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PubMed1994

Recycling the single cell to detect specific chromosomes and to investigate specific gene sequences.

We have developed a new procedure, called cell recycling, which combines the two powerful techniques of polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on the same single cell. A fixed cell is used as the DNA template for PCR, prior to the FISH analysis. Using single blastomeres from mouse embryos as a model system, cell recycling procedures detect the single-copy beta-haemoglobin gene sequence at an efficiency of 70% as well as sex chromosome constitution at an efficiency of 74% in the same single cell. Cell recycling will increase the success rate of pregnancy following preimplantation diagnosis for a specific gene defect by identifying embryos with chromosomal abnormalities and eliminating them from the transfer procedure.

Animals↗

PubMed1998

Analysis of HLA-DQ alpha sequences for prenatal diagnosis in single fetal cells from maternal blood.

We have extended a previously developed method that allows prenatal DNA diagnosis of female fetuses through the isolation of single nucleated erythrocytes from maternal blood by developing a method that can distinguish between maternal and fetal nucleated erythrocytes. Nucleated erythrocytes were separated by a density-gradient method and then collected by micromanipulation. Sex was determined after primer extension preamplification (PEP) of the entire genome of a single cell, and human leukocyte antigen (HLA)-DQ alpha type was determined after further amplification of this gene. The HLA-DQ alpha genotype of fetal erythrocytes in maternal blood samples and their corresponding paternal and maternal lymphocytes were successfully determined in all cases. The accuracy of the method was determined by using single nucleated erythrocytes from umbilical cord blood from five normal deliveries. This is the first demonstration that the fetal HLA-DQ alpha gene sequences can be identified in a small aliquot of a single nucleated erythrocyte in maternal blood. We believe that this method ushers in a new era in which the reliability and accuracy of noninvasive prenatal DNA diagnosis from maternal blood is markedly improved.

Alleles↗

PubMed1999

The first dose of a Haemophilus influenzae type b conjugate vaccine reactivates memory B cells: evidence for extensive clonal selection, intraclonal affinity maturation, and multiple isotype switches to IgA2.

The Ab response of a healthy adult to the first dose of a Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugate vaccine was studied at the level of Ig gene usage by circulating Ab-secreting cells. Forty-one IgA and 17 IgG mRNA sequences were obtained. The major part of the response was confined to IgA Ab-secreting cells, and 72% of the IgA sequences were derived from the progeny of a single rearranged B cell. These sequences could be arranged in a genealogical tree showing multiple somatic mutations and at least two intraclonal isotype switches to IgA2. Fourteen somatic mutations were shared by this clonal progeny, indicating that extreme clonal selection had occurred early in the clonal development. Taking into account the frequency of somatic mutations and the clone size, it was evident that the responding cell population must have originated from a mutated, highly selected, and expanded population of cells existing before vaccination, i.e., memory B cells. The dominating heavy and light chains of the response were combined in a Fab that bound HibCP. It was shown that the shared heavy and light chain mutations increased the affinity for HibCP considerably, indicating that the clonal selection had been driven by affinity. Pre-existing memory cells in unvaccinated adults may explain several features of Ab responses to polysaccharide vaccines and may play a role in acquiring the ability to respond to pure polysaccharides during infancy.

Adult↗

PubMed1983

Two homologous cytochromes b5 in a single cell.

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Ozols and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704, 163-173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight microsomal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine-15 by arginine. This paper brings the final proof that two iso-cytochromes b5 exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed.

Amino Acid Sequence↗

PubMed1998

Localization of trinucleotide repeat sequences in myotonic dystrophy cells using a single fluorochrome-labeled PNA probe.

A labeled peptide nucleic acid (PNA) antisense probe was used to study the spatial distribution of triplet repeats (CTG) in human myotonic dystrophy (DM) cells by high-resolution fluorescence in situ hybridization (FISH). It was found that transcripts containing triplet repeats were present as a number of discrete foci in the DM nuclei. Greater numbers of foci were visible with the PNA probe than a comparable DNA probe. The PNA probe was also used to visualize the triplet-repeat expansion within the DM gene located on chromosome 19. Using the intensity of the expanded triplet-repeat on the chromosomes as a reference, it was estimated there were between 15-230 RNA molecules in each focus observed in DM nuclei.

Cell Nucleus↗

PubMed2025

Coalescing single-cell genomes and transcriptomes to decode breast cancer progression.

Understanding epithelial lineages of breast cancer and genotype-phenotype relationships requires direct measurements of the genome and transcriptome of the same single cells at scale. To achieve this, we developed wellDR-seq, a high-genomic-resolution, high-throughput method to simultaneously profile the genome and transcriptome of thousands of single cells. We profiled 33,646 single cells from 12 estrogen-receptor-positive breast cancers and identified ancestral subclones in multiple patients that showed a luminal hormone-responsive lineage, indicating a potential cell of origin. In contrast to bulk studies, wellDR-seq enabled the study of subclone-level gene-dosage relationships, which showed near-linear correlations in large chromosomal segments and extensive variation at the single-gene level. We identified dosage-sensitive and dosage-insensitive genes, including many breast cancer genes as well as sporadic copy-number aberrations in non-cancer cells. Overall, these data reveal complex relationships between copy number and gene expression in single cells, improving our understanding of breast cancer progression.

