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At least 217 records · Page 12Linked to original sources
PubMed2026

Predicting gene-specific regulation with transcriptomic and epigenetic single-cell data.

MOTIVATION: Analysis of single cell ATAC-seq and RNA-seq data has allowed to gain unprecedented insights into gene regulation by allowing to define cell type-specific regulatory regions and their effects on gene expression. While powerful, such analysis is challenging due to the inherent sparsity of single cell data. RESULTS: We present a new approach, MetaFR, to learn gene-specific models that link open-chromatin variation from scATAC-seq data to gene expression from scRNA-seq. Using efficient regression trees, we illustrate that accurate expression prediction models can be learned on the single-cell or meta-cell level. Validation was done using fine-mapped eQTLs. Meta-cell models were found to outperform single-cell models for most genes. Comparison to the SOTA method SCARlink revealed advantages of MetaFR in terms of runtime and prediction performance. MetaFR thus allows time-efficient analysis and obtains reliable models of gene expression prediction, which can be used to study gene regulation in any organism for which scRNA-seq and scATAC-seq data is available. AVAILABILITY AND IMPLEMENTATION: MetaFR is available under https://github.com/SchulzLab/MetaFR.

Single-Cell Analysis↗
PubMed1991

Contextual analysis and intermediate cell markers enhance high-resolution cell image analysis for automated cervical smear diagnosis.

Until now, efforts to automate cervical smear diagnosis have focused on analyzing features of individual cells. In a complex specimen such as that obtained from a cervical scrape, diagnostically significant cells may not be adequately represented or may elude detection by the automated technology. An approach is needed that extracts additional quantitative information from cervical smears beyond what the cell-by-cell approach can provide. A new methodology, contextual analysis, was developed to extract global quantitative information about cells, cell clusters, and background debris. This pilot study was designed to compare the efficacy of contextual analysis with high-resolution, single cell analysis and the analysis of intermediate cell markers. Thirty-four samples prepared as monolayers and stained with the Feulgen-Thionin/Congo Red stain were measured. Contextual analysis alone was able to classify 91% of the smears correctly; single cell analysis classified 94% of the cells correctly; and the intermediate cell analysis correctly identified the smear diagnosis for 84% of the cells. When all three analysis methods were combined into a simple smear level classifier, the overall smear classification accuracy was improved over those obtained using the three methodologies alone.

Biomarkers↗
PubMed1993

Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.

Germinal centres are areas of intense B lymphocyte proliferation inside primary B cell follicles in spleen and lymph nodes. Rearranged V genes from single human B cells, isolated from histological sections of two such structures by micromanipulation, were amplified and sequenced. Cells from the follicular mantle were clonally diverse and largely expressed germline V genes. Germinal centres were dominated by a few large B cell clones dispersed throughout these structures and exhibiting intraclonal diversity by ongoing somatic hypermutation. Pronounced counterselection of replacement mutations seen in one of the germinal centres may indicate a late phase of the germinal centre reaction. A polyclonal population of activated B cells expressing unmutated antibodies in the dark zone of the other germinal centre may represent the initial founder cells.

Adult↗
PubMed2002

Rat parietal cells express CCK(2) receptor mRNA: gene expression analysis of single cells isolated by laser-assisted microdissection.

Gastrin plays a crucial role in maintaining a normal cellular composition and function of the oxyntic mucosa. It has been debated for decades whether parietal cells possess cholecystokinin-2 (CCK(2)) receptors and interact directly with gastrin. We investigated whether parietal cells express CCK(2) receptor mRNA by using new molecular biology techniques. Rat oxyntic mucosal cells were dispersed and enriched by elutriation, and single parietal and ECL cells were isolated from cell smears by means of laser microbeam microdissection and laser pressure catapulting. The mRNA from each single cell was isolated and subjected to one-step multiplex or conventional reverse transcription-polymerase chain reaction and subsequent nested PCR. Specific primers for the CCK(2) receptor were used in combination with primers for H,K-ATPase and histidine decarboxylase, specific markers for parietal and ECL cells, respectively. CCK(2) receptor mRNA was detected in 25% of the rat parietal cells and 40% of the ECL cells examined.

