Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Cognitive behavioral therapy for suicide prevention among Veterans receiving substance use disorder treatment: Results from a randomized trial.

Veterans receiving substance use disorder (SUD) treatment are at a clear elevated risk for engaging in suicidal behaviors. Intervening to reduce suicide risk during an episode of SUD treatment could meaningfully target a key high-risk group of Veterans. Cognitive Behavioral Therapy for Suicide Prevention (CBT-SP) was developed to reduce the frequency and duration of suicidal ideation, as well as decrease suicidal behaviors. The form of CBT-SP in this study progressed from building an understanding of the cognitive model to practicing new skills, and highlighted the links between substance use, craving, self-efficacy and suicidal ideation and attempts. CBT-SP was compared to an attention matched 8-session control condition (termed Supportive Psychoeducational Control [SPC]) during a multi-site randomized controlled trial for 299 Veterans receiving outpatient SUD treatment services within the Veterans Health Administration. The frequency of suicidal ideation remained relatively constant over 24-months of follow-up, however the duration of suicidal ideation decreased, and suicide attempts decreased relative to baseline in both conditions. Forty-two participants (14%) reported at least one suicide attempt during the 2-year follow-up period. No statistically significant differences were found between CBT-SP and SPC on any of these outcomes. Analyses of secondary outcomes indicate that preparatory behaviors for suicide were less common among those in the CBT-SP condition than SPC across the 24-month follow-up (OR, 95%CI = 0.44 (0.25, 0.79); p = 0.02). Veterans in SUD treatment are a high-risk group and delivery of suicide-specific interventions is feasible during SUD care. However, results did not indicate that CBT-SP was superior to SPC on any primary outcomes, underscoring the importance of identifying and testing alternative approaches that support suicide reduction in this group.

Esophageal Bougienage for Management of Lodged Esophageal Coins: Safe, Effective, Efficient, and Underused.

OBJECTIVES: The aims of the study are to evaluate outcomes of esophageal bougienage for management of lodged esophageal coins and to assess the extent of bougienage usage and barriers among emergency providers. METHODS: We performed a retrospective chart review of pediatric patients with single lodged esophageal coins presenting to our single academic pediatric emergency department between November 2012 and December 2018. We compared procedural outcomes, complications, length of stay (LOS), and cost between those managed with bougienage and with endoscopy. We further surveyed emergency physicians to assess the extent of bougienage usage and barriers to utilization across different institutions. RESULTS: We identified 205 patients with single lodged esophageal coins presenting during our study window. One hundred forty-seven patients ultimately underwent bougienage with 97% success and no major complications. Fifty-six patients were managed by endoscopy with 100% success and one major complication. Bougienage had significantly lower LOS (median 2.18 vs 11.92 hours, P < 0.001) and hospital charges (median $3533 vs $12,679, P < 0.001) compared with endoscopy. We received 242 completed surveys representing 38 states from primarily academic pediatric emergency physicians. The majority of respondents (90%) used specialist consult with only 4.5% performing bougienage. A total of 36.4% of respondents had never heard of the procedure and only 16.1% had ever performed it. Barriers to usage included lack of provider training (95.6%), perceived risk of complications (94.4%), and perceived lack of success (80.5%). CONCLUSIONS: Bougienage is safe and effective with significant LOS and cost benefits compared with endoscopy. Despite these advantages, the procedure is underused, because of lack of provider education and concerns regarding safety, efficacy, and both family and specialist preference, which are not supported by current literature. These data support the need for broader education regarding the bougienage technique, as well as larger prospective studies of its safety and outcomes.

Encouraging the use of the Veterans Crisis Line among high-risk Veterans: A randomized trial of a Crisis Line Facilitation intervention.

