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1.
Clin Chem ; 69(7): 724-733, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37228223

RESUMO

BACKGROUND: Cannabis is increasingly used both medically and recreationally. With widespread use, there is growing concern about how to identify cannabis-impaired drivers. METHODS: A placebo-controlled randomized double-blinded protocol was conducted to study the effects of cannabis on driving performance. One hundred ninety-one participants were randomized to smoke ad libitum a cannabis cigarette containing placebo or delta-9-tetrahydrocannabinol (THC) (5.9% or 13.4%). Blood, oral fluid (OF), and breath samples were collected along with longitudinal driving performance on a simulator (standard deviation of lateral position [SDLP] and car following [coherence]) over a 5-hour period. Law enforcement officers performed field sobriety tests (FSTs) to determine if participants were impaired. RESULTS: There was no relationship between THC concentrations measured in blood, OF, or breath and SDLP or coherence at any of the timepoints studied (P > 0.05). FSTs were significant (P < 0.05) for classifying participants into the THC group vs the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired at this same timepoint. Combining a 2 ng/mL THC cutoff in OF with positive findings on FSTs reduced the number of controls classified as impaired to zero, 86 minutes after smoking the placebo. CONCLUSIONS: Requiring a positive toxicology result in addition to the FST observations substantially improved the classification accuracy regarding possible driving under the influence of THC by decreasing the percentage of controls classified as impaired.


Assuntos
Condução de Veículo , Cannabis , Dirigir sob a Influência , Alucinógenos , Fumar Maconha , Humanos , Dronabinol , Agonistas de Receptores de Canabinoides
2.
Nucleic Acids Res ; 46(9): e54, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29514223

RESUMO

Publicly available genomic data are a valuable resource for studying normal human variation and disease, but these data are often not well labeled or annotated. The lack of phenotype information for public genomic data severely limits their utility for addressing targeted biological questions. We develop an in silico phenotyping approach for predicting critical missing annotation directly from genomic measurements using well-annotated genomic and phenotypic data produced by consortia like TCGA and GTEx as training data. We apply in silico phenotyping to a set of 70 000 RNA-seq samples we recently processed on a common pipeline as part of the recount2 project. We use gene expression data to build and evaluate predictors for both biological phenotypes (sex, tissue, sample source) and experimental conditions (sequencing strategy). We demonstrate how these predictions can be used to study cross-sample properties of public genomic data, select genomic projects with specific characteristics, and perform downstream analyses using predicted phenotypes. The methods to perform phenotype prediction are available in the phenopredict R package and the predictions for recount2 are available from the recount R package. With data and phenotype information available for 70,000 human samples, expression data is available for use on a scale that was not previously feasible.


Assuntos
Perfilação da Expressão Gênica , Fenótipo , Análise de Sequência de RNA , Simulação por Computador , Feminino , Humanos , Masculino , Software
3.
Proteomics ; 19(15): e1800315, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30983154

RESUMO

Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry, and computational methods allow us to carry out multiomics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, next-generation sequencing and high-resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs is employed. Analysis of 145 million paired-end reads from next-generation sequencing confirmed expression of 12 186 protein-coding genes (FPKM ≥0.1), 439 novel long non-coding RNAs, and revealed 6089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identifies 6477 proteins including confirmation of N-termini for 1091 proteins, isoforms for 149 proteins, and 1034 phosphosites. A database search to specifically identify other post-translational modifications provide evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, and mono-, di- and tri-methylation events. Evidence for 11 "missing proteins," which are proteins for which there was insufficient or no protein level evidence, is provided. Peptides supporting missing protein and novel events are validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing are identified. Overall, it is believed that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.


Assuntos
Proteômica/métodos , Transcriptoma/genética , Processamento Alternativo/genética , Células Endoteliais da Veia Umbilical Humana , Humanos
4.
BMC Genomics ; 14: 892, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24341889

RESUMO

BACKGROUND: RNA-Sequencing (RNA-Seq) experiments have been optimized for library preparation, mapping, and gene expression estimation. These methods, however, have revealed weaknesses in the next stages of analysis of differential expression, with results sensitive to systematic sample stratification or, in more extreme cases, to outliers. Further, a method to assess normalization and adjustment measures imposed on the data is lacking. RESULTS: To address these issues, we utilize previously published eQTLs as a novel gold standard at the center of a framework that integrates DNA genotypes and RNA-Seq data to optimize analysis and aid in the understanding of genetic variation and gene expression. After detecting sample contamination and sequencing outliers in RNA-Seq data, a set of previously published brain eQTLs was used to determine if sample outlier removal was appropriate. Improved replication of known eQTLs supported removal of these samples in downstream analyses. eQTL replication was further employed to assess normalization methods, covariate inclusion, and gene annotation. This method was validated in an independent RNA-Seq blood data set from the GTEx project and a tissue-appropriate set of eQTLs. eQTL replication in both data sets highlights the necessity of accounting for unknown covariates in RNA-Seq data analysis. CONCLUSION: As each RNA-Seq experiment is unique with its own experiment-specific limitations, we offer an easily-implementable method that uses the replication of known eQTLs to guide each step in one's data analysis pipeline. In the two data sets presented herein, we highlight not only the necessity of careful outlier detection but also the need to account for unknown covariates in RNA-Seq experiments.


