Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Emergence of Novel Norovirus GII.4 Variant.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Alternative splicing redefines landscape of commonly mutated genes in acute myeloid leukemia.

Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

Pediatric Sedation/Anesthesia for MRI: Results From the Pediatric Sedation Research Consortium.

BACKGROUND: Magnetic resonance imaging (MRI) is the most common imaging procedure requiring sedation/anesthesia in children. Understanding adverse events associated with sedation/anesthesia is important in making decisions regarding MRI vs. other imaging modalities. No large studies have evaluated the practice of pediatric sedation/anesthesia for MRI by a variety of pediatric specialists. PURPOSE: Utilize a large pediatric sedation database to characterize the patients and adverse events associated with sedation/anesthesia for pediatric MRI. STUDY TYPE: Retrospective analysis of prospectively collected data. SUBJECTS: The Pediatric Sedation Research Consortium (PSRC) has 109,947 entries for sedations for MRI from November 10, 2011 through December 18, 2017. ASSESSMENT: Patient demographics, sedative medications, interventions, and adverse events are described. Associations with adverse events were assessed. Trends in sedative medications used over time are examined. STATISTICAL TESTS: Descriptive statistics, Chi-Squared and Fisher's Exact tests for categorical variables, logistic regression and assessment of trend using logistic regression and other method. RESULTS: A total of 109,947 MRI-related sedations were examined. Most subjects (66.2%) were 5 years old or younger. Seizure or other neurologic issue prompted MRI in 63.7% of cases. Providers responsible for sedation/anesthesia included intensivists (49.3%), emergency medicine physicians (28.2%), hospitalists (10.2%), and anesthesiologists (9.8%). The most commonly used sedative agent was propofol (89.1%). The most common airway intervention was supplemental oxygen (71.7%), followed by head/airway repositioning (20.6%). Airway-related adverse events occurred in 8.4% of patients. Serious adverse events occurred in only 0.06% of patients, including three cases of cardiac arrest. No mortality was recorded. There was a statistically significant increase in the use of dexmedetomidine over time. DATA CONCLUSIONS: Overall, adverse event rates were low. Sedation/anesthesia with propofol infusion and natural airway was the most common method used by this varied group of sedation providers. The use of dexmedetomidine increased over time. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 5.

Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation.

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells.

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.

Sedatives used in children to obtain head CT in the emergency department.

OBJECTIVES: Children in the emergency department who require computerized tomography (CT) of the head often are given sedative medications to facilitate completion of the study with adequate imaging. A prior study found the two most common medications used to obtain head CT in children were pentobarbital and chloral hydrate; however, these medications have become less popular. We hypothesized that there was variability in medication choice amongst providers in the emergency department and there has been a change in the preferred sedatives used in the last decade. METHODS: We conducted a retrospective multicenter cross-sectional study of children 0-18 years old who received a medication with sedative properties and underwent head CT while in the emergency department from 2007 to 2018, using the Pediatric Health Information System (PHIS) database. The primary outcome measure was the frequency of administration of drugs within an individual sedative class. RESULTS: We analyzed 24,418 patient encounters, of whom 53% received an opioid and 41% received a benzodiazepine. There were statistically significant decreases in the use of barbiturates, chloral hydrate, anti-emetic sedatives, and opioids, while increases in barbiturate combination drugs, benzodiazepines and dexmedetomidine were observed over the study period. The majority of medications were administered parenterally. CONCLUSION: There is wide variability in sedatives used in children to obtain head CT and the preferred drugs have shifted over the last decade.

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes.

Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic coregulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets, including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these coregulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues, including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, Drosophila, and Caenorhabditis elegans suggests this antagonism is conserved throughout evolution.

Human Norovirus Epitope D Plasticity Allows Escape from Antibody Immunity without Loss of Capacity for Binding Cellular Ligands.

Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.IMPORTANCE Human norovirus causes ∼20% of all acute gastroenteritis and ∼200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

The Use of Intranasal Dexmedetomidine and Midazolam for Sedated Magnetic Resonance Imaging in Children: A Report From the Pediatric Sedation Research Consortium.

OBJECTIVE: The objective of this study was to describe the use of intranasal dexmedetomidine (IN DEX) for sedated magnetic resonance imaging (MRI) examinations in children. The use of IN DEX for MRI in children has not been well described in the literature. MATERIALS AND METHODS: The Pediatric Sedation Research Consortium (PSRC) is a collaborative and multidisciplinary group of sedation practitioners dedicated to understanding and improving the process of pediatric sedation. We searched the 2007 version of the PSRC database solely for instances in which IN DEX was used for MRI diagnostic studies. Patients receiving intravenous medications were excluded. Patient demographics, IN DEX dose, adjunct medications and dose, as well as procedure completion, complications, interventions, and monitoring providers were analyzed. RESULTS: A total of 224 sedation encounters were included in our primary analysis. There were no major adverse events. Most sedations (88%) required no intervention. Registered nurses were the monitoring provider in over 99% of cases. The median (interquartile range) dose of dexmedetomidine was 3 (2.5-3) mcg/kg. Adjunctive midazolam was used in 219/224 (98%) of the cases. All procedures were completed. CONCLUSIONS: This report from the PSRC shows that IN DEX in combination with midazolam is an effective medication regimen for children who require an MRI with sedation.

