| 1. |
- Akula, Srinivas, et al.
(författare)
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Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:22
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Tidskriftsartikel (refereegranskat)abstract
- Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.
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| 2. |
- Arvidsson, Gustav, et al.
(författare)
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Multimodal Single-Cell Sequencing of B Cells in Primary Sjögren's Syndrome
- 2024
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Ingår i: Arthritis & Rheumatology. - 2326-5191 .- 2326-5205. ; 76:2, s. 255-267
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Tidskriftsartikel (refereegranskat)abstract
- Objective. B cells are important in the pathogenesis of primary Sjögren's syndrome (pSS). Patients positive for Sjögren's syndrome antigen A/Sjögren syndrome antigen B (SSA/SSB) autoantibodies are more prone to systemic disease manifestations and adverse outcomes. We aimed to determine the role of B cell composition, gene expression, and B cell receptor usage in pSS subgroups stratified for SSA/SSB antibodies.Methods. Over 230,000 B cells were isolated from peripheral blood of patients with pSS (n = 6 SSA−, n = 8 SSA+ single positive and n = 10 SSA/SSB+ double positive) and four healthy controls and processed for single-cell RNA sequencing (scRNA-seq) and single-cell variable, diversity, and joining (VDJ) gene sequencing (scVDJ-seq).Results. We show that SSA/SSB+ patients present the highest and lowest proportion of naïve and memory B cells, respectively, and the highest up-regulation of interferon-induced genes across all B cell subtypes. Differential usage of IGHV showed that IGHV1-69 and IGHV4-30-4 were more often used in all pSS subgroups compared with controls. Memory B cells from SSA/SSB+ patients displayed a higher proportion of cells with unmutated VDJ transcripts compared with other pSS patient groups and controls, indicating altered somatic hypermutation processes. Comparison with previous studies revealed heterogeneous clonotype pools, with little overlap in CDR3 sequences. Joint analysis using scRNA-seq and scVDJ-seq data allowed unsupervised stratification of patients with pSS and identified novel parameters that correlated to disease manifestations and antibody status.Conclusion. We describe heterogeneity and molecular characteristics in B cells from patients with pSS, providing clues to intrinsic differences in B cells that affect the phenotype and outcome and allowing stratification of patients with pSS at improved resolution.
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| 3. |
- Diamanti, Klev, 1987-, et al.
(författare)
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Single nucleus transcriptomics data integration recapitulates the major cell types in human liver
- 2021
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Ingår i: Hepatology Research. - : Wiley. - 1386-6346 .- 1872-034X. ; 51:2, s. 233-238
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Tidskriftsartikel (refereegranskat)abstract
- Hepatology Research published by John Wiley & Sons Australia, Ltd on behalf of Japan Society of Hepatology Aim: The aim of this study was to explore the benefits of data integration from different platforms for single nucleus transcriptomics profiling to characterize cell populations in human liver. Methods: We generated single-nucleus RNA sequencing data from Chromium 10X Genomics and Drop-seq for a human liver sample. We utilized state of the art bioinformatics tools to undertake a rigorous quality control and to integrate the data into a common space summarizing the gene expression variation from the respective platforms, while accounting for known and unknown confounding factors. Results: Analysis of single nuclei transcriptomes from both 10X and Drop-seq allowed identification of the major liver cell types, while the integrated set obtained enough statistical power to separate a small population of inactive hepatic stellate cells that was not characterized in either of the platforms. Conclusions: Integration of droplet-based single nucleus transcriptomics data enabled identification of a small cluster of inactive hepatic stellate cells that highlights the potential of our approach. We suggest single-nucleus RNA sequencing integrative approaches could be utilized to design larger and cost-effective studies.
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| 4. |
- Feiner, Nathalie, et al.
(författare)
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Environmentally induced DNA methylation is inherited across generations in an aquatic keystone species
- 2022
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Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 25:5
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Tidskriftsartikel (refereegranskat)abstract
- Transgenerational inheritance of environmentally induced epigenetic marks can have significant impacts on eco-evolutionary dynamics, but the phenomenon remains controversial in ecological model systems. We used whole-genome bisulfite sequencing of individual water fleas (Daphnia magna) to assess whether environmentally induced DNA methylation is transgenerationally inherited. Genetically identical females were exposed to one of three natural stressors, or a de-methylating drug, and their offspring were propagated clonally for four generations under control conditions. We identified between 70 and 225 differentially methylated CpG positions (DMPs) in F1 individuals whose mothers were exposed to a natural stressor. Roughly half of these environmentally induced DMPs persisted until generation F4. In contrast, treatment with the drug demonstrated that pervasive hypomethylation upon exposure is reset almost completely after one generation. These results suggest that environmentally induced DNA methylation is non-random and stably inherited across generations in Daphnia, making epigenetic inheritance a putative factor in the eco-evolutionary dynamics of freshwater communities.