The Veterans Crisis Line (VCL) is a core component of VA's suicide prevention strategy. Despite the availability and utility of the VCL, many Veterans do not utilize this resource during times of crisis. A brief, psychoeducational behavioral intervention (termed Crisis Line Facilitation [CLF]) was developed to increase utilization of the VCL and reduce suicidal behaviors in high-risk Veterans. The therapist-led session includes educational information regarding the VCL, as well as a chance to discuss the participant's perceptions of contacting the VCL during periods of crisis. The final component of the session is a practice call placed to the VCL by both the therapist and the participant. The CLF intervention was compared to Enhanced Usual Care (EUC) during a multi-site randomized clinical trial for 307 Veteran participants recently hospitalized for a suicidal crisis who reported no contact with the VCL in the prior 12 months. Initial analyses indicated that participants randomized to the CLF intervention were less likely to report suicidal behaviors, including suicide attempts compared to participants randomized to receive EUC over 12-months of follow-up (χ2 = 18.48/p < 0.0001), however this effect was not sustained when analyses were conducted on an individual level. No significant differences were found between conditions on VCL utilization. Initial evidence suggests a brief CLF intervention has an impact on preventing suicidal behaviors in Veterans treated in inpatient mental health programs; however, it may not change use of the VCL. This brief intervention could be easily adapted into clinical settings to be delivered by standard clinical staff.

Pediatric Ovarian Torsion: Should You Go With the Flow?

OBJECTIVES: Ovarian torsion (OT) is an emergency that mandates early detection and surgical detorsion to avoid catastrophic consequences of further adnexal injury. Prompt ultrasound is critical for accurate diagnosis. Traditionally, evaluation of arterial and venous flow was used as a diagnostic tool for OT, but recent radiologic research has indicated that ovarian size and size discrepancy between sides is a better diagnostic criterion. This study seeks to determine whether ovarian size discrepancy or vascular flow to the ovary is more accurate in the diagnosis of OT in the pediatric emergency population and to better describe symptoms that distinguish OT from other abdominal and pelvic pathology. METHODS: This was a retrospective, cross-sectional study evaluating all female pediatric patients, aged 1 to 18 years, who underwent a pelvic ultrasound to evaluate for OT over a 2-year period in our pediatric emergency department. Patients suitable for inclusion were identified via Nuance mPowerTM, a search engine that provides clinical analytics based on radiology reports generated within our institution. RESULTS: We reviewed the medical records of 193 female patients aged 1 to 18 years, all of whom had a pelvic ultrasound (with or without Doppler) to evaluate for OT during the study period. In comparing ovarian size on ultrasound, patients with OT had a significantly larger magnitude of difference in ovarian volume than patients without torsion (5.57× [interquartile range, 3-12.5] vs 1.56× [interquartile range, 1.24-2.25; P < 0.001]). Ovarian torsion was associated with a 33-fold increased risk of lack of arterial flow (relative risk, 33.33) and with a 9-fold increased risk of lack of venous flow (relative risk, 9.27), when compared with those patients without OT. Patients with OT were significantly more likely to have emesis and peritoneal signs on examination, as well as previous history of OT (P = 0.01, 0.02, and 0.002, respectively) than those without OT. All patients with OT reported abdominal pain. CONCLUSIONS: We found that a large size discrepancy between ovaries is indicative of OT. Our data also suggest that presence of Doppler flow on ultrasound cannot be used to exclude OT but that lack of Doppler flow on ultrasound is a significant diagnostic marker. As previous studies have also found, clinical symptoms of OT are nonspecific and do not offer any certainty in differentiating OT from other pathologies.

Sticking It Straight: Pediatric Procedure Curriculum Initiative.

