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.

High-field MRI using standard pulse sequences has moderate to substantial interobserver agreement and good accuracy for differentiation between intracranial extra-axial histiocytic sarcoma and meningioma in dogs.

Intracranial extra-axial histiocytic sarcoma shares common MRI features with meningioma. As histiocytic sarcoma carries a generally worse prognosis than meningioma, the ability to differentiate between these two neoplasms is of clinical value. The aim of this retrospective diagnostic accuracy and observer agreement study was to evaluate the accuracy and reliability of high-field MRI to differentiate between these two tumors, using standard pulse sequences and published MRI features. A total of 51 dogs were included (26 meningiomas and 25 histiocytic sarcomas). Magnetic resonance imaging examinations were independently assessed by three experienced board-certified radiologists, evaluating 18 imaging features. They were asked to assign each case to one of three categories (meningioma, histiocytic sarcoma, and undetermined). Agreement for the MRI diagnosis across all three reviewers was moderate (κ 0.54) while paired interobserver agreement ranged from moderate to substantial (κ 0.58-0.74) with percent agreement ranging between 86.1% and 87.7%. Overall, the probability of correctly diagnosing meningioma in a dog with this tumor ranged between 79.2% and 94.4%, and the probability of correctly diagnosing histiocytic sarcoma in a dog with this tumor ranged between 76.0% and 92.3%. The overall probability to diagnose the correct tumor, irrespective of type, ranged between 79.2% and 89.7%. Histiocytic sarcomas tended to have more extensive edema and more often had combined perilesional and distant meningeal enhancement affecting both pachy- and leptomeninges, while for meningiomas, meningeal enhancement tended to more commonly be perilesional and pachymeningeal. Imaging features that seemed more useful to make a correct diagnosis included "location/type of meningeal enhancement," "osseous changes in the adjacent neurocranium," "cystic changes," and "herniation severity."

SPEAQeasy: a scalable pipeline for expression analysis and quantification for R/bioconductor-powered RNA-seq analyses.

BACKGROUND: RNA sequencing (RNA-seq) is a common and widespread biological assay, and an increasing amount of data is generated with it. In practice, there are a large number of individual steps a researcher must perform before raw RNA-seq reads yield directly valuable information, such as differential gene expression data. Existing software tools are typically specialized, only performing one step-such as alignment of reads to a reference genome-of a larger workflow. The demand for a more comprehensive and reproducible workflow has led to the production of a number of publicly available RNA-seq pipelines. However, we have found that most require computational expertise to set up or share among several users, are not actively maintained, or lack features we have found to be important in our own analyses. RESULTS: In response to these concerns, we have developed a Scalable Pipeline for Expression Analysis and Quantification (SPEAQeasy), which is easy to install and share, and provides a bridge towards R/Bioconductor downstream analysis solutions. SPEAQeasy is portable across computational frameworks (SGE, SLURM, local, docker integration) and different configuration files are provided ( http://research.libd.org/SPEAQeasy/ ). CONCLUSIONS: SPEAQeasy is user-friendly and lowers the computational-domain entry barrier for biologists and clinicians to RNA-seq data processing as the main input file is a table with sample names and their corresponding FASTQ files. The goal is to provide a flexible pipeline that is immediately usable by researchers, regardless of their technical background or computing environment.

Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods.

BACKGROUND: RNA sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of these biases have focused on adapter ligation bias with limited evaluation of reverse transcription bias or amplification bias. Furthermore, evaluations of the quantification of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited. No study had yet evaluated the quantification of isomiRs of altered length or compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. RESULTS: All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. CONCLUSIONS: Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

A genome-scale DNA methylation study in women with interstitial cystitis/bladder pain syndrome.

