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At least 163 records · Page 9Linked to original sources
PubMed1975

Rapid chemical dehydration of samples for electron microscopic examinations.

Acidified 2,2-dimethoxypropane was used to chemically dehydrate biologic tissues for examination in the electron microscope. The ultrastructural integrities of single-celled algae, plant tissues (cotyledon, root, leaf) and animal tissues (liver, pancreas, muscle, cartilage) were maintained. Our technique was simpler and quicker than physically exchanging water for organic solvents (e.g., acetone, ethanol) as generally performed in microscopy.

Animals↗
PubMed1993

Multiple cell interactions are required for fate specification during male spicule development in Caenorhabditis elegans.

The B blast cell divides postembryonically in C. elegans males to produce 47 progeny that include all of the cells of the copulatory spicules. During the early development of the B lineage, the anterior daughter of B, B.a, generates eight cells. These cells migrate to form four pairs of cells that flank the developing cloaca (ventral, dorsal, and two identical lateral pairs). For each pair, the more anterior cell produces a distinct lineage ('anterior fate') from the posterior cell ('posterior fate'). For the ventral and dorsal pairs, either cell can migrate to the anterior position and produce the anterior lineage, and the other cell migrates posterior and produces the posterior lineage (Sulston and Horvitz, 1977, Dev. Biol. 56, 110-156). The migration is variable, although the resultant fate pattern is invariant. In the two lateral pairs, both the migration and fate pattern are invariant. Using a laser microbeam to selectively ablate neighboring cells we have found that the cells of the lateral pair also respond to positional cues. For all four pairs other male-specific blast cells provide extracellular cues. In general, F and U promote anterior fates, Y promotes some posterior fates, and the B.a progeny promote posterior fates. Several of these cues are redundant. By ablating combinations of cells we have deduced how these signals may act in concert to specify the fates of the B.a progeny. We propose that fate specification in these pairs depends on three general classes of extracellular cues: positional cues, modulators of positional cues, and lateral signals. The B lineage thus provides an opportunity to study with single cell resolution the integration of multiple intercellular signals.

Animals↗
PubMed2002

Synaptic convergence of motor and somatosensory cortical afferents onto GABAergic interneurons in the rat striatum.

Cortical afferents to the basal ganglia, and in particular the corticostriatal projections, are critical in the expression of basal ganglia function in health and disease. The corticostriatal projections are topographically organized but also partially overlap and interdigitate. To determine whether projections from distinct cortical areas converge at the level of single interneurons in the striatum, double anterograde labeling from the primary motor (M1) and primary somatosensory (S1) cortices in the rat, was combined with immunolabeling for parvalbumin (PV), to identify one population of striatal GABAergic interneurons. Cortical afferents from M1 and S1 gave rise to distinct, but partially overlapping, arbors of varicose axons in the striatum. PV-positive neurons were often apposed by cortical terminals and, in many instances, apposed by terminals from both cortical areas. Frequently, individual cortical axons formed multiple varicosities apposed to the same PV-positive neuron. Electron microscopy confirmed that the cortical terminals formed asymmetric synapses with the dendrites and perikarya of PV-positive neurons as well as unlabelled dendritic spines. Correlated light and electron microscopy revealed that individual PV-positive neurons received synaptic input from axon terminals derived from both motor and somatosensory cortices. These results demonstrate that, within areas of overlap of functionally distinct projections, there is synaptic convergence at the single cell level. Sensorimotor integration in the basal ganglia is thus likely to be mediated, at least in part, by striatal GABAergic interneurons. Furthermore, our findings suggest that the pattern of innervation of GABAergic interneurons by cortical afferents is different from the cortical innervation of spiny projection neurons.

Animals↗
PubMed1998

[Alteration of genomic structure and/or expression of cancer associated genes in hepatocellular carcinoma].