OBJECTIVES: Literature demonstrates that pediatric residents are not graduating with procedural confidence and competency. This was confirmed with our own institution's Accreditation Council for Graduate Medical Education and internal surveys. Our primary objective was to improve procedural confidence among pediatric residents with the introduction of a mandatory longitudinal pediatric procedural curriculum, including simulation in combination with online modules. METHODS: We performed a quality improvement intervention to increase resident comfort level performing Accreditation Council for Graduate Medical Education-required procedures. This study involved pediatric residents, postgraduation year (PGY) 1-3, at an academic, tertiary care hospital. Between April 2015 and June 2017, the combination of online self-directed learning modules and hands-on simulation curriculum was implemented for pediatric residents. Surveys were administered at 1-year intervals to assess self-reported comfort level on 12 procedures using a Likert scale (1 for "strongly disagree" to 5 for "strongly agree, maximum score of 60 for all procedures). RESULTS: Forty (63%) of 63 participant presurveys and 45 (71%) of 63 postsurveys were available for analysis. The mean comfort level for all procedures demonstrated a statistically significant increase from 32.4 to 37.1, or 12.7% (P = 0.005). By PGY level, the score increased from 24.4 to 30.9 (21%) for PGY1, 34.4 to 37.5 (8.3%) for PGY2, and 38.6 to 42.8 (9.8%) for PGY3 (P < 0.005). Overall, pediatric residents rated the simulation experience very favorably. CONCLUSIONS: A mandatory longitudinal procedure curriculum improved procedural comfort level among pediatric residents. Iterative curriculum designs found the most productive combination to be deliberate practice within mastery learning simulation sessions with required precourse online modules.

One Health Investigation of SARS-CoV-2 Infection and Seropositivity among Pets in Households with Confirmed Human COVID-19 Cases-Utah and Wisconsin, 2020.

Approximately 67% of U.S. households have pets. Limited data are available on SARS-CoV-2 in pets. We assessed SARS-CoV-2 infection in pets during a COVID-19 household transmission investigation. Pets from households with ≥1 person with laboratory-confirmed COVID-19 were eligible for inclusion from April-May 2020. We enrolled 37 dogs and 19 cats from 34 households. All oropharyngeal, nasal, and rectal swabs tested negative by rRT-PCR; one dog's fur swabs (2%) tested positive by rRT-PCR at the first sampling. Among 47 pets with serological results, eight (17%) pets (four dogs, four cats) from 6/30 (20%) households had detectable SARS-CoV-2 neutralizing antibodies. In households with a seropositive pet, the proportion of people with laboratory-confirmed COVID-19 was greater (median 79%; range: 40-100%) compared to households with no seropositive pet (median 37%; range: 13-100%) (p = 0.01). Thirty-three pets with serologic results had frequent daily contact (≥1 h) with the index patient before the person's COVID-19 diagnosis. Of these 33 pets, 14 (42%) had decreased contact with the index patient after diagnosis and none were seropositive; of the 19 (58%) pets with continued contact, four (21%) were seropositive. Seropositive pets likely acquired infection after contact with people with COVID-19. People with COVID-19 should restrict contact with pets and other animals.

Dazed and Confused: Altered Mental Status in an Adolescent Male.

BACKGROUND: Absence status epilepticus (ASE) is a form of generalized nonconvulsive status epilepticus. ASE is characterized by impairment in consciousness, which can vary widely, making the diagnosis more difficult. The typical patient with ASE will be confused yet responsive and in a "trance-like state" with delayed speech, clumsy gait, and the ability to perform simple tasks after prompting. With treatment, typical ASE has an excellent prognosis and does not appear to be associated with significant neuronal damage. CASE PRESENTATION: An 11-year-old boy with history of febrile seizures presented to the emergency department (ED) with altered mental status without trauma or ingestion. His vital signs and physical examination were normal, with the exception of appearing intoxicated with sparse verbalization and inappropriate emotional responses. All laboratory results and imaging were unremarkable. While in the ED, his neurologic examination trended toward normal, returning almost to baseline. He was admitted to the hospital for video electroencephalogram, which revealed status epilepticus. After benzodiazepine therapy, epileptic electrical activity ceased and the patient's symptoms resolved. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: ASE is a rare condition that is uncommonly described in the pediatric population. These patients are frequently misdiagnosed on initial presentation as their alteration in mental status can be easily confused with ingestion, trauma, psychiatric illness, or infectious etiologies. Overturning the long-standing emergency dogma of "if they're talking to you, it's not a seizure" is undoubtedly difficult, but both pediatric and adult providers should be aware of this clinical entity.

Genome-wide sequencing-based identification of methylation quantitative trait loci and their role in schizophrenia risk.