Animals↗
PubMed2005

Sequential application of interphase-FISH and CGH to single cells.

A comprehensive genomic analysis of single cells is needed for numerous scenarios in tumor genetics, clinical diagnostics and forensic application. PCR protocols were developed which allow an unbiased amplification of the whole genome of a single cell for subsequent analyses by comparative genomic hybridization (CGH). However, verification of single-cell CGH results has been impossible as the procedure naturally involves the destruction of the respective cell. Here we show that the genome of individual cells can be analyzed by two different single cell techniques applied sequentially to the same cell. In a first step, interphase fluorescence in situ hybridization (FISH) is applied. After evaluation of the interphase-FISH signals, cells of interest can be selected for a further analysis. Single cells are collected by laser microdissection, the DNA is amplified by linker-adaptor PCR and subjected to CGH-analysis. This strategy offers new opportunities for a sophisticated selection of cells based on interphase-FISH signals. Furthermore, the sequential application of two different single-cell approaches to the same single-cell represents the only option to control and verify the single-cell CGH results. We demonstrate the feasibility of this approach with a series of experiments including cells from pre- and postnatal diagnostics, for example, cells with trisomies 13, 18, or 21, respectively, leukemia and tumor cells and tissue sections.

In Situ Hybridization, Fluorescence↗
PubMed1997

[Lecture of principles and applications of capillary electrophoresis (V) Applications of capillary electrophoresis in pharmaceutical analysis and clinical chemistry].

A review with 68 references is presented on the application and new developments of CE in pharmaceutical analysis, clinical chemistry and single cell analysis etc. The pharmaceutical analysis includes main component assay, determination of drug-related impurities, stoichiometric determination and quantitative determinations. The Chinese herb analysis includes determinations of main components and prescriptions of Chinese herb. The chiral separation includes separation mechanism and some new chiral selectors. The application of CE in clinical chemistry includes determinations of serum and protein, monitoring of drug metabolism, etc. Single cell analysis includes determinations of component in neurocells and human erythrocyte.

Animals↗
PubMed1997

Single-cell cytokine analysis of gamma delta T cell responses to nonpeptide mycobacterial antigens.

TCR gamma delta T cells are considered important in the rapid immune response to intracellular infection. We investigated the early response of peripheral blood gamma delta T cells to the nonpeptide Ag isopentenyl pyrophosphate and to its synthetic analogue ethyl pyrophosphate. In healthy donors, an increase in the number of gamma delta T cells was detected as soon as 4 days after stimulation with the nonpeptide Ags. Single-cell analysis of cytokine production was performed by intracellular staining of IFN-gamma and IL-4. gamma delta T cells were found to rapidly expand and produce IFN-gamma in response to nonpeptide Ags. Furthermore, IL-12 augmented the IFN-gamma response. In contrast, gamma delta T cells from the majority of HIV+ donors did not expand or express IFN-gamma in response to nonpeptide Ags, even in the presence of IL-12. These findings indicate a role for nonpeptide-reactive gamma delta T cells in effective cell-mediated immunity for intracellular pathogens.

Antigens, Bacterial↗
PubMed1997

Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.

In this study, we show that direct mutational analysis of genomic DNA can be performed on single somatic cells extracted from a frozen, immunohistochemically stained tissue section using laser-assisted capture microscopy. Eighty-nine single tumor cells were separately dissected from one case of human basal cell cancer (BCC) and p53 mutations were analyzed by direct semi-automated sequencing of PCR fragments. Amplification was obtained for at least one of the two analyzed exons from approximately 50% of the single tumor cells. Identical p53 mutations were found in widely spread areas of the tumor, suggesting a clonal proliferation originating from one cell. Interestingly, comparison between results of immunohistochemistry and genetic analysis of the single cells revealed the same p53 mutations irrespective of the p53 immunoreactivity. We propose that this approach has a great potential to allow investigation of genotypic differences in single cells and more specifically to resolve important and fundamental questions determining cancer heterogeneity.