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| 5. |
- Foox, Jonathan, et al.
(författare)
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The SEQC2 epigenomics quality control (EpiQC) study
- 2021
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Ingår i: Genome Biology. - : BioMed Central (BMC). - 1465-6906 .- 1474-760X. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundCytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group.ResultsEach sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms.ConclusionsThe data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.
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| 6. |
- Gezelius, Henrik, PhD, 1977-, et al.
(författare)
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Comparison of high-throughput single-cell RNA-seq methods for ex vivo drug screening
- 2024
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Ingår i: NAR Genomics and Bioinformatics. - : Oxford University Press. - 2631-9268. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Functional precision medicine (FPM) aims to optimize patient-specific drug selection based on the unique characteristics of their cancer cells. Recent advancements in high throughput ex vivo drug profiling have accelerated interest in FPM. Here, we present a proof-of-concept study for an integrated experimental system that incorporates ex vivo treatment response with a single-cell gene expression output enabling barcoding of several drug conditions in one single-cell sequencing experiment. We demonstrate this through a proof-of-concept investigation focusing on the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) E/R+ Reh cell line. Three different single-cell transcriptome sequencing (scRNA-seq) approaches were evaluated, each exhibiting high cell recovery and accurate tagging of distinct drug conditions. Notably, our comprehensive analysis revealed variations in library complexity, sensitivity (gene detection), and differential gene expression detection across the methods. Despite these differences, we identified a substantial transcriptional response to fludarabine, a highly relevant drug for treating high-risk ALL, which was consistently recapitulated by all three methods. These findings highlight the potential of our integrated approach for studying drug responses at the single-cell level and emphasize the importance of method selection in scRNA-seq studies. Finally, our data encompassing 27 327 cells are freely available to extend to future scRNA-seq methodological comparisons.
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| 7. |
- Han, Yanan, et al.
(författare)
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Comparison of EM-seq and PBAT methylome library methods for low-input DNA
- 2022
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Ingår i: Epigenetics. - : Informa UK Limited. - 1559-2294 .- 1559-2308. ; 17:10, s. 1195-1204
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Tidskriftsartikel (refereegranskat)abstract
- DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole-genome methylation quantification of low input samples.
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| 8. |
- Lindqvist, C. Mårten, et al.
(författare)
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Deep targeted sequencing in pediatric acute lymphoblastic leukemia unveils distinct mutational patterns between genetic subtypes and novel relapse-associated genes
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:39, s. 64071-64088
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Tidskriftsartikel (refereegranskat)abstract
- To characterize the mutational patterns of acute lymphoblastic leukemia (ALL) we performed deep next generation sequencing of 872 cancer genes in 172 diagnostic and 24 relapse samples from 172 pediatric ALL patients. We found an overall greater mutational burden and more driver mutations in T-cell ALL (T-ALL) patients compared to B-cell precursor ALL (BCP-ALL) patients. In addition, the majority of the mutations in T-ALL had occurred in the original leukemic clone, while most of the mutations in BCP-ALL were subclonal. BCP-ALL patients carrying any of the recurrent translocations ETV6-RUNX1, BCR-ABL or TCF3-PBX1 harbored few mutations in driver genes compared to other BCP-ALL patients. Specifically in BCP-ALL, we identified ATRX as a novel putative driver gene and uncovered an association between somatic mutations in the Notch signaling pathway at ALL diagnosis and increased risk of relapse. Furthermore, we identified EP300, ARID1A and SH2B3 as relapse-associated genes. The genes highlighted in our study were frequently involved in epigenetic regulation, associated with germline susceptibility to ALL, and present in minor subclones at diagnosis that became dominant at relapse. We observed a high degree of clonal heterogeneity and evolution between diagnosis and relapse in both BCP-ALL and T-ALL, which could have implications for the treatment efficiency.
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