Cancer is thought to arise from the accumulation of several genetic mutations in a single cell. These include integration of viral genomes, activation of protooncogenes and inactivation of tumor suppressor genes. HCC is one of the most common cancers in Asia and Africa. Various studies have revealed its association with hepatitis B or C viral infection. While activation of known protooncogenes, such as ras genes does not seem to play an important role, frequent allelic loss on specific chromosomal arms, 4q, 13q, 16q and 17p, indicates that dysfunction of diverse tumor suppressor genes located on these chromosome arms is involved in the development of HCC. An informative p53 mutational spectrum of frequent G to T transversions in codon 249 is found in HCCs from either Qidong, People's Republic of China, or southern Africa. This observation links exposure to aflatoxin B1, a known cancer risk factor in these geographic regions. Recently, we found that expression of syndecan-1, which is a transmembrane heparan sulfate proteoglycan involved in cell matrix interactions and growth factor bindings, was inversely associated with metastatic potential in human hepatocellular carcinoma as like nm23-H1 expression was. Transfection with syndecan-1 gene suppresses invasive activity of hepatoma cells. These data support our hypothesis that syndecan-1 is one of important metastasis suppressor factors in hepatoma cells. PR-39 is a proline-rich antimicrobial peptide which was isolated from a pig small intestine and has been reported to induced syndecan-1 on mouse mesenchymal cells. Transfection with PR-39 gene caused induction of syndecan-1 and altered invasive phenotype and actin structure on hepatoma cells. Syndecan-1 and PR-39 may serve as a basis for design of drug or gene therapy effective against metastasis of hepatocellular carcinomas.

Amino Acid Sequence↗
PubMed1994

Integration site of human papillomavirus type-18 DNA in chromosome band 8q22.1 of C4-I cervical carcinoma: DNase I hypersensitivity and methylation of cellular flanking sequences.

The C4-I cell line derived from a non-keratinizing squamous cell carcinoma of the uterine cervix contains integrated human papillomavirus-18 DNA. Fluorescence in situ hybridization of C4-I cells demonstrated a single viral integration site at 8q22.1 on a derivative chromosome originating from an 8q;12q translocation. 8q22 is a site of chromosome fragility and is also recombinogenic in several human malignancies. DNase I hypersensitivity of the integration site was studied with a cellular flanking probe. A hypersensitive site was detected within 3 kb from viral DNA. The integration sites are undermethylated in C4-I and HeLa cells and fully methylated in tumor cell lines of other origin, such as lymphoid cells.

Carcinoma, Squamous Cell↗
PubMed2004

Cell cycle regulation of human immunodeficiency virus type 1 integration in T cells: antagonistic effects of nuclear envelope breakdown and chromatin condensation.

We examined the influence of mitosis on the kinetics of human immunodeficiency virus type 1 integration in T cells. Single-round infection of cells arrested in G1b or allowed to synchronously proceed through division showed that mitosis delays virus integration until 18-24 h postinfection, whereas integration reaches maximum levels by 15 h in G1b-arrested cells. Subcellular fractionation of metaphase-arrested cells indicated that, while nuclear envelope disassembly facilitates docking of viral DNA to chromatin, chromosome condensation directly antagonizes and therefore delays integration. As a result of the balance between the two effects, virus integration efficiency is eventually up to threefold greater in dividing cells. At the single-cell level, using a green fluorescent protein-expressing reporter virus, we found that passage through mitosis leads to prominent asymmetric segregation of the viral genome in daughter cells without interfering with provirus expression.

Animals↗
PubMed2002

Ultra-low-volume, real-time measurements of lactate from the single heart cell using microsystems technology.

The fabrication of microelectrodes integrated within ultra-low-volume microtiter chambers for the amperometric determination of metabolites continues to be of interest in the subject of single-cell and high-throughput screening. The microsystem described in this paper consists of a two-microelectrode sensor with a microfluidic dispensation technology, which is able to deliver both very low titers (6.5 pL) and single heart cells into a low-volume microphotoelectrochemical cell. Devices were fabricated using photolithography and liftoff giving reproducible sensors integrated within high aspect ratio titer chambers (with a volume of 360 pL), made of the photoepoxy SU8. In this paper, the determination of lactate was optimized using an enzyme-linked assay based upon lactate oxidase, involving the amperometric determination of hydrogen peroxide at +640 mV versus an internal Ag/AgCl pseudoreference. The microsystem (including the microfluidic dispensers and structures as well as the microsensor) was subsequently used to measure the lactate content of single heart cells. Dynamic electrochemical measurements of lactate during cell permeabilization are presented. We also show the use of respiratory uncouplers to simulate ischemia in the single myocyte and show that, as expected, the rate of lactate production from the hypoxic heart cell is greater than that within the normoxic healthy myocyte.