Aged↗
PubMed2003

Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.

Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.

Animals↗
PubMed1999

Voltage-gated sodium and potassium channels in radial glial cells of trout optic tectum studied by patch clamp analysis and single cell RT-PCR.

Radial glial cells in the visual center of trout were analyzed immunocytochemically and with the whole cell mode of the patch-clamp technique in combination with RT-PCR. By immunostaining with anti-GFAP antibodies radially oriented cell processes spanning the entire width of the tectum were brightly labeled, while with anti-S-100 antiserum the cell bodies residing in a discrete layer close to the ventricular border became most clearly visible. Virtually all radial glial cells examined in brain slices exhibited voltage-gated sodium inward currents that were activated above -40 mV, blocked by micromolar concentrations of TTX and totally eliminated if sodium was substituted for Tris in the bath solution. In contrast with adjacent nerve cells of the same slices radial glial cells did not exhibit spontaneous electrical activity and could not be stimulated to generate action potentials by depolarizing current injections. Two types of voltage-gated potassium outward currents were elicited by depolarizing voltage steps: a sustained current with delayed rectifier properties and a superimposed transient "A"-type current, both being activated at a threshold potential of -40 mV. In cultured radial glial cells subtle differences were noticed regarding current density, inactivation kinetics, and TEA-sensitivity of the potassium currents. Inwardly rectifying potassium currents activating at hyperpolarized voltages were not observed. By single cell RT-PCR the transcripts of two shaker-related potassium channel genes (termed tsha1-a fish homologue to Kv1.2- and tsha3) were amplified, while transcripts for tsha 2 and tsha 4 were not detected.

Animals↗
PubMed1996

Estimation of the frequency of self-reactive T cells in health and inflammatory diseases by limiting dilution analysis and single cell cloning.

Autoreactive T cells have recently been detected not only in autoimmune diseases but also in healthy individuals, but their frequency is thought to be low. The aim of our study was to estimate the frequency of self-reactive T cells by using limiting dilution analyses of peripheral blood lymphocytes. Assessment of self-reactivity in this study was defined as T-cell proliferation to autologous non-T cells in the absence of foreign antigens. When culture conditions were optimized by adding interleukin 2, healthy individuals showed a frequency of self-reactive T cells ranging from 1/60 to 1/600. These results were confirmed by using unseparated peripheral blood leukocytes or Epstein-Barr virus transformed B-cell blasts as stimulators. All cultures were performed exclusively in autologous serum. Single cell cloning from a healthy donor yielded 568 T-cell clones, 12 of which showed self-reactivity giving a frequency of more than 1 in 50 T cells. Eight of these 12 T-cell clones were inhibited by MHC-class II antibodies. Frequency analyses of self-reactive T cells in patients with autoimmune diseases (rheumatoid arthritis, autoimmune hepatitis or primary biliary cirrhosis), with viral hepatitis or with inflammatory bowel diseases showed similar frequencies in all patient groups and no significant differences from normal individuals. In conclusion, we have found a high frequency of self-reactive T cells in both health and disease. We postulate that self-reactive T cells constitute an important part of the physiological T-cell repertoire.

Adolescent↗
PubMed1991

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction.

