohort (N=1515), a 5-class model was preferred based on model fit indices, clinical interpretability and class size: class 1 (16%) characterized by alcohol use disorder and homelessness; class 2 (14%) characterized by medical conditions, MH/substance use disorders and homelessness; class 3 (25%) characterized primarily by medical conditions; class 4 (13%) characterized by more serious MH disorders, drug use disorder and homelessness; and class 5 (32%) characterized by medical conditions with some MH and substance use. Patient demographics, utilization, charges and mortality also varied by class. CONCLUSIONS: The overall cohort had high rates of multiple chronic medical conditions, MH, substance use disorders, and homelessness. However, the patterns of these conditions were different between subgroups, providing important information for tailoring interventions.

Chromatin-remodeling SWI/SNF complex regulates coenzyme Q6 synthesis and a metabolic shift to respiration in yeast.

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q6 (ubiquinone or CoQ6) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism.

The chromatin remodeling complex Swi/Snf regulates splicing of meiotic transcripts in Saccharomyces cerevisiae.

Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae, such as splicing of meiotic transcripts. Like other eukaryotes, S. cerevisiae undergoes a dramatic reprogramming of gene expression during meiosis, including regulated splicing of a number of crucial meiosis-specific RNAs. Splicing of a subset of these is dependent upon the splicing activator Mer1. Here we show a crucial role for the chromatin remodeler Swi/Snf in regulation of splicing of meiotic genes and find that the complex affects meiotic splicing in two ways. First, we show that Swi/Snf regulates nutrient-dependent downregulation of ribosomal protein encoding RNAs, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the ribosomal protein genes) to Mer1-regulated transcripts. We also demonstrate that Mer1 expression is dependent on Snf2, its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus. Hence, Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae. We also reveal an evolutionarily conserved mechanism whereby the cell redirects its energy from maintaining its translational capacity to the process of meiosis.

The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae.

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

Appointment reminders by text message in a safety net health care system: a pragmatic investigation.

INTRODUCTION: Short Message Service (SMS) appointment reminders may provide a wide-reaching, low cost approach to reducing operational inefficiencies and improving access to care. Previous studies indicate this modality may improve attendance rates, yet there is a need for large-scale, pragmatic studies that include unintended consequences and operational costs. METHODS: This pragmatic investigation was a before-after analysis that compared visit attendance outcomes among patients who opted into SMS appointment reminders with outcomes among those who declined over an 18-month evaluation period from March 25, 2013, to September 30, 2014. Eligibility in our integrated safety net health care system included age greater than 17, English or Spanish as a primary language, and a cell phone number in our scheduling system. RESULTS: 47,390 patients were invited by SMS to participate, of which 20,724 (43.7 percent) responded with 18,138 opting in (81.5 percent of respondents). Participants received SMS reminders for 77,783 scheduled visits; comparison group patients (N=72,757) were scheduled for 573,079 visits during the evaluation period. Intervention and comparison groups had, respectively, attendance rates of 72.8 percent versus 66.1 percent (p<0.001), cancellation rates of 13.2 percent versus 18.6 percent (p<0.001), and no show rates of 14.0 percent versus 15.3 percent. Patient satisfaction with text messaging ranged from 77 percent to 96 percent. Implementation challenges included a low rate of inaccurate reminders due to non-standard use of the scheduling system across clinical departments. DISCUSSION: SMS appointment reminders improve patient satisfaction and provide a low operating cost approach to reducing operational inefficiencies through improved attendance rates in an integrated safety net health care system.

Posttranscriptional Regulation of Gcr1 Expression and Activity Is Crucial for Metabolic Adjustment in Response to Glucose Availability.

The transcription factor Gcr1 controls expression of over 75% of the genes in actively growing yeast. Yet despite its widespread effects, regulation of Gcr1 itself remains poorly understood. Here, we show that posttranscriptional Gcr1 regulation is nutrient dependent. Moreover, GCR1 RNA contains a long, highly conserved intron, which allows the cell to generate multiple RNA and protein isoforms whose levels change upon glucose depletion. Intriguingly, an isoform generated by intron retention is exported from the nucleus, and its translation is initiated from a conserved, intronic translation start site. Expression of gene products from both the spliced and unspliced RNAs is essential, as cells expressing only one isoform cannot adjust their metabolic program in response to environmental changes. Finally, we show that the Gcr1 proteins form dimers, providing an elegant mechanism by which this one gene, through its regulation, can perform the repertoire of transcriptional activities necessary for fine-tuned environmental response.

SMITten by the Speed of Splicing.