Indicators and Reagents↗
PubMed2003

Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population.

Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.

Cell Culture Techniques↗
PubMed2002

Four cell types with distinctive membrane properties and morphologies in lamina I of the spinal dorsal horn of the adult rat.

Lamina I of the spinal dorsal horn plays an important role in the processing and relay of nociceptive information. Signal processing depends, in part, on neuronal membrane properties. Intrinsic membrane properties of lamina I neurons were therefore investigated using whole cell patch clamp recordings in a slice preparation of adult rat spinal cord. Based on responses to somatic current injection, four cell types were identified: tonic, which fire comparatively slowly but continuously throughout stimulation; phasic, which fire a high frequency burst of variable duration; delayed onset, which fire irregularly and with a marked delay to the first spike; and single spike, which typically fire only one action potential even when strongly depolarised. Classification by spiking pattern was further refined by identification of characteristic stimulus-response curves and quantification of several response parameters. Objectivity of the classification was confirmed by cluster analysis. Responses to stimulus trains and synaptic input as well as the kinetics of spontaneous synaptic events revealed differences in the signal processing characteristics of the cell types: tonic and delayed onset cells appeared to act predominantly as integrators whereas phasic and single spike cells acted as coincidence detectors. Intracellular labelling revealed a significant correlation between morphological and physiological cell types: tonic cells were typically fusiform, phasic cells were pyramidal, and delayed onset and single spike cells were multipolar. Thus, there are multiple physiological cells types in lamina I with specific morphological correlates and distinctive signal processing characteristics that confer significant differences in the transduction of input into spike trains.

Animals↗
PubMed2026

A CFH- and SPINT2-based prognostic signature for cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant tumor with a poor prognosis, and reliable biomarkers for postoperative risk stratification remain limited. This study aimed to develop and validate a CFH- and SPINT2-based prognostic signature to support postoperative risk stratification and inform adjuvant therapy selection in CCA through integrative machine learning and single-cell transcriptomics. METHODS: Differentially expressed genes were screened from GSE26566. Integrative machine learning (least absolute shrinkage and selection operator-Cox, random forest, and univariate Cox regression) was performed in the training cohort (GSE89749; n=115) to construct a risk model, which was externally validated in two independent cohorts: cohort 1 (E-MTAB-6389; n=75) and cohort 2 [The Cancer Genome Atlas Cholangiocarcinoma (TCGA-CHOL) data set; n=36]. Systematic analysis was conducted and included examinations of immune infiltration [via single-sample gene set enrichment analysis (ssGSEA)], pathway enrichment (via hallmark GSEA), cellular localization (via single-cell RNA sequencing), and drug sensitivity (via the Genomics of Drug Sensitivity in Cancer 2 database). RESULTS: Two genes, CFH and SPINT2, were identified and incorporated into a prognostic risk score. High-risk patients in the training cohort had a significantly worse overall survival (log-rank P=0.02). External validation was performed in two independent cohorts. In validation cohort 1, the risk group was an independent prognostic factor [hazard ratio =2.27, 95% confidence interval (CI): 1.18-4.37; P=0.01]. In validation cohort 2, the model demonstrated acceptable discriminative ability (concordance index =0.721; 3-year area under the curve =0.692). The high-risk group exhibited an immunosuppressive microenvironment characterized by increased infiltration of macrophages and myeloid-derived suppressor cells, along with the activation of epithelial-mesenchymal transition, inflammatory response, and NF-κB signaling pathways. Single-cell analysis revealed a cell-type-specific expression pattern: CFH was predominantly expressed in fibroblasts, while SPINT2 was mainly expressed in malignant cells. Drug sensitivity analysis demonstrated that the high-risk group was more sensitive to gemcitabine, cisplatin, poly(ADP-ribose) polymerase (PARP) inhibitors, and mammalian target of rapamycin (mTOR) inhibitors, whereas the low-risk group was more sensitive to lapatinib. CONCLUSIONS: The CFH- and SPINT2-based prognostic signature may serve as an independent biomarker for postoperative risk stratification in CCA. High-risk patients, characterized by fibroblast-derived CFH enrichment and malignant-cell SPINT2 loss, exhibit an immunosuppressive microenvironment and may be more suitable for gemcitabine-based chemotherapy or PARP/mTOR inhibitors, whereas low-risk patients may benefit from less intensive adjuvant strategies or HER2/EGFR-targeted lapatinib. Prospective validation is warranted before clinical implementation.