Breast Neoplasms↗

PubMed2020

Genomic Analysis of Circulating Tumor Cells at the Single-Cell Level.

Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification-based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non-small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non-small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.

Carcinoma, Non-Small-Cell Lung↗

PubMed2026

Immature Neutrophil Programs Associate With Burn Mortality and Extend Across Critical Illnesses.

Severe burns provoke a systemic "genomic storm," yet cell states associated with divergent outcomes remain unclear. We profiled blood cells by single-cell RNA-Sequencing (73 014 cells) from adult patients with burn injuries within postburn day 17 (n = 4) and healthy donors (n = 5), integrated data with bulk signatures of burn size, inhalation injury, and mortality, and evaluated clinical associations in the American Burn Association National Burn Repository. Burn was associated with emergency hematopoiesis marked by expansion of hematopoietic stem/progenitor-like cells, immature neutrophils, and plasmablast/plasma cell states, alongside depletion of naïve CD4+/CD8+ T cells and dendritic cells. Larger burns (>20% TBSA) showed enrichment of humoral transcriptional programs, including plasmablast/plasma cell activation and suppression of cytotoxic CD8+ T-cell states. In multivariable models, inhalation injury was a stronger predictor of death (adjusted odds ratio [OR] 1.9) than burn size (adjusted OR 1.1) and shared greater overlap with the most perturbed single cells in non-survivors; 55% of co-perturbed cells were neutrophils, implicating granulocyte dysregulation as a common lethal axis. We identified a neutrophil-specific 5-gene panel (OLFM4, RETN, LCN2, ARG1, and BTNL3) that discriminated survivors vs non-survivors after burns (area under the curve [AUC] > 0.9) and generalized to trauma (n = 158; AUC 0.81) and intensive care unit COVID-19 (n = 103; AUC 0.75), providing information orthogonal to conventional biomarkers and severity scores. Cytomorphology corroborated transcriptomic immaturity, with ~2-fold higher band neutrophils and larger neutrophil size in a fatal case. Computational drug-reversal analysis highlighted galectin-1 inhibition as a candidate modulator of mortality-associated neutrophil programs. Together, our findings suggest that immature neutrophils represent a shared immune feature across severe burns and other forms of critical illness.

Humans↗

PubMed2002

Very low frequencies of human normal CD34+ haematopoietic progenitor cells express the Wilms' tumour gene WT1 at levels similar to those in leukaemia cells.

The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT-PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1.3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT-PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1.1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1.2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+CD38- and CD34+CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38- and lineage-committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells.

Antigens, CD34↗

PubMed1986

Eukaryotic initiation factor 4D. Purification from human red blood cells and the sequence of amino acids around its single hypusine residue.

Eukaryotic initiation factor 4D (eIF-4D) was purified from human red blood cells by a simple 5-step procedure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the preparations of eIF-4D were composed of variable amounts of two closely migrating forms of the factor, each of which contained a single residue of the unique amino acid hypusine. The structural similarity of the two forms of human eIF-4D was evidenced by the indistinguishable patterns of radioactivity on peptide maps of tryptic digests prepared from radioiodinated samples. A peptide containing the single hypusine residue was readily isolated from a tryptic digest of human eIF-4D by virtue of its high positive charge and hydrophilic character. Amino acid sequence determination on this peptide revealed the following primary structure around hypusine: Thr-Gly-hypusine-His-Gly-His-Ala-Lys.

Amino Acid Sequence↗

PubMed1996

RhD genotype determination by single sperm cell analysis.

OBJECTIVE: An Rh-negative woman with preexisting anti-D antibodies may affect some or all subsequent fetuses, depending on the genotype of her Rh-positive partner. Currently, a reliable technique for an absolute determination of RhD genotype is not available. This study was initiated to develop an accurate method for RhD genotyping in men. STUDY DESIGN: RhD genotype was determined by deoxyribonucleic acid amplification of a D-specific sequence in single sperm cell samples. Micromanipulation techniques were used for sampling of single sperm cells, which were further amplified by multiplex nested polymerase chain reaction at the RhD locus. A RhD sequence amplification product was expected in all of the successfully amplified samples from Rh-positive homozygotes, in some of the samples from heterozygotes, and in none of the samples form Rh-negative subjects. RESULTS: RhD genotype was accurately determined in 10 of 10 donors. A total of 132 single sperm cells were analyzed (8 to 17 samples per donor), of which 96 were successfully amplified as assessed by an internal control. As expected, the specific region of the RhD gene was amplified in all, some, and none of the signal-positive sperm samples from Rh-positive homozygotes, heterozygotes, and Rh-negative subjects, respectively, allowing accurate determination of the genotype. CONCLUSION: An accurate diagnosis of the RhD genotype can be attained from single sperm cell analysis by means of polymerase chain reaction and may have major clinical applications in the management of Rh isoimmunization.

Base Sequence↗

PubMed1995

Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin.

The cDNA encoding the receptor for gonadoliberin (GnRH or LH-RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK-. By using a dicistronic expression strategy utilizