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Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)‐induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow‐sense heritability of OXY‐induced locomotor activity traits ranged from 0.22 to 0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7%–12% of the variance in OXY locomotion and anxiety‐like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near Gabra2 (alpha‐2 subunit of GABA‐A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163–181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine‐mapped a 2.45‐Mb region (170.16–172.61 Mb). Transcriptome analysis identified five, localized striatal cis‐eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9 codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder. We exploited near‐isogenic C57BL/6 substrains to fine‐map a 2.45‐Mb region containing DNA variants underlying oxycodone behaviors. We mapped a genomic region on distal chromosome 1 and identified genotype‐driven, differentially expressed mRNAs and proteins within this region and DNA variants. Atp1a2 and Kcnj9 as two likely candidate genes underlying oxycodone behavior.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)-induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow-sense heritability was estimated at 0.22-0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7-12% of the variance in OXY locomotion and anxiety-like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near (alpha-2 subunit of GABA-A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163-181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). Transcriptome analysis identified five, localized striatal cis-eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder.

This study examines VA chaplains' understandings of moral injury (MI) and preferred intervention strategies. Drawing qualitative responses with a nationally-representative sample, content analyses indicated that chaplains' definitions of MI comprised three higher order clusters: (1) MI events, (2) mechanisms in development of MI, and (3) warning signs of MI. Similarly, chaplains' intervention foci could be grouped into three categories: (1) pastoral/therapeutic presence, (2) implementing specific interventions, and (3) therapeutic processes to promote moral repair. Findings are discussed related to emerging conceptualizations of MI, efforts to adapt existing evidence-based interventions to better address MI, and the potential benefits of better integrating chaplains into VA mental health service delivery.

Computerized decision support decreases the number of computed tomography pulmonary angiograms (CTPA) for pulmonary embolism (PE) ordered in emergency departments, but it is not always well accepted by emergency physicians. We studied a department-endorsed, evidence-based clinical protocol that included the PE rule-out criteria (PERC) rule, multi-modal education using principles of knowledge translation (KT), and clinical decision support embedded in our order entry system, to decrease the number of unnecessary CTPA ordered. We performed a historically controlled observational before-after study for one year pre- and post-implementation of a departmentally-endorsed protocol. We included patients > 18 in whom providers suspected PE and who did not have a contraindication to CTPA. Providers entered clinical information into a diagnostic pathway via computerized order entry. Prior to protocol implementation, we provided education to ordering providers. The primary outcome measure was the number of CTPA ordered per 1,000 visits one year before vs. after implementation. CTPA declined from 1,033 scans for 98,028 annual visits (10.53 per 1,000 patient visits (95% CI [9.9-11.2]) to 892 scans for 101,172 annual visits (8.81 per 1,000 patient visits (95% CI [8.3-9.4]) p<0.001. The absolute reduction in PACT ordered was 1.72 per 1,000 visits (a 16% reduction). Patient characteristics were similar for both periods. Knowledge translation clinical decision support using the PERC rule significantly reduced the number of CTPA ordered.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

BackgroundIdentification and characterization of the antigen specificity of tumor-infiltrating lymphocytes (TILs) are key to understand anti-tumor immunity and can guide the development of novel and effective immunotherapies. Our DECODE® platform enables the identification of TCR and peptide-MHC (pMHC) across the immune synapse of TILs.MethodsTo investigate the T cell response directed against the melanoma antigen landscape, we performed a comprehensive analysis of tumor-infiltrating lymphocytes (TILs) derived from 21 melanoma patients. We employed two complementary approaches that support our DECODE ® platform: (1) an engineered reporter cell system expressing chimeric pMHC-TCR (MCR) hybrid molecules, loaded with tens of thousands of peptides derived from antigens highly expressed in melanoma; and (2) a high-throughput TIL screening platform using barcoded peptide-HLA tetramers. These approaches incorporated diverse libraries that allowed us to broadly screen hundreds of antigens across 68 HLA alleles, enabling the in-depth discovery, validation, and application of TCR-epitope pairs. Immunogenicity of the identified epitopes was tested by assessing their ability to activate T cells in vitro.ResultsBy using our TCR-pMHC DECODE® technologies, we identified a broad repertoire of pMHC-TCR pairs from TILs derived from melanoma patients. The epitopes originated from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), personalized neoantigens, and Dark Antigens, a group of cancer targets derived from otherwise non-coding genetic regions. In-depth analysis of one patient revealed tumor-specific dark antigens recognized by TIL-derived TCRs, which were also detectable in peripheral blood, suggesting systemic immunologic prevalence.Epitopes from these antigens were tested for immunogenicity using in vitro expansion approaches; we observed Dark Antigen epitope-specific T cell expansion, supporting a role for integration of these epitopes into cancer vaccine approaches. Likewise, decoded TCRs were expressed in primary T cells, and the recombinant TCR-T cells were activated by cell lines derived from multiple solid tumor types, highlighting the therapeutic potential of these TCRs for both cell therapy and bispecific engager formats.ConclusionsThese findings showcase the power of our DECODE® technology to uncover clinically relevant, TIL-derived epitopes and TCRs from TAAs, TSAs, personalized neoantigens and, dark antigens for next-generation cancer immunotherapies that include TCR bispecifics and cancer vaccines. Application of this approach will provide more effective immunotherapies for many solid tumor indications.