Cholangiocarcinoma (CCA)↗
PubMed1991

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed2025

PreDigs: A Database of Context-specific Cell Type Markers and Precise Cell Subtypes for Digestive Cell Annotation.

Research on cell type markers helps investigators explore the diverse cellular composition of gastrointestinal tumors, thereby enhancing our understanding of tumor heterogeneity and its impact on disease progression and treatment response. However, the integration of large-scale datasets and the standardization of cell type identification remain challenging. Here, we developed PreDigs, a user-friendly database of predicted signatures for the digestive system, which offers 124 curated single-cell RNA sequencing datasets, covering over 3.4 million cells, all available for download. After unsupervised clustering, we unified the identification and nomenclature of cell subtype labels, constructing a cell ontology tree with 142 cell types across 8 hierarchical levels. Meanwhile, we calculated three different context-specific cell type markers, including "Cell Markers", "Subtype Markers", and "TPN Markers", based on various application requirements within or across tissues. Through the integrated analysis of PreDigs data, we identified distinct cell subpopulations exclusive to tumors, one of which corresponds to tumor-specific endothelial cells. Additionally, PreDigs offers online cell annotation tools, allowing users to classify single cells with greater flexibility. PreDigs is accessible at https://www.biosino.org/predigs/.

Humans↗
PubMed1990

Heterogeneity of polyoma tumors in hamsters: analysis of cytoskeletal proteins and viral gene expression.

Murine polyomavirus-induced hamster tumors revealed an unexpected heterogeneity with respect to patterns of cytoskeletal proteins expressed in different visceral and subcutaneous tumors and with respect to viral gene expression early during tumor outgrowth. All tumors analyzed expressed vimentin. Desmin was found in all heart tumors, to variable degrees in kidney tumors and in trace amounts only in 1 out of 4 s.c. tumors. The alpha-smooth-muscle actin isoform was observed in heart tumors only and was restricted to structures that we interpret as being proliferating pericytes or proliferating smooth-muscle cells of the media. In kidneys of infected newborn animals and before the appearance of macroscopic tumors, viral early mRNAs were transcribed from free viral genomes. In tumor tissue the size of the viral transcripts was altered, suggesting that they were transcribed from integrated viral DNA. Since in each tumor discrete bands of viral RNAs were detected, individual tumors arose presumably from single cells and one functional integration event. In contrast, in situ hybridizations of kidney tissue and tumors showed large quantitative differences in viral gene expression not only between different tumors but also between individual cells of the same tumor.

Animals↗
PubMed2025

Network-based drug repurposing for psychiatric disorders using single-cell genomics.

Neuropsychiatric disorders lack effective treatments due to a limited understanding of the underlying cellular and molecular mechanisms. To address this, we integrated population-scale single-cell genomics data and analyzed 23 cell-type-level gene regulatory networks across schizophrenia, bipolar disorder, and autism. Our analysis revealed potential druggable transcription factors co-regulating known risk genes that converge into cell-type-specific co-regulated modules. We applied graph neural networks on those modules to prioritize novel risk genes and leveraged them in a network-based drug repurposing framework to identify 220 drug molecules with the potential for targeting specific cell types. We found evidence for 37 of these drugs in reversing disorder-associated transcriptional phenotypes. Additionally, we discovered 335 drug-cell quantitative trait loci (eQTLs), revealing genetic variation's influence on drug target expression at the cell-type level. Our results provide a single-cell network medicine resource that provides potential mechanistic insights for advancing treatment options for neuropsychiatric disorders.

Drug Repositioning↗
PubMed1983

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.

