C57BL/6J (B6) and DBA/2J (D2) are two of the most commonly used inbred mouse strains in neuroscience research. However, the only currently available mouse genome is based entirely on the B6 strain ...sequence. Subsequently, oligonucleotide microarray probes are based solely on this B6 reference sequence, making their application for gene expression profiling comparisons across mouse strains dubious due to their allelic sequence differences, including single nucleotide polymorphisms (SNPs). The emergence of next-generation sequencing (NGS) and the RNA-Seq application provides a clear alternative to oligonucleotide arrays for detecting differential gene expression without the problems inherent to hybridization-based technologies. Using RNA-Seq, an average of 22 million short sequencing reads were generated per sample for 21 samples (10 B6 and 11 D2), and these reads were aligned to the mouse reference genome, allowing 16,183 Ensembl genes to be queried in striatum for both strains. To determine differential expression, 'digital mRNA counting' is applied based on reads that map to exons. The current study compares RNA-Seq (Illumina GA IIx) with two microarray platforms (Illumina MouseRef-8 v2.0 and Affymetrix MOE 430 2.0) to detect differential striatal gene expression between the B6 and D2 inbred mouse strains. We show that by using stringent data processing requirements differential expression as determined by RNA-Seq is concordant with both the Affymetrix and Illumina platforms in more instances than it is concordant with only a single platform, and that instances of discordance with respect to direction of fold change were rare. Finally, we show that additional information is gained from RNA-Seq compared to hybridization-based techniques as RNA-Seq detects more genes than either microarray platform. The majority of genes differentially expressed in RNA-Seq were only detected as present in RNA-Seq, which is important for studies with smaller effect sizes where the sensitivity of hybridization-based techniques could bias interpretation.
FLT3 mutations are prevalent in AML patients and confer poor prognosis. Crenolanib, a potent type I pan-FLT3 inhibitor, is effective against both internal tandem duplications and ...resistance-conferring tyrosine kinase domain mutations. While crenolanib monotherapy has demonstrated clinical benefit in heavily pretreated relapsed/refractory AML patients, responses are transient and relapse eventually occurs. Here, to investigate the mechanisms of crenolanib resistance, we perform whole exome sequencing of AML patient samples before and after crenolanib treatment. Unlike other FLT3 inhibitors, crenolanib does not induce FLT3 secondary mutations, and mutations of the FLT3 gatekeeper residue are infrequent. Instead, mutations of NRAS and IDH2 arise, mostly as FLT3-independent subclones, while TET2 and IDH1 predominantly co-occur with FLT3-mutant clones and are enriched in crenolanib poor-responders. The remaining patients exhibit post-crenolanib expansion of mutations associated with epigenetic regulators, transcription factors, and cohesion factors, suggesting diverse genetic/epigenetic mechanisms of crenolanib resistance. Drug combinations in experimental models restore crenolanib sensitivity.
Of the more than 100 studies that have examined relationships between excessive ethanol consumption and the brain transcriptome, few rodent studies have examined chronic consumption. Heterogeneous ...stock collaborative cross mice freely consumed ethanol vs. water for 3 months. Transcriptional differences were examined for the central nucleus of the amygdala, a brain region known to impact ethanol preference. Early preference was modestly predictive of final preference and there was significant escalation of preference in females only. Genes significantly correlated with female preference were enriched in annotations for the primary cilium and extracellular matrix. A single module in the gene co-expression network was enriched in genes with an astrocyte annotation. The key hub node was the master regulator, orthodenticle homeobox 2 (Otx2). These data support an important role for the extracellular matrix, primary cilium and astrocytes in ethanol preference and consumption differences among individual female mice of a genetically diverse population.
•Chronic alcohol consumption alters the brain transcriptome.•Highly diverse genetically heterogeneous mice vary in alcohol preference.•Chronically drinking females but not males escalate preference.•Primary cilium and extracellular matrix transcriptome impact female preference.•A gene co-expression network for alcohol preference was enriched in astrocyte genes.