Splicing occurs co-transcriptionally, but relative rates of splicing and transcription that might reveal mechanisms of their coordinated control have remained mysterious. Now, Carrillo Oesterreich et al. show that the fastest introns are gone nearly as soon as the 3' splice site is transcribed and that introns have distinct splicing kinetics with respect to polymerase progression along the gene.

For many patients who use large amounts of health care services, the need is intense yet temporary.

Patients who accumulate multiple emergency department visits and hospital admissions, known as super-utilizers, have become the focus of policy initiatives aimed at preventing such costly use of the health care system through less expensive community- and primary care-based interventions. We conducted cross-sectional and longitudinal analyses of 4,774 publicly insured or uninsured super-utilizers in an urban safety-net integrated delivery system for the period May 1, 2011-April 30, 2013. Our analysis found that consistently 3 percent of adult patients met super-utilizer criteria and accounted for 30 percent of adult charges. Fewer than half of super-utilizers identified as such on May 1, 2011, remained in the category seven months later, and only 28 percent remained at the end of a year. This finding has important implications for program design and for policy makers because previous studies may have obscured this instability at the individual level. Our study also identified clinically relevant subgroups amenable to different interventions, along with their per capita utilization and costs before and after being identified as super-utilizers. Future solutions include improving predictive modeling to identify individuals likely to experience sustained levels of avoidable utilization, better classifying subgroups for whom interventions are needed, and implementing stronger program evaluation designs.

Augmenting Predictive Modeling Tools with Clinical Insights for Care Coordination Program Design and Implementation.

CONTEXT: The Center for Medicare and Medicaid Innovation (CMMI) awarded Denver Health's (DH) integrated, safety net health care system $19.8 million to implement a "population health" approach into the delivery of primary care. This major practice transformation builds on the Patient Centered Medical Home (PCMH) and Wagner's Chronic Care Model (CCM) to achieve the "Triple Aim": improved health for populations, care to individuals, and lower per capita costs. CASE DESCRIPTION: This paper presents a case study of how DH integrated published predictive models and front-line clinical judgment to implement a clinically actionable, risk stratification of patients. This population segmentation approach was used to deploy enhanced care team staff resources and to tailor care-management services to patient need, especially for patients at high risk of avoidable hospitalization. Developing, implementing, and gaining clinical acceptance of the Health Information Technology (HIT) solution for patient risk stratification was a major grant objective. FINDINGS: In addition to describing the Information Technology (IT) solution itself, we focus on the leadership and organizational processes that facilitated its multidisciplinary development and ongoing iterative refinement, including the following: team composition, target population definition, algorithm rule development, performance assessment, and clinical-workflow optimization. We provide examples of how dynamic business intelligence tools facilitated clinical accessibility for program design decisions by enabling real-time data views from a population perspective down to patient-specific variables. CONCLUSIONS: We conclude that population segmentation approaches that integrate clinical perspectives with predictive modeling results can better identify high opportunity patients amenable to medical home-based, enhanced care team interventions.

Considering the kinetics of mRNA synthesis in the analysis of the genome and epigenome reveals determinants of co-transcriptional splicing.

When messenger RNA splicing occurs co-transcriptionally, the potential for kinetic control based on transcription dynamics is widely recognized. Indeed, perturbation studies have reported that when transcription kinetics are perturbed genetically or pharmacologically splice patterns may change. However, whether kinetic control is contributing to the control of splicing within the normal range of physiological conditions remains unknown. We examined if the kinetic determinants for co-transcriptional splicing (CTS) might be reflected in the structure and expression patterns of the genome and epigenome. To identify and then quantitatively relate multiple, simultaneous CTS determinants, we constructed a scalable mathematical model of the kinetic interplay of RNA synthesis and CTS and parameterized it with diverse next generation sequencing (NGS) data. We thus found a variety of CTS determinants encoded in vertebrate genomes and epigenomes, and that these combine variously for different groups of genes such as housekeeping versus regulated genes. Together, our findings indicate that the kinetic basis of splicing is functionally and physiologically relevant, and may meaningfully inform the analysis of genomic and epigenomic data to provide insights that are missed when relying on statistical approaches alone.

Introduction to cotranscriptional RNA splicing.

The discovery that many intron-containing genes can be cotranscriptionally spliced has led to an increased understanding of how splicing and transcription are intricately intertwined. Cotranscriptional splicing has been demonstrated in a number of different organisms and has been shown to play roles in coordinating both constitutive and alternative splicing. The nature of cotranscriptional splicing suggests that changes in transcription can dramatically affect splicing, and new evidence suggests that splicing can, in turn, influence transcription. In this chapter, we discuss the mechanisms and consequences of cotranscriptional splicing and introduce some of the tools used to measure this process.