Animals↗
PubMed2026

Integrative evidence-knowledge marker selection enhances LLM-based cell type annotation in single-cell RNA-seq analysis.

BACKGROUND: Cell type annotation is essential for gaining biological insight from single-cell RNA sequencing data, yet manual labeling remains time-consuming and difficult to reproduce. Various computational approaches have been developed to automate this process, and recent studies suggest that large language models can infer cell types with promising accuracy in single-cell analysis. However, most workflows still rely on cluster-specific markers derived from gene expression alone or manual curation. As a result, marker selection can be sensitive to statistical criteria and dataset-dependent bias, which may lead to the selection of less informative genes or missing important markers, while providing limited biological context. RESULTS: To address this limitation, we introduce CELLIA, an LLM-based workflow for automated and robust cell type annotation. CELLIA employs an integrative evidence-knowledge marker selection strategy that combines statistical differential expression criteria with curated tissue-specific marker resources to identify informative marker genes. In benchmarking analyses of 102 cell types, this approach improved agreement with manual annotations. In addition, CELLIA achieved higher agreement in subtype-level analyses of closely related immune populations and was further evaluated in a non-immune stromal subtype setting, covering 25 cell types in total. CONCLUSION: By integrating evidence-knowledge from gene expression with curated biological prior knowledge, CELLIA provides a more stable marker selection and improves the reliability of LLM-cell type annotation.

Cell type annotation↗
PubMed1984

Rearrangements of host and viral DNA in mouse cells transformed by simian virus 40.

We have determined the structure of host DNA and viral DNA at the site of integration of Simian virus 40 (SV40) in a line of transformed Balb/c-3T3 cells (SVB400) isolated by single cell cloning after virus infection. Recombinant phage containing integrated viral DNA and flanking host DNA were purified from a genomic library and, in conjunction with restriction endonuclease cleavage analysis of the transformed cell DNA, were used to determine the organization of the integrated viral sequences. There is heterogeneity in the arrangement of the viral sequences resulting from tandem duplications of all or part of the SV40 genome with preservation of the viral-host junctions. The predominant arrangement is the result of tandem duplication of 41% of the SV40 genome from 0.64 to 0.23. Analysis of the structure of integrated viral DNA in SVB400 at different passage numbers and in single cell clones derived from the 20th passage indicated that rearrangements of viral DNA occur after the integration event and continue with passage of the cells. The organization of host sequences before and after the integration of SV40 was determined by restriction endonuclease cleavage analysis of parental 3T3 DNA and SVB400 DNA, and by analysis of recombinant phage isolated from genomic libraries. A deletion of at least 15 X 10(3) bases of host DNA occurred at the site of integration, which indicates that viral integration was not a result of a simple insertion of SV40. Nucleotide sequence analysis of the virus-host junctions showed that retained SV40 sequences were colinear with the viral genome, and that the junctions with SV40 DNA occurred at nucleotide numbers 1377 and 3610. There was no evidence of duplications of viral or host sequences at the junctions, and a comparison of the flanking mouse sequences with the deleted SV40 sequences revealed no significant homology at the point of joining of the two genomes.

Animals↗
PubMed2025

Spatial transcriptomics-aided localization for single-cell transcriptomics with STALocator.

Single-cell RNA-sequencing (scRNA-seq) techniques can measure gene expression at single-cell resolution but lack spatial information. Spatial transcriptomics (ST) techniques simultaneously provide gene expression data and spatial information. However, the data quality of the spatial resolution or gene coverage is still much lower than the quality of the single-cell transcriptomics data. To this end, we develop a ST-Aided Locator for single-cell transcriptomics (STALocator) to localize single cells to corresponding ST data. Applications on simulated data showed that STALocator performed better than other localization methods. When applied to the human brain and squamous cell carcinoma data, STALocator could robustly reconstruct the relative spatial organization of critical cell populations. Moreover, STALocator could enhance gene expression patterns for Slide-seqV2 data and predict genome-wide gene expression data for fluorescence in situ hybridization (FISH) and Xenium data, leading to the identification of more spatially variable genes and more biologically relevant Gene Ontology (GO) terms compared with the raw data. A record of this paper's transparent peer review process is included in the supplemental information.

Single-Cell Analysis↗