Radiologic Imaging in Trauma Patients with Cervical Spine Immobilization at a Pediatric Trauma Center.

BACKGROUND: Pediatric trauma patients with cervical spine (CS) immobilization using a cervical collar often require procedural sedation (PS) for radiologic imaging. The limited ability to perform airway maneuvers while CS immobilized with a cervical collar is a concern for emergency department (ED) staff providing PS. OBJECTIVE: To describe the use of PS and analgesia for radiologic imaging acquisition in pediatric trauma patients with CS immobilization. METHODS: Retrospective medical record review of all trauma patients with CS immobilization at a high-volume pediatric trauma center was performed. Patient demographics, imaging modality, PS success, sedative and analgesia medications, and adverse events were analyzed. Patients intubated prior to arrival to the ED were excluded. RESULTS: A total of 1417 patients with 1898 imaging encounters met our inclusion criteria. A total of 398 patients required more than one radiographic imaging procedure. The median age was 8 years (range 3.8-12.75 years). Computed tomography of the head was used in 974 of the 1898 patients (51.3%). A total of 956 of the 1898 patients (50.4%) required sedatives or analgesics for their radiographic imaging, with 875 (91.5%) requiring a single sedative or analgesic agent, and 81 (8.5%) requiring more than one medication. Airway obstruction was the most common adverse event, occurring in 5 of 956 patients (0.3%). All imaging procedures were successfully completed. CONCLUSION: Only 50% of CS immobilized, nonintubated patients required a single sedative or analgesic medication for their radiologic imaging. Procedural success was high, with few adverse events.

Antigenic Characterization of a Novel Recombinant GII.P16-GII.4 Sydney Norovirus Strain With Minor Sequence Variation Leading to Antibody Escape.

Background: Human noroviruses are the leading cause of acute gastroenteritis. Strains of the GII.4 genotype cause pandemic waves associated with viral evolution and subsequent antigenic drift and ligand-binding modulation. In November 2015, a novel GII.4 Sydney recombinant variant (GII.P16-GII.4 Sydney) emerged and replaced GII.Pe-GII.4 Sydney as the predominant cause of acute gastroenteritis in the 2016-2017 season in the United States. Methods: Virus-like particles of GII.4 2012 and GII.4 2015 were compared for ligand binding and antibody reactivity, using a surrogate neutralization assay. Results: Residue changes in the capsid between GII.4 2012 and GII.4 2015 decreased the potency of human polyclonal sera and monoclonal antibodies. A change in epitope A resulted in the complete loss of reactivity of a class of blockade antibodies and reduced levels of a second antibody class. Epitope D changes modulated monoclonal antibody potency and ligand-binding patterns. Conclusions: Substitutions in blockade antibody epitopes between GII.4 2012 and GII.4 2015 influenced antigenicity and ligand-binding properties. Although the impact of polymerases on fitness remains uncertain, antigenic variation resulting in decreased potency of antibodies to epitope A, coupled with altered ligand binding, likely contributed significantly to the spread of GII.4 2015 and its replacement of GII.4 2012 as the predominant norovirus outbreak strain.

Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3-dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3-dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3ß phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing.

Induced transcription and stability of CELF2 mRNA drives widespread alternative splicing during T-cell signaling.

Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3'UTR length and contributes to the total signal-induced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.

Emergence of Novel Human Norovirus GII.17 Strains Correlates With Changes in Blockade Antibody Epitopes.

Background: Human norovirus is a significant public health burden, with >30 genotypes causing endemic levels of disease and strains from the GII.4 genotype causing serial pandemics as the virus evolves new ligand binding and antigenicity features. During 2014-2015, genotype GII.17 cluster IIIb strains emerged as the leading cause of norovirus infection in select global locations. Comparison of capsid sequences indicates that GII.17 is evolving at previously defined GII.4 antibody epitopes. Methods: Antigenicity of virus-like particles (VLPs) representative of clusters I, II, and IIIb GII.17 strains were compared by a surrogate neutralization assay based on antibody blockade of ligand binding. Results: Sera from mice immunized with a single GII.17 VLP identified antigenic shifts between each cluster of GII.17 strains. Ligand binding of GII.17 cluster IIIb VLP was blocked only by antisera from mice immunized with cluster IIIb VLPs. Exchange of residues 393-396 from GII.17.2015 into GII.17.1978 ablated ligand binding and altered antigenicity, defining an important varying epitope in GII.17. Conclusions: The capsid sequence changes in GII.17 strains result in loss of blockade antibody binding, indicating that viral evolution, specifically at residues 393-396, may have contributed to the emergence of cluster IIIb strains and the persistence of GII.17 in human populations.