DNA methylation (DNAm) is an epigenetic regulator of gene expression and a hallmark of gene-environment interaction. Using whole-genome bisulfite sequencing, we have surveyed DNAm in 344 samples of human postmortem brain tissue from neurotypical subjects and individuals with schizophrenia. We identify genetic influence on local methylation levels throughout the genome, both at CpG sites and CpH sites, with 86% of SNPs and 55% of CpGs being part of methylation quantitative trait loci (meQTLs). These associations can further be clustered into regions that are differentially methylated by a given SNP, highlighting the genes and regions with which these loci are epigenetically associated. These findings can be used to better characterize schizophrenia GWAS-identified variants as epigenetic risk variants. Regions differentially methylated by schizophrenia risk-SNPs explain much of the heritability associated with risk loci, despite covering only a fraction of the genomic space. We provide a comprehensive, single base resolution view of association between genetic variation and genomic methylation, and implicate schizophrenia GWAS-associated variants as influencing the epigenetic plasticity of the brain.

ACEP SimBox: A Pediatric Simulation-Based Training Innovation.

Thirty million pediatric visits (<18 years old) occur across 5,000 US emergency departments (EDs) each year, with most of these cases presenting to community EDs. Simulation-based training is an effective method to improve and sustain EDs' readiness to triage and stabilize critically ill infants and children, but large simulation centers are mostly concentrated at academic hospitals. The use of pediatric simulation-based training has been limited in the community ED setting due to the high cost of equipment and limited access to content experts in pediatric critical care. We designed an innovative "off-the-shelf" simulation-based training resource, "American College of Emergency Physicians (ACEP) SimBox," that provides a free low-technology manikin along with teaching aids and train-the-trainer materials to community EDs to run a simulation drill in their own workspaces with local educators. The goal was to develop an "off-the-shelf," free, open-access, simulation-based resource to improve the readiness of community EDs to triage, resuscitate, and transfer critically ill infants as measured by presimulation and postsimulation surveys measuring opinions regarding the scenario, session experience, and most valuable aspect of the session. Between January 2018 and December 2019, 179 ACEP SimBoxes were shipped across the United States, reaching 36 of 50 states. Facilitators and participants who completed the postsimulation survey evaluated the session as a valuable use of their time. All facilitator respondents reported that the low-technology manikins, paired with their institution-specific equipment, were sufficient for learning, thus reducing costs. All participant respondents reported an increased commitment to pediatric readiness for their ED after completing the simulation session. This innovation resulted in the implementation of a unique simulation-based training intervention across many community EDs in the United States. The ACEP SimBox innovation demonstrates that an easy to use and unique simulation-based training tool can be developed, distributed, and implemented across many community EDs in the United States to help improve community ED pediatric readiness.

Point-of-Care Ultrasound Facilitates Management of Ruptured Ectopic Pregnancy.

ABSTRACT: Abdominal pain in the pregnant adolescent presents a diagnostic dilemma with potential life-threatening etiologies. We present a case where point-of-care ultrasound was used to facilitate diagnosis and expedite lifesaving management of a ruptured ectopic pregnancy. We further review the technique and literature for first-trimester transabdominal point-of-care ultrasound.

The Development and Pilot Testing of an Evidence-Based Cardiovascular Disease Risk-Prevention Program to Promote Healthy Lifestyle Behaviors Among African-American College Students - Rams Have HEART.

Objectives: Cardiovascular disease (CVD) is the leading cause of death in the United States. Among the risk factors for college students, obesity and physical inactivity are disproportionately high among African Americans (AAs), and while studies of the obesity epidemic have increased in recent years, few target AA college-aged students. This study developed and piloted an evidence-based, 15-week, 3-credit hour, CVD risk-prevention and intervention course, Rams Have HEART that used e-learning, web-based technologies, and a mobile application and compared its effects against a control course. Methods: Two cohorts were recruited in a two-year period; 124 AA college students voluntarily consented to participate in the study, with n = 63 representing the control group and n = 61 representing the intervention. CVD risk factors were assessed by examining blood markers and anthropometric measurements. Demographic, clinical, and survey data (physical measures, blood marker investigation, and self-report surveys) were collected at baseline, post-intervention, and follow-up over the academic year. Results: The mean blood markers for lipid panel and glucose results were within the established optimal range. Intake of fruits and vegetables increased along with knowledge of CVD risk factors; 86% of students enrolled in the intervention passed the course; 100% (n = 61) would recommend it to future students. Conclusion: Developing and offering a healthy lifestyle-behavior CVD intervention course to AA college students is feasible and effective in optimizing their awareness of chronic disease risk factors and prompting behavior change.