Mast cell-fibroblast interactions have been extensively investigated in the last few years. Fibroblasts support the in vitro survival but not proliferation of mouse connective-tissue type mast cells. However, the factor(s) that allow their survival on fibroblast monolayers has not been identified. We have investigated the presence of mRNA for IL-3 and granulocyte-macrophage-CSF in single mouse mast cells, before and after co-culture with 3T3 fibroblasts, using the polymerase chain reaction technique. The system was calibrated first by using in vitro generated population of mouse bone-marrow derived mast cells (BMMC). Significant differences in the amplification of IL-3 cDNA were observed in each of the BMMC cells examined, whereas the amplification of cDNA for the alpha-subunit of the Fc epsilon RI were similar. Inasmuch as murine cultured IL-3-dependent mast cells differentiate into connective tissue-like mast cells when co-cultured with 3T3 fibroblasts without any exogenous supply of growth factors, it was of interest to determine whether these connective tissue-like mast cells produce IL-3 message. Separation of the differentiated BMMC from the fibroblast monolayer, by either trypsinization or by single cell manipulation revealed the synthesis of a detectable amount of IL-3 mRNA in these mast cells. Whether this IL-3 mRNA was induced by fibroblasts was further investigated using connective tissue mast cells freshly purified from the mouse peritoneal cavity. Only about 20% of these connective tissue mast cells produced detectable amount of granulocyte-macrophage-CSF mRNA whereas in less than 10% of the cells IL-3 mRNA was detected. However, when these connective tissue mast cells were co-cultured with 3T3 fibroblasts for 18 hours and then separated, IL-3 mRNA were detected in most of the cells whereas no such mRNA was detected in tissue mast cells incubated for 18 h with medium derived from 3T3 fibroblasts. Therefore we conclude that fibroblasts induce the accumulation of IL-3 mRNA in connective tissue mast cells. The production of IL-3 may play a role in the survival of this type of mast cells on the fibroblast monolayer.

Animals↗
PubMed2026

The role of KIAA1467 in breast cancer: insights from pan-cancer and single-cell sequencing analysis.

BACKGROUND: Improving the response rate of single-agent immune checkpoint blockade (ICB) urgently requires the discovery of new therapeutic targets for combinatorial regimens. Analyses of tumor microenvironment (TME)-associated biomarkers have verified that KIAA1467 drives the formation of an immune-excluded, non-inflamed TME in breast cancer (BRCA). This study systematically explores the expression pattern, prognostic value, immune regulatory function, biological effects, and drug resistance relevance of FAM234B (also known as KIAA1467) in BRCA. METHODS: We performed pan-cancer survival analysis using The Cancer Genome Atlas (TCGA) datasets. Multi-omics bioinformatics analyses were conducted to evaluate KIAA1467 expression across malignancies. Single-cell RNA sequencing (scRNA-seq) data from GSE176078 was utilized to localize KIAA1467 expression at the cellular level. Immunohistochemistry and western blot assays validated KIAA1467 expression in BRCA clinical specimens. Correlation analyses were implemented to assess relationships between KIAA1467 expression, clinicopathological features, immune modulators, tumor-infiltrating immune cells, and p53 mutation status. Functional enrichment analysis uncovered relevant signaling pathways. Bioinformatic half maximal inhibitory concentration (IC50) prediction and in vitro cellular experiments were applied to evaluate associations between KIAA1467 and chemotherapeutic drug sensitivity. RESULTS: TCGA pan-cancer survival analysis demonstrated that elevated KIAA1467 expression significantly predicted shortened overall survival in BRCA and multiple other tumor types. KIAA1467 displayed distinct expression patterns across cancers, with prominent upregulation in BRCA. scRNA-seq confirmed enriched KIAA1467 expression within BRCA cells, and its upregulation in BRCA tissues was further verified by immunohistochemistry and western blot. High KIAA1467 expression was positively correlated with advanced tumor grade and lymphatic metastasis. KIAA1467 showed negative correlations with most immune modulators and core immune checkpoint molecules, as well as tumor-infiltrating immune cells in the TME, implying its potential function in tumor immune evasion. Low KIAA1467 expression was tightly linked to p53 mutations. Enrichment analysis indicated participation of KIAA1467 in epithelial-mesenchymal transition, apoptosis and cell cycle arrest. Furthermore, high KIAA1467 expression corresponded to higher estimated IC50 values of cisplatin, gefitinib, paclitaxel and gemcitabine, consistent with reduced chemosensitivity observed in vitro. CONCLUSIONS: This study reveals the multifaceted oncogenic role of KIAA1467 in BRCA. KIAA1467 participates in remodeling an immunosuppressive TME, correlates with malignant progression and chemoresistance, and may serve as a promising candidate target to optimize ICB-based combination therapy for BRCA. These findings offer new perspectives for the clinical treatment and comprehensive management of BRCA.