Assuntos
Locos de Características Quantitativas , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/normas , Sangue , Encéfalo , Mapeamento Cromossômico , Técnicas de Genotipagem , Humanos , Padrões de Referência , Reprodutibilidade dos Testes
5.
Fluids Barriers CNS ; 16(1): 20, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31303172

RESUMO

BACKGROUND: Blood-brain barrier dysfunction is associated with many late-stage neurodegenerative diseases. An emerging question is whether the mutations associated with neurodegenerative diseases can independently lead to blood-brain barrier (BBB) dysfunction. Studies from patient-derived induced pluripotent stem cells suggest that mutations associated with neurodegenerative disease are non-cell autonomous, resulting in gain of toxic function in derived neurons and astrocytes. Here we assess whether selected mutations associated with neurodegenerative diseases can contribute to impairment of the blood-brain barrier. METHODS: We assessed barrier function of confluent monolayers of human brain microvascular endothelial cells (hBMECs) derived from induced pluripotent stem cells (iPSC) from three healthy individuals and eight individuals with neurodegenerative disease. We systematically assessed protein and gene expression of BBB biomarkers, transendothelial resistance (TEER), permeability of Lucifer yellow, permeability of D-glucose, permeability of rhodamine 123, the efflux ratio of rhodamine 123, and P-gp inhibition using Tariquidar for confluent monolayers of human brain microvascular endothelial cell (hBMECs). RESULTS: We provide evidence supporting the hypothesis that mutations associated with neurodegenerative disease can independently cause BBB dysfunction. These functional changes are not catastrophic since barrier breakdown would result in BBB impairment during development. Synergistic interactions between non-cell autonomous cerebrovascular dysfunction and the effects of gain-of-toxic function in neurons (e.g. toxic oligomers) are likely to increase disease burden through a positive feedback mechanism. CONCLUSIONS: These results suggest that the accumulation of defects in brain microvascular endothelial cells may ultimately lead to impairment of the BBB. Small changes in barrier function over time could lead to accumulated defects that result in positive feedback to unrelated central nervous system diseases.


Assuntos
Barreira Hematoencefálica/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação/fisiologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Adulto , Idoso , Barreira Hematoencefálica/patologia , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia
6.
Am Stat ; 72(1): 53-57, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-32981941

RESUMO

Within the statistics community, a number of guiding principles for sharing data have emerged; however, these principles are not always made clear to collaborators generating the data. To bridge this divide, we have established a set of guidelines for sharing data. In these, we highlight the need to provide raw data to the statistician, the importance of consistent formatting, and the necessity of including all essential experimental information and pre-processing steps carried out to the statistician. With these guidelines we hope to avoid errors and delays in data analysis.

7.
Mol Autism ; 8: 6, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28316770

RESUMO

BACKGROUND: The etiology of autism, a complex, heritable, neurodevelopmental disorder, remains largely unexplained. Given the unexplained risk and recent evidence supporting a role for epigenetic mechanisms in the development of autism, we explored the role of CpG and CpH (H = A, C, or T) methylation within the autism-affected cortical brain tissue. METHODS: Reduced representation bisulfite sequencing (RRBS) was completed, and analysis was carried out in 63 post-mortem cortical brain samples (Brodmann area 19) from 29 autism-affected and 34 control individuals. Analyses to identify single sites that were differentially methylated and to identify any global methylation alterations at either CpG or CpH sites throughout the genome were carried out. RESULTS: We report that while no individual site or region of methylation was significantly associated with autism after multi-test correction, methylated CpH dinucleotides were markedly enriched in autism-affected brains (~2-fold enrichment at p < 0.05 cutoff, p = 0.002). CONCLUSIONS: These results further implicate epigenetic alterations in pathobiological mechanisms that underlie autism.


Assuntos
Transtorno Autístico/genética , Metilação de DNA , Análise de Sequência de DNA/métodos , Transtorno Autístico/patologia , Autopsia , Composição de Bases , Química Encefálica , Epigênese Genética , Humanos , Masculino
8.
Nat Commun ; 8(1): 1011, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29066808

RESUMO

Integration of emerging epigenetic information with autism spectrum disorder (ASD) genetic results may elucidate functional insights not possible via either type of information in isolation. Here we use the genotype and DNA methylation (DNAm) data from cord blood and peripheral blood to identify SNPs associated with DNA methylation (meQTL lists). Additionally, we use publicly available fetal brain and lung meQTL lists to assess enrichment of ASD GWAS results for tissue-specific meQTLs. ASD-associated SNPs are enriched for fetal brain (OR = 3.55; P < 0.001) and peripheral blood meQTLs (OR = 1.58; P < 0.001). The CpG targets of ASD meQTLs across cord, blood, and brain tissues are enriched for immune-related pathways, consistent with other expression and DNAm results in ASD, and reveal pathways not implicated by genetic findings. This joint analysis of genotype and DNAm demonstrates the potential of both brain and blood-based DNAm for insights into ASD and psychiatric phenotypes more broadly.


Assuntos
Transtorno do Espectro Autista/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética , Transtorno do Espectro Autista/sangue , Encéfalo/embriologia , Encéfalo/metabolismo , Estudos de Casos e Controles , Pré-Escolar , Epigenômica/métodos , Sangue Fetal/metabolismo , Seguimentos , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Lactente , Recém-Nascido , Pulmão/embriologia , Pulmão/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Cordão Umbilical/metabolismo
9.
Nat Commun ; 5: 5748, 2014 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25494366

RESUMO

Recent studies of genomic variation associated with autism have suggested the existence of extreme heterogeneity. Large-scale transcriptomics should complement these results to identify core molecular pathways underlying autism. Here we report results from a large-scale RNA sequencing effort, utilizing region-matched autism and control brains to identify neuronal and microglial genes robustly dysregulated in autism cortical brain. Remarkably, we note that a gene expression module corresponding to M2-activation states in microglia is negatively correlated with a differentially expressed neuronal module, implicating dysregulated microglial responses in concert with altered neuronal activity-dependent genes in autism brains. These observations provide pathways and candidate genes that highlight the interplay between innate immunity and neuronal activity in the aetiology of autism.

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