AIMS: To assess the feasibility of using voided urine samples to perform a DNA methylation study in females with interstitial cystitis/bladder pain syndrome (IC/BPS) as compared to age- and race-matched controls. A unique methylation profile could lead to a non-invasive, reproducible, and objective biomarker that would aid clinicians in the diagnosis of IC/BPS. METHODS: Nineteen IC/BPS patients and 17 controls were included. IC/BPS patients had an Interstitial Cystitis Symptom Index score of >8; controls had no bladder symptoms. DNA was extracted from pelleted urine sediment. Samples with >500 ng of genomic DNA underwent quantitative DNA methylation assessment using the Illumina Infinium MethylationEPIC BeadChip. Age- and race-matching was applied prior to analysis. Linear regression models were used to compare average methylation between IC/BPS cases and controls at each cytosine guanine dinucleotide site (loci where methylation can occur). RESULTS: Sixteen participants (eight IC/BPS age- and race-matched to eight controls) had adequate DNA for methylation analysis. The median age was 43.5 years (interquartile range 33.8, 65.0), the median BMI was 27.1 (IQR 22.7, 31.4), and 14 were Caucasian (87.5%). A total of 688 417 CpG sites were analyzed. In exploratory pathway analysis utilizing the top 1000 differentially methylated CpG sites, the mitogen-activated protein kinase (MAPK) pathway was overrepresented by member genes. CONCLUSIONS: The results demonstrate the feasibility of using voided urine specimens from women with IC/BPS to perform DNA methylation assessments. Additionally, the data suggest genes within or downstream of the MAPK pathway exhibit altered methylation in IC/BPS.

Establishing Benchmarks For Minimum Competence With Dry Lab Robotic Surgery Drills.

The Robotic Objective Structured Assessment of Technical Skills (R-OSATS) is a previously validated assessment tool that is used to assess 5 standardized inanimate robotic surgery drills. R-OSATS is used to evaluate performance on surgical drills, with scores of 0 to 20 for each drill. Our objective was to establish the minimum threshold score that denotes competence on these drills. Thus, we performed a standard setting study using data from surgeons and trainees in 8 academic medical centers. Cutoff scores for the minimal level of competence using R-OSATS were established using 2 techniques: the modified Angoff and the contrasting groups methods. For the modified Angoff method, 8 content experts met and, in an iterative process, derived the scores that a minimally competent trainee should receive. After 2 iterative rounds of scoring and discussion with the modified Angoff method, we established a minimum competence score per drill with high agreement (rWG range, 0.92-0.98). There was unanimous consensus that a trainee needs to achieve competence on each independent drill. A second method, the contrasting groups method, was used to verify our results. In this method, we compared R-OSATS scores from "inexperienced" (34 postgraduate year 1 and 2 trainees) with "experienced" (22 faculty and fellow) robotic surgeons. The distributions of scores from both groups were plotted, and a cutoff score for each drill was determined from the intersection of the 2 curves. Using this method, the minimum score for competence would be 14 per drill, which is slightly more stringent but confirms the results obtained from the modified Angoff approach. In conclusion, using 2 well-described standard setting techniques, we have established minimum benchmarks designating trainee competence for 5 dry lab robotic surgery drills.

Amygdala and anterior cingulate transcriptomes from individuals with bipolar disorder reveal downregulated neuroimmune and synaptic pathways.

Recent genetic studies have identified variants associated with bipolar disorder (BD), but it remains unclear how brain gene expression is altered in BD and how genetic risk for BD may contribute to these alterations. Here, we obtained transcriptomes from subgenual anterior cingulate cortex and amygdala samples from post-mortem brains of individuals with BD and neurotypical controls, including 511 total samples from 295 unique donors. We examined differential gene expression between cases and controls and the transcriptional effects of BD-associated genetic variants. We found two coexpressed modules that were associated with transcriptional changes in BD: one enriched for immune and inflammatory genes and the other with genes related to the postsynaptic membrane. Over 50% of BD genome-wide significant loci contained significant expression quantitative trait loci (QTL) (eQTL), and these data converged on several individual genes, including SCN2A and GRIN2A. Thus, these data implicate specific genes and pathways that may contribute to the pathology of BP.

Analysis of the caudate nucleus transcriptome in individuals with schizophrenia highlights effects of antipsychotics and new risk genes.

Most studies of gene expression in the brains of individuals with schizophrenia have focused on cortical regions, but subcortical nuclei such as the striatum are prominently implicated in the disease, and current antipsychotic drugs target the striatum's dense dopaminergic innervation. Here, we performed a comprehensive analysis of the genetic and transcriptional landscape of schizophrenia in the postmortem caudate nucleus of the striatum of 443 individuals (245 neurotypical individuals, 154 individuals with schizophrenia and 44 individuals with bipolar disorder), 210 from African and 233 from European ancestries. Integrating expression quantitative trait loci analysis, Mendelian randomization with the latest schizophrenia genome-wide association study, transcriptome-wide association study and differential expression analysis, we identified many genes associated with schizophrenia risk, including potentially the dopamine D2 receptor short isoform. We found that antipsychotic medication has an extensive influence on caudate gene expression. We constructed caudate nucleus gene expression networks that highlight interactions involving schizophrenia risk. These analyses provide a resource for the study of schizophrenia and insights into risk mechanisms and potential therapeutic targets.