Characterizing the dynamic and functional DNA methylation landscape in the developing human cortex.

DNA methylation (DNAm) is a key epigenetic regulator of gene expression across development. The developing prenatal brain is a highly dynamic tissue, but our understanding of key drivers of epigenetic variability across development is limited. We, therefore, assessed genomic methylation at over 39 million sites in the prenatal cortex using whole-genome bisulfite sequencing and found loci and regions in which methylation levels are dynamic across development. We saw that DNAm at these loci was associated with nearby gene expression and enriched for enhancer chromatin states in prenatal brain tissue. Additionally, these loci were enriched for genes associated with neuropsychiatric disorders and genes involved with neurogenesis. We also found autosomal differences in DNAm between the sexes during prenatal development, though these have less clear functional consequences. We lastly confirmed that the dynamic methylation at this critical period is specifically CpG methylation, with generally low levels of CpH methylation. Our findings provide detailed insight into prenatal brain development as well as clues to the pathogenesis of psychiatric traits seen later in life.

Cortical cellular diversity and development in schizophrenia.

While a definitive understanding of schizophrenia etiology is far from current reality, an increasing body of evidence implicates perturbations in early development that alter the trajectory of brain maturation in this disorder, leading to abnormal function in early childhood and adulthood. This atypical development likely arises from an interaction of many brain cell types that follow distinct developmental paths. Because both cellular identity and development are governed by the transcriptome and epigenome, two levels of gene regulation that have the potential to reflect both genetic and environmental influences, mapping "omic" changes over development in diverse cells is a fruitful avenue for schizophrenia research. In this review, we provide a survey of human brain cellular composition and development, levels of genomic regulation that determine cellular identity and developmental trajectories, and what is known about how genomic regulation is dysregulated in specific cell types in schizophrenia. We also outline technical challenges and solutions to conducting cell type-specific functional genomic studies in human postmortem brain.

Rams Have Heart, a Mobile App Tracking Activity and Fruit and Vegetable Consumption to Support the Cardiovascular Health of College Students: Development and Usability Study.

BACKGROUND: With the increasing use of mobile devices to access the internet and as the main computing system of apps, there is a growing market for mobile health apps to provide self-care advice. Their effectiveness with regard to diet and fitness tracking, for example, needs to be examined. The majority of American adults fail to meet daily recommendations for healthy behavior. Testing user engagement with an app in a controlled environment can provide insight into what is effective and not effective in an app focused on improving diet and exercise. OBJECTIVE: We developed Rams Have Heart, a mobile app, to support a cardiovascular disease (CVD) intervention course. The app tracks healthy behaviors, including fruit and vegetable consumption and physical activity, throughout the day. This paper aimed to present its functionality and evaluated adherence among the African American college student population. METHODS: We developed the app using the Personal Health Informatics and Intervention Toolkit, a software framework. Rams Have Heart integrates self-reported health screening with health education, diary tracking, and user feedback modules to acquire data and assess progress. The parent study, conducted at a historically black college and university-designated institution in southeastern United States, consisted of a semester-long intervention administered as an academic course in the fall, for 3 consecutive years. Changes were made after the cohort 1 pilot study, so results only include cohorts 2 and 3, comprising a total of 115 students (n=55 intervention participants and n=54 control participants) aged from 17 to 24 years. Data collected over the study period were transferred using the secure Hypertext Transfer Protocol Secure protocol and stored in a secure Structured Query Language server database accessible only to authorized persons. SAS software was used to analyze the overall app usage and the specific results collected. RESULTS: Of the 55 students in the intervention group, 27 (49%) students in cohort 2 and 25 (45%) in cohort 3 used the Rams Have Heart app at least once. Over the course of the fall semester, app participation dropped off gradually until exam week when most students no longer participated. The average fruit and vegetable intake increased slightly, and activity levels decreased over the study period. CONCLUSIONS: Rams Have Heart was developed to allow daily tracking of fruit and vegetable intake and physical activity to support a CVD risk intervention for a student demographic susceptible to obesity, heart disease, and type 2 diabetes. We conducted an analysis of app usage, function, and user results. Although a mobile app provides privacy and flexibility for user participation in a research study, Rams Have Heart did not improve compliance or user outcomes. Health-oriented research studies relying on apps in support of user goals need further evaluation.