Rationale Histamine H 3 receptor antagonists, such as ABT-288, have been shown to possess cognitive-enhancing and wakefulness-promoting effects. On the surface, this might suggest that H 3 antagonists possess psychomotor stimulant-like effects and, as such, may have the potential for abuse. Objectives The aim of the present study was to further characterize whether ABT-288 possesses stimulant-like properties and whether its pharmacology gives rise to abuse liability. Methods The locomotor-stimulant effects of ABT-288 were measured in mice and rats, and potential development of sensitization was addressed. Drug discrimination was used to assess amphetamine-like stimulus properties, and drug self-administration was used to evaluate reinforcing effects of ABT-288. The potential development of physical dependence was also studied. Results ABT-288 lacked locomotor-stimulant effects in both rats and mice. Repeated administration of ABT-288 did not result in cross-sensitization to the stimulant effects of d-amphetamine in mice, suggesting that there is little overlap in circuitries upon which the two drugs interact for motor activity. ABT-288 did not produce amphetamine-like discriminative stimulus effects in drug discrimination studies nor was it self-administered by rats trained to self-administer cocaine. There were no signs of physical dependence upon termination of repeated administration of ABT-288 for 30 days. Conclusions The sum of these preclinical data, the first of their kind applied to H 3 antagonists, indicates that ABT-288 is unlikely to possess a high potential for abuse in the human population and suggests that H 3 antagonists, as a class, are similar in this regard.

IntroductionCardiovascular collapse is a common complication during tracheal intubation of critically ill adults. Whether administration of an intravenous fluid bolus prevents cardiovascular collapse during tracheal intubation remains uncertain. A prior randomised trial found fluid bolus administration to be ineffective overall but suggested potential benefit for patients receiving positive pressure ventilation during tracheal intubation.Methods and analysisThe PREventing cardiovascular collaPse with Administration of fluid REsuscitation during Induction and Intubation (PREPARE II) trial is a prospective, multi-centre, non-blinded randomised trial being conducted in 13 academic intensive care units in the USA. The trial will randomise 1065 critically ill adults undergoing tracheal intubation with planned use of positive pressure ventilation (non-invasive ventilation or bag-mask ventilation) between induction and laryngoscopy to receive 500 mL of intravenous crystalloid or no intravenous fluid bolus. The primary outcome is cardiovascular collapse, defined as any of: systolic blood pressure <65 mm Hg, new or increased vasopressor administration between induction and 2 min after intubation, or cardiac arrest or death between induction and 1 hour after intubation. The primary analysis will be an unadjusted, intention-to-treat comparison of the primary outcome between patients randomised to fluid bolus administration and patients randomised to no fluid bolus administration using a χ2 test. The sole secondary outcome is 28-day in-hospital mortality. Enrolment began on 1 February 2019 and is expected to conclude in June 2020.Ethics and disseminationThe trial was approved by either the central institutional review board at Vanderbilt University Medical Center or the local institutional review board at each trial site. Results will be submitted for publication in a peer-reviewed journal and presented at scientific conferences.Trial registration numberNCT03787732.

Rationale: Histamine H sub(3) receptor antagonists, such as ABT-288, have been shown to possess cognitive-enhancing and wakefulness-promoting effects. On the surface, this might suggest that H sub(3) antagonists possess psychomotor stimulant-like effects and, as such, may have the potential for abuse. Objectives: The aim of the present study was to further characterize whether ABT-288 possesses stimulant-like properties and whether its pharmacology gives rise to abuse liability. Methods: The locomotor-stimulant effects of ABT-288 were measured in mice and rats, and potential development of sensitization was addressed. Drug discrimination was used to assess amphetamine-like stimulus properties, and drug self-administration was used to evaluate reinforcing effects of ABT-288. The potential development of physical dependence was also studied. Results: ABT-288 lacked locomotor-stimulant effects in both rats and mice. Repeated administration of ABT-288 did not result in cross-sensitization to the stimulant effects of d-amphetamine in mice, suggesting that there is little overlap in circuitries upon which the two drugs interact for motor activity. ABT-288 did not produce amphetamine-like discriminative stimulus effects in drug discrimination studies nor was it self-administered by rats trained to self-administer cocaine. There were no signs of physical dependence upon termination of repeated administration of ABT-288 for 30 days. Conclusions: The sum of these preclinical data, the first of their kind applied to H sub(3) antagonists, indicates that ABT-288 is unlikely to possess a high potential for abuse in the human population and suggests that H sub(3) antagonists, as a class, are similar in this regard.

Histamine H^sub 3^ receptor antagonists, such as ABT-288, have been shown to possess cognitive-enhancing and wakefulness-promoting effects. On the surface, this might suggest that H^sub 3^ antagonists possess psychomotor stimulant-like effects and, as such, may have the potential for abuse. The aim of the present study was to further characterize whether ABT-288 possesses stimulant-like properties and whether its pharmacology gives rise to abuse liability. The locomotor-stimulant effects of ABT-288 were measured in mice and rats, and potential development of sensitization was addressed. Drug discrimination was used to assess amphetamine-like stimulus properties, and drug self-administration was used to evaluate reinforcing effects of ABT-288. The potential development of physical dependence was also studied. ABT-288 lacked locomotor-stimulant effects in both rats and mice. Repeated administration of ABT-288 did not result in cross-sensitization to the stimulant effects of d-amphetamine in mice, suggesting that there is little overlap in circuitries upon which the two drugs interact for motor activity. ABT-288 did not produce amphetamine-like discriminative stimulus effects in drug discrimination studies nor was it self-administered by rats trained to self-administer cocaine. There were no signs of physical dependence upon termination of repeated administration of ABT-288 for 30 days. The sum of these preclinical data, the first of their kind applied to H^sub 3^ antagonists, indicates that ABT-288 is unlikely to possess a high potential for abuse in the human population and suggests that H^sub 3^ antagonists, as a class, are similar in this regard.[PUBLICATION ABSTRACT]