Developmental Profile of Psychiatric Risk Associated With Voltage-Gated Cation Channel Activity.

BACKGROUND: Recent breakthroughs in psychiatric genetics have implicated biological pathways onto which genetic risk for psychiatric disorders converges. However, these studies do not reveal the developmental time point(s) at which these pathways are relevant. METHODS: We aimed to determine the relationship between psychiatric risk and developmental gene expression relating to discrete biological pathways. We used postmortem RNA sequencing data (BrainSeq and BrainSpan) from brain tissue at multiple prenatal and postnatal time points, with summary statistics from recent genome-wide association studies of schizophrenia, bipolar disorder, and major depressive disorder. We prioritized gene sets for overall enrichment of association with each disorder and then tested the relationship between the association of their constituent genes with their relative expression at each developmental stage. RESULTS: We observed relationships between the expression of genes involved in voltage-gated cation channel activity during early midfetal, adolescence, and early adulthood time points and association with schizophrenia and bipolar disorder, such that genes more strongly associated with these disorders had relatively low expression during early midfetal development and higher expression during adolescence and early adulthood. The relationship with schizophrenia was strongest for the subset of genes related to calcium channel activity, while for bipolar disorder, the relationship was distributed between calcium and potassium channel activity genes. CONCLUSIONS: Our results indicate periods during development when biological pathways related to the activity of calcium and potassium channels may be most vulnerable to the effects of genetic variants conferring risk for psychiatric disorders. Furthermore, they indicate key time points and potential targets for disorder-specific therapeutic interventions.

T1ρ, T2 mapping, and EPIC-µCT Imaging in a Canine Model of Knee Osteochondral Injury.

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020.

Profiling gene expression in the human dentate gyrus granule cell layer reveals insights into schizophrenia and its genetic risk.

Specific cell populations may have unique contributions to schizophrenia but may be missed in studies of homogenate tissue. Here laser capture microdissection followed by RNA sequencing (LCM-seq) was used to transcriptomically profile the granule cell layer of the dentate gyrus (DG-GCL) in human hippocampus and contrast these data to those obtained from bulk hippocampal homogenate. We identified widespread cell-type-enriched aging and genetic effects in the DG-GCL that were either absent or directionally discordant in bulk hippocampus data. Of the ~9 million expression quantitative trait loci identified in the DG-GCL, 15% were not detected in bulk hippocampus, including 15 schizophrenia risk variants. We created transcriptome-wide association study genetic weights from the DG-GCL, which identified many schizophrenia-associated genetic signals not found in transcriptome-wide association studies from bulk hippocampus, including GRM3 and CACNA1C. These results highlight the improved biological resolution provided by targeted sampling strategies like LCM and complement homogenate and single-nucleus approaches in human brain.

A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndrome models and human autism spectrum disorder.

Autism spectrum disorder (ASD) is genetically heterogeneous with convergent symptomatology, suggesting common dysregulated pathways. In this study, we analyzed brain transcriptional changes in five mouse models of Pitt-Hopkins syndrome (PTHS), a syndromic form of ASD caused by mutations in the TCF4 gene, but not the TCF7L2 gene. Analyses of differentially expressed genes (DEGs) highlighted oligodendrocyte (OL) dysregulation, which we confirmed in two additional mouse models of syndromic ASD (Ptenm3m4/m3m4 and Mecp2tm1.1Bird). The PTHS mouse models showed cell-autonomous reductions in OL numbers and myelination, functionally confirming OL transcriptional signatures. We also integrated PTHS mouse model DEGs with human idiopathic ASD postmortem brain RNA-sequencing data and found significant enrichment of overlapping DEGs and common myelination-associated pathways. Notably, DEGs from syndromic ASD mouse models and reduced deconvoluted OL numbers distinguished human idiopathic ASD cases from controls across three postmortem brain data sets. These results implicate disruptions in OL biology as a cellular mechanism in ASD pathology.

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.

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.

Regional Heterogeneity in Gene Expression, Regulation, and Coherence in the Frontal Cortex and Hippocampus across Development and Schizophrenia.