Impact of Pre-exposure History and Host Genetics on Antibody Avidity Following Norovirus Vaccination.

Background: Development of high avidity, broadly neutralizing antibodies (Abs) is a priority after vaccination against rapidly evolving, widely disseminated viruses like human norovirus. After vaccination with a multivalent GI.1 and GII.4c norovirus virus-like particle (VLP) vaccine candidate adjuvanted with alum and monophosphoryl lipid A (MPL), blockade Ab titers peaked early, with no increase in titer following a second vaccine dose. Methods: Blockade Ab relative avidity was evaluated by measuring the slope of blockade Ab neutralization curves. Results: Blockade Ab avidity to the GI.1 vaccine component peaked at day 35 (7 days after dose 2). Avidities to heterotypic genogroup I VLPs were not sustained at day 35 after vaccination or GI.1 infection, as measured from archived sera. Only secretor-positive participants maintained high avidity blockade Ab to GI.1 at day 180. Avidity to the GII.4c vaccine component peaked at day 7, remained elevated through day 180, and was not secretor dependent. Avidity to an immunologically novel GII.4 strain VLP correlated with preexisting Ab titer to an ancestral strain Epitope A. Conclusions: Host genetics and pre-exposure history shape norovirus vaccine Ab responses, including blockade Ab avidity. Avidity of potentially neutralizing Ab may be an important metric for evaluating vaccine responses to highly penetrant viruses with cross-reactive serotypes.

Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons.

HnRNP L is a ubiquitous splicing-regulatory protein that is critical for the development and function of mammalian T cells. Previous work has identified a few targets of hnRNP L-dependent alternative splicing in T cells and has described transcriptome-wide association of hnRNP L with RNA. However, a comprehensive analysis of the impact of hnRNP L on mRNA expression remains lacking. Here we use next-generation sequencing to identify transcriptome changes upon depletion of hnRNP L in a model T-cell line. We demonstrate that hnRNP L primarily regulates cassette-type alternative splicing, with minimal impact of hnRNP L depletion on transcript abundance, intron retention, or other modes of alternative splicing. Strikingly, we find that binding of hnRNP L within or flanking an exon largely correlates with exon repression by hnRNP L. In contrast, exons that are enhanced by hnRNP L generally lack proximal hnRNP L binding. Notably, these hnRNP L-enhanced exons share sequence and context features that correlate with poor nucleosome positioning, suggesting that hnRNP may enhance inclusion of a subset of exons via a cotranscriptional or epigenetic mechanism. Our data demonstrate that hnRNP L controls inclusion of a broad spectrum of alternative cassette exons in T cells and suggest both direct RNA regulation as well as indirect mechanisms sensitive to the epigenetic landscape.

Evaluation of methohexital as an alternative to propofol in a high volume outpatient pediatric sedation service.

BACKGROUND: Propofol is a preferred agent for many pediatric sedation providers because of its rapid onset and short duration of action. It allows for quick turn around times and enhanced throughput. Occasionally, intravenous (IV) methohexital (MHX), an ultra-short acting barbiturate is utilized instead of propofol. OBJECTIVE: Describe the experience with MHX in a primarily propofol driven outpatient sedation program and to see if it serves as an acceptable alternative when propofol is not the preferred pharmacologic option. METHODS: Retrospective chart review from 2012 to 2015 of patients receiving IV MHX as their primary sedation agent. Data collected included demographics, reason for methohexital use, dosing, type of procedure, success rate, adverse events (AE), duration of the procedure, and time to discharge. RESULTS: Methohexital was used in 240 patient encounters. Median age was 4years (IQR 2-7), 71.8% were male, and 80.4% were ASA-PS I or II. Indications for MHX use: egg+soy/peanut allergy in 93 (38.8%) and mitochondrial disorder 9 (3.8%). Median induction bolus was 2.1mg/kg (IQR, 1.9-2.8), median maintenance infusion was 4.5mg/kg/h (IQR, 3.0-6.0). Hiccups 15 (6.3%), secretions requiring intervention 14 (5.8%), and cough 12 (5.0%) were the most commonly occurring minor AEs. Airway obstruction was seen in 28 (11.6%). Overall success rate was 94%. Median time to discharge after procedure completion was 40.5min (IQR 28-57). CONCLUSION: Methohexital can be used with a high success rate and AEs that are not inconsistent with propofol administration. Methohexital should be considered when propofol is not a preferred option.

TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells.

PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.