Computational biology has increasingly turned to agent-based modeling to explore complex biological systems. Biological diffusion (diffusion, decay, secretion, and uptake) is a key driver of ...biological tissues. GPU computing can vastly accelerate the diffusion and decay operators in the partial differential equations used to represent biological transport in an agent-based biological modeling system. In this article, we utilize OpenACC to accelerate the diffusion portion of PhysiCell, a cross-platform agent-based biosimulation framework. We demonstrate an almost 40× speedup on the state-of-the-art NVIDIA Ampere 100 GPU compared to a serial run on AMD’s EPYC 7742. We also demonstrate 9× speedup on the 64-core AMD EPYC 7742 multicore platform. By using OpenACC for both the CPUs and the GPUs, we maintain a single source code base, thus creating a portable yet performant solution. With the simulator’s most significant computational bottleneck significantly reduced, we can continue cancer simulations over much longer times.
•Uveitis is a heterogeneous collection of diseases.•Despite the heterogeneity of uveitis, specific RNA transcripts expressed by cells in peripheral blood as well as pathways involving multiple genes ...are common to different forms of uveitis.•The shared expression of mRNA from different forms of uveitis is consistent with a prior publication based on peripheral blood and thus independently validated.•Transcripts that are implicated in multiple forms of uveitis could suggest therapeutic targets that could benefit multiple forms of uveitis.
Uveitis is a heterogeneous collection of diseases. We tested the hypothesis that despite the diversity of uveitides, there could be common mechanisms shared by multiple subtypes, and that evidence of these common mechanisms may be detected as gene expression profiles in whole blood.
Cohort study.
Ninety subjects with uveitis including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulointerstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy controls were enrolled, predominantly at Oregon Health & Science University. RNA-Seq data generated from peripheral, whole blood identified 19,859 unique transcripts. We analyzed gene expression pathways via Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). We validated our list of upregulated genes by comparison to a previously published study on peripheral blood gene expression among 50 subjects with diverse forms of uveitis.
Both the Kyoto Encyclopedia of Genes and Genomes and GO analysis identified multiple shared pathways or GO terms with a P value of <.0001. Almost all pathways related to the immune response and/or response to an infection. A total of 119 individual transcripts were upregulated by at least 1.5-fold and false discovery rate <.05, and 61 were downregulated by similar criteria. Comparing mRNA from our study with a false discovery rate <.05 and the prior report, we identified 10 common gene transcripts: ICAM1, IL15RA, IL15, IRF1, IL10RB, GSK3A, TYK2, MEF2A, MEF2B, and MEF2D.
Many forms of uveitis share overlapping mechanisms. These data support the concept that a single therapeutic approach could benefit diverse forms of this disease.
RNA-Seq experiments have shown great potential for transcriptome profiling. While sequencing increases the level of biological detail, integrative data analysis is also important. One avenue is the ...construction of coexpression networks. Because the capacity of RNA-Seq data for network construction has not been previously evaluated, we constructed a coexpression network using striatal samples, derived its network properties and compared it with microarray-based networks.
The RNA-Seq coexpression network displayed scale-free, hierarchical network structure. We detected transcripts groups (modules) with correlated profiles; modules overlap distinct ontology categories. Neuroanatomical data from the Allen Brain Atlas reveal several modules with spatial colocalization. The network was compared with microarray-derived networks; correlations from RNA-Seq data were higher, likely because greater sensitivity and dynamic range. Higher correlations result in higher network connectivity, heterogeneity and centrality. For transcripts present across platforms, network structure appeared largely preserved. From this study, we present the first RNA-Seq data de novo network inference.
To test the hypothesis that idiopathic uveitis can be categorized into subtypes based on gene expression from blood.
Case control study.
We applied RNA-Seq to peripheral blood from patients with ...uveitis associated with 1 of 4 systemic diseases, including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulo-interstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy control subjects evaluated predominantly at Oregon Health and Science University. A high-dimensional negative binomial regression model implemented in the edgeR R package compared each disease group with the control subjects. The 20 most distinctive genes for each diagnosis were extracted. Of 80 genes, there were 75 unique genes. A classification algorithm was developed by fitting a gradient boosting tree with 5-fold cross-validation. Messenger RNA from subjects with idiopathic uveitis were analyzed to see if any fit clinically and by gene expression pattern with one of the diagnosable entities.
For uveitis associated with a diagnosable systemic disease, gene expression profiling achieved an overall accuracy of 85% (balanced average of sensitivity plus specificity, P < .001). Although most patients with idiopathic uveitis presumably have none of these 4 associated systemic diseases, gene expression profiles helped to reclassify 11 of 38 subjects.