KIAA1467↗
PubMed1989

Microcolumn separations and the analysis of single cells.

Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.

Amino Acids↗
PubMed1995

Histometric texture analysis of DNA in thin sections from breast biopsies. Application to the detection of malignancy-associated changes in carcinoma in situ.

OBJECTIVE: To evaluate histometric measurement of nuclear texture in breast biopsy sections in order to detect malignancy-associated changes in apparently normal tissue in the vicinity of carcinoma in situ. STUDY DESIGN: We previously showed that image cytometry measurements of nuclear features--foremost, texture features, describing the organization of Feulgenstained DNA in the cell--can be used to distinguish normal-appearing, diploid epithelial cells from patients with invasive carcinoma of the breast from those with benign biopsies. In that study, referred to as the "single cell analysis," images of at least 200 epithelial cells were acquired for each slide, and substantial user interaction was required to segment cells from each field. Location of isolated cells and interactive segmentation are both time-consuming procedures, particularly in breast tissue, where nuclei can be tightly clustered within a duct. With histometric texture analysis on the same specimens, segmentation of individual cells was ignored, and texture measurements were performed over the entire cluster of relevant cells. With this approach, ploidy information is not available, and touching and overlapping nuclei are included in the measurements. Measurement of histometric texture properties requires substantially less time (at least an order of magnitude) than individual cell measurement and, if ploidy information is not significant, may therefore provide a more practical means of analysis for tissue sections. RESULTS: Seventeen cases of invasive carcinoma and 17 cases of nonproliferative breast disease were examined. Using stepwise discriminant function analysis, slides were classified into one of the two groups with an accuracy of 88.6% in the case of single cell analysis and with an accuracy of 88.2% using histometric analysis. CONCLUSION: The existence of malignancy-associated changes in the breast was confirmed by an independent analysis of the same specimens. Although the two methods are not directly comparable, we found that histometric texture analysis performs at least as well as single-cell analysis for the detection of malignancy-associated changes in breast carcinoma.

Biopsy↗
PubMed2000

Instrumentation for chemical cytometry.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 microns in diameter) can be analyzed by injecting the intact cell into a capillary, lysing the cell, separating and detecting the cellular components, and reconditioning the capillary prior to the next injection. In this paper, we report on technical improvements to single-cell analysis. We designed an inexpensive multipurpose single-cell injector that facilitates the following: (i) monitoring of injection, (ii) reproducible pressure- or electrokinetic-driven injection of the cell, (iii) complete cell lysis by SDS within 30 s of injection, and (iv) pressure-driven capillary reconditioning. Furthermore, we report on the analysis of glycosylation and glycolysis in single human carcinoma cells (HT29 cell line). The reliability and quality of the analysis is confirmed by comparing electropherograms from single cells and those from purified cell extracts.

Electrophoresis, Capillary↗
PubMed1999

Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.

A PCR strategy is described for global amplification of DNA from a single eukaryotic cell that enables the comprehensive analysis of the whole genome. By comparative genomic hybridization, not only gross DNA copy number variations, such as monosomic X and trisomic 21 in single male cells and cells from Down's syndrome patients, respectively, but multiple deletions and amplifications characteristic for human tumor cells are reliably retrieved. As a model of heterogeneous cell populations exposed to selective pressure, we have studied single micrometastatic cells isolated from bone marrow of cancer patients. The observed congruent pattern of comparative genomic hybridization data, loss of heterozygosity, and mutations as detected by sequencing attests to the technique's fidelity and demonstrates its usefulness for assessing clonal evolution of genetic variants in complex populations.

Bone Marrow Cells↗
PubMed2005

Single cell studies of the cell cycle and some models.

Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models.

Cell Cycle↗