Efficacy of a Psychosocial Pain Management Intervention for Men and Women With Substance Use Disorders and Chronic Pain: A Randomized Clinical Trial.

Importance: Chronic pain is common in those with substance use disorders (SUDs) and predicts poorer addiction treatment outcomes. A critical challenge for addiction treatment is to develop effective methods to improve pain-related and substance use-related outcomes for those in treatment for SUDs. Objective: To examine the efficacy of an integrated behavioral pain management intervention (Improving Pain During Addiction Treatment [ImPAT]) for men and women with SUDs to treat pain, functioning, and substance use. Design, Setting, and Participants: In this randomized clinical trial, 8 sessions of ImPAT were compared with 8 sessions of a supportive psychoeducational control (SPC) condition for adults with pain treated at a large residential SUD treatment program. Follow-up occurred at 3, 6, and 12 months postbaseline. A total of 1372 adults were screened, including 960 men and 412 women, and 510 adults were randomized, including 264 men and 246 women. The goal was to recruit approximately equal numbers of men and women to examine results separately in men and women. A total of 470 of 510 participants (92.2%) completed at least 1 follow-up assessment. Data were collected from October 3, 2011, to January 14, 2016. Data were analyzed from February 1, 2016, to May 1, 2020. Interventions: ImPAT focused on how a psychosocial model of pain was associated with functioning and relapse prevention and provides skills to manage pain. SPC served as the active control condition and involved discussions of topics like nutrition and the course of addiction, which were intended to be relevant to the patient population and to have face validity but be distinct from the content of ImPAT. Main Outcomes and Measurements: The primary outcomes were pain intensity, pain-related functioning, and behavioral pain tolerance at 12 months. Secondary outcomes were frequency of alcohol and drug use over 12 months. Results: Of the 510 included participants, the mean (SD) age was 34.8 (10.3) years. A total of 133 men and 122 women were assigned to ImPAT, and a total of 131 men and 124 women were assigned to the SPC condition. Over 12 months of follow-up, randomization to the ImPAT intervention was associated with higher tolerance of pain among men, higher by a mean score of 0.11 (95% CI, 0.03 to 0.18; P = .004; Cohen d = 0.40) at 3 months and by 0.07 (95% CI, -0.01 to 0.19; P = .11; Cohen d = 0.25) at 12 months. Women receiving the ImPAT intervention experienced a reduction in pain intensity from 3 to 12 months, while women receiving the SPC condition experienced an increase in pain intensity, resulting in lower pain in the ImPAT condition by a mean score of 0.58 (95% CI, -0.07 to 1.22; P = .08; Cohen d = -0.22) at 12 months. No differences were found between the ImPAT and SPC conditions on alcohol or drug use. Conclusions and Relevance: Behavioral pain management is not typically included in addiction treatment, but the present results indicate that this type of intervention was associated with better pain-related outcomes, including pain tolerance in men and pain intensity in women. Improvements in substance use-related outcomes beyond that achieved by treatment as usual were not observed. Treatment programs should consider providing psychosocial pain management services to augment standard addiction treatment. Trial Registration: ClinicalTrials.gov Identifier: NCT01372267.

Dissecting transcriptomic signatures of neuronal differentiation and maturation using iPSCs.

Human induced pluripotent stem cells (hiPSCs) are a powerful model of neural differentiation and maturation. We present a hiPSC transcriptomics resource on corticogenesis from 5 iPSC donor and 13 subclonal lines across 9 time points over 5 broad conditions: self-renewal, early neuronal differentiation, neural precursor cells (NPCs), assembled rosettes, and differentiated neuronal cells. We identify widespread changes in the expression of both individual features and global patterns of transcription. We next demonstrate that co-culturing human NPCs with rodent astrocytes results in mutually synergistic maturation, and that cell type-specific expression data can be extracted using only sequencing read alignments without cell sorting. We lastly adapt a previously generated RNA deconvolution approach to single-cell expression data to estimate the relative neuronal maturity of iPSC-derived neuronal cultures and human brain tissue. Using many public datasets, we demonstrate neuronal cultures are maturationally heterogeneous but contain subsets of neurons more mature than previously observed.

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

Characterizing the nuclear and cytoplasmic transcriptomes in developing and mature human cortex uncovers new insight into psychiatric disease gene regulation.

Transcriptome compartmentalization by the nuclear membrane provides both stochastic and functional buffering of transcript activity in the cytoplasm, and has recently been implicated in neurodegenerative disease processes. Although many mechanisms regulating transcript compartmentalization are also prevalent in brain development, the extent to which subcellular localization differs as the brain matures has yet to be addressed. To characterize the nuclear and cytoplasmic transcriptomes during brain development, we sequenced both RNA fractions from homogenate prenatal and adult human postmortem cortex using poly(A)+ and Ribo-Zero library preparation methods. We find that while many genes are differentially expressed by fraction and developmental expression changes are similarly detectable in nuclear and cytoplasmic RNA, the compartmented transcriptomes become more distinct as the brain matures, perhaps reflecting increased utilization of nuclear retention as a regulatory strategy in adult brain. We examined potential mechanisms of this developmental divergence including alternative splicing, RNA editing, nuclear pore composition, RNA-binding protein motif enrichment, and RNA secondary structure. Intron retention is associated with greater nuclear abundance in a subset of transcripts, as is enrichment for several splicing factor binding motifs. Finally, we examined disease association with fraction-regulated gene sets and found nuclear-enriched genes were also preferentially enriched in gene sets associated with neurodevelopmental psychiatric disorders. These results suggest that although gene-level expression is globally comparable between fractions, nuclear retention of transcripts may play an underappreciated role in developmental regulation of gene expression in brain, particularly in genes whose dysregulation is related to neuropsychiatric disorders.

Divergent neuronal DNA methylation patterns across human cortical development reveal critical periods and a unique role of CpH methylation.

BACKGROUND: DNA methylation (DNAm) is a critical regulator of both development and cellular identity and shows unique patterns in neurons. To better characterize maturational changes in DNAm patterns in these cells, we profile the DNAm landscape at single-base resolution across the first two decades of human neocortical development in NeuN+ neurons using whole-genome bisulfite sequencing and compare them to non-neurons (primarily glia) and prenatal homogenate cortex. RESULTS: We show that DNAm changes more dramatically during the first 5 years of postnatal life than during the entire remaining period. We further refine global patterns of increasingly divergent neuronal CpG and CpH methylation (mCpG and mCpH) into six developmental trajectories and find that in contrast to genome-wide patterns, neighboring mCpG and mCpH levels within these regions are highly correlated. We integrate paired RNA-seq data and identify putative regulation of hundreds of transcripts and their splicing events exclusively by mCpH levels, independently from mCpG levels, across this period. We finally explore the relationship between DNAm patterns and development of brain-related phenotypes and find enriched heritability for many phenotypes within identified DNAm features. CONCLUSIONS: By profiling DNAm changes in NeuN-sorted neurons over the span of human cortical development, we identify novel, dynamic regions of DNAm that would be masked in homogenate DNAm data; expand on the relationship between CpG methylation, CpH methylation, and gene expression; and find enrichment particularly for neuropsychiatric diseases in genomic regions with cell type-specific, developmentally dynamic DNAm patterns.