Peripheral blood gene expression profiling is a potential adjunct in accurate differential diagnosis of the cause of uveitis. Validation of these results and characterization of the gene expression profile from additional discrete diagnoses could enhance the value of these observations.
•Idiopathic uveitis is the most frequent subtype of uveitis in a referral clinic, but the term idiopathic fails to convey prognostic or therapeutic implications to providers or patients.•Analysis of gene expression from cells in peripheral blood shows that subtypes of uveitis associated with systemic disease display distinctive patterns of gene expression.•The pattern of gene expression can help classify some examples of idiopathic uveitis.
Background
Genome‐wide profiling to examine brain transcriptional features associated with excessive ethanol (EtOH) consumption has been applied to a variety of species including rodents, nonhuman ...primates (NHPs), and humans. However, these data were obtained from cross‐sectional samples which are particularly vulnerable to individual variation when obtained from small outbred populations typical of human and NHP studies. In the current study, a novel within‐subject design was used to examine the effects of voluntary EtOH consumption on prefrontal cortex (PFC) gene expression in a NHP model.
Methods
Two cohorts of cynomolgus macaques (n = 23) underwent a schedule‐induced polydipsia procedure to establish EtOH self‐administration followed by 6 months of daily open access to EtOH (4% w/v) and water. Individual daily EtOH intakes ranged from an average of 0.7 to 3.7 g/kg/d. Dorsal lateral PFC area 46 (A46) brain biopsies were collected in EtOH‐naïve and control monkeys; contralateral A46 biopsies were collected from the same monkeys following the 6 months of fluid consumption. Gene expression changes were assessed using RNA‐Seq paired analysis, which allowed for correction of individual baseline differences in gene expression.
Results
A total of 675 genes were significantly down‐regulated following EtOH consumption; these were functionally enriched for immune response, cell adhesion, plasma membrane, and extracellular matrix. A total of 567 genes that were up‐regulated following EtOH consumption were enriched in microRNA target sites and included target sites associated with Toll‐like receptor pathways. The differentially expressed genes were also significantly enriched in transcription factor binding sites.
Conclusions
The data presented here are the first to use a longitudinal biopsy strategy to examine how chronic EtOH consumption affects gene expression in the primate PFC. Prominent effects were seen in both cell adhesion and neuroimmune pathways; the latter contained both pro‐ and antiinflammatory genes. The data also indicate that changes in miRNAs and transcription factors may be important epigenetic regulators of EtOH consumption.
Walter et al. used a unique within subject design to detect changes in the brain prefrontal cortex transcriptome associated with chronic ethanol exposure in non‐human primates. The data obtained showed that broad categories of genes were affected, especially those associated with neuro‐immune function, cell adhesion and the extracellular matrix. The data suggest that chronic ethanol exposure has marked functional and structural effects on the prefrontal cortex.
Formalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with ...patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry.
True archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses.
We report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases.
Our results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.
Peripheral blood is a highly accessible biofluid providing a rich source of information about human physiology and health status. However, for studies of the blood transcriptome with RNA sequencing ...(RNA-Seq) techniques, high levels of hemoglobin mRNAs (hgbRNA) present in blood can occupy valuable sequencing space, impacting detection and quantification of non-hgbRNAs. In this study, we evaluated two methods for preparing ribosomal RNA (rRNA)-depleted sequencing libraries for RNA-Seq of whole blood, one of which is also designed to deplete hgbRNAs. Two experiments were performed: one evaluating library performance across 6 human blood samples and the other examining library reproducibility and performance in a two-subject subset. We find that addition of hgbRNA depletion to the rRNA-depletion protocol for library preparation from blood RNA effectively reduces highly abundant hgbRNA reads; however, it does not result in a statistically significant increase in differentially expressed genes in our patient-control study. Bioinformatic removal of globin gene counts in non-hgbRNA depleted libraries provides improvement in overall performance of these libraries. We conclude that use of a standard ribosomal RNA depletion method for library preparation coupled with bioinformatic removal of globin gene counts is sufficient for reproducible and sensitive measurement of both coding and noncoding RNAs in the blood transcriptome.
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