The hippocampus formation, although prominently implicated in schizophrenia pathogenesis, has been overlooked in large-scale genomics efforts in the schizophrenic brain. We performed RNA-seq in hippocampi and dorsolateral prefrontal cortices (DLPFCs) from 551 individuals (286 with schizophrenia). We identified substantial regional differences in gene expression and found widespread developmental differences that were independent of cellular composition. We identified 48 and 245 differentially expressed genes (DEGs) associated with schizophrenia within the hippocampus and DLPFC, with little overlap between the brain regions. 124 of 163 (76.6%) of schizophrenia GWAS risk loci contained eQTLs in any region. Transcriptome-wide association studies in each region identified many novel schizophrenia risk features that were brain region-specific. Last, we identified potential molecular correlates of in vivo evidence of altered prefrontal-hippocampal functional coherence in schizophrenia. These results underscore the complexity and regional heterogeneity of the transcriptional correlates of schizophrenia and offer new insights into potentially causative biology.

The effect of platelet-rich plasma on osseous healing in dogs undergoing high tibial osteotomy.

OBJECTIVES: The purpose of this study was to investigate whether platelet-rich plasma (PRP) enhances osseous healing in conjunction with a high tibial osteotomy in dogs. STUDY DESIGN: Randomized controlled trial. METHODS: Sixty-four client-owned pet dogs with naturally occurring rupture of the anterior cruciate ligament and that were to be treated with a high tibial osteotomy (tibial plateau leveling osteotomy) were randomized into the treatment or control group. Dogs in the treatment group received autologous platelet-rich plasma activated with calcium chloride and bovine thrombin to produce a well-formed PRP gel that was placed into the osteotomy at the time of surgery. Dogs in the control group received saline lavage of the osteotomy. All dogs had the osteotomy stabilized with identical titanium alloy implants and all aspects of the surgical procedure and post-operative care were identical among dogs of the two groups. Bone healing was assessed at exactly 28, 49, and 70 days after surgery with radiography and ultrasonography and with MRI at day 28. The effect of PRP on bone healing was assessed using a repeated measures analysis of covariance with radiographic and ultrasonographic data and using a t-test with the MRI data. RESULTS: Sixty dogs completed the study. There were no significant differences in age, weight, or gender distribution between the treatment and control groups. Twenty-seven dogs were treated with PRP and 33 were in the control group. The average platelet concentration of the PRP was 1.37x106 platelets/µL (±489x103) with a leukocyte concentration of 5.45x103/µL (±3.5x103). All dogs demonstrated progressive healing over time and achieved clinically successful outcomes. Time since surgery and patient age were significant predictors of radiographic healing and time since surgery was a significant predictor of ultrasonographic assessment of healing. There was no significant effect of PRP treatment as assessed radiographically, ultrasonographically, or with MRI. CONCLUSION: The PRP used in this study did not hasten osseous union in dogs treated with a high tibial osteotomy.

Utility of MRI for Characterizing Articular Cartilage Pathology in Dogs with Medial Coronoid Process Disease.

OBJECTIVE: To determine whether assessment of morphological MRI sequences or delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) would have strong correlations with arthroscopic assessment of cartilage pathology in dogs with naturally occurring medial compartment pathology of the elbow. METHODS: Dogs tentatively diagnosed with medial coronoid disease had evaluation of their affected elbows using radiography, morphological MRI sequences, and dGEMRIC MRI evaluation prior to arthroscopy. Elbow radiographs were graded 0-6 for severity of changes. Cartilage of the medial coronoid process (MCP) and humeral trochlea (HT) were scored on a 0-3 scale using anatomical MRI sequences. The T1 relaxation times for the MCP and trochlea were quantified using dGEMRIC. Cartilage pathology was graded arthroscopically using a modified Outerbridge score (MOS) by a surgeon blinded to MRI assessment. Correlations between radiography and MOS, and between MRI and MOS, were quantified. RESULTS: Twenty-six elbows in 14 dogs were evaluated. There were statistically significant (p < 0.05) moderate correlations between radiographic scores and MOS for the MCP (r = 0.71) and HT (0.57). There was a statistically significant moderate correlation between morphological MRI scoring and MOS for the HT (r = 0.54; p < 0.05), but not for the MCP (p > 0.05). There was a weak, but significant correlation, between the dGEMRIC value and MOS of the MCP (r = 0.41; p < 0.05), but no correlation between the dGEMRIC values and MOS for the HT (p > 0.05). CLINICAL RELEVANCE: Statistically significant correlations to MOS were identified for both radiography and MRI but neither diagnostic modality provided sufficiently strong correlations to serve as a substitute for arthroscopic evaluation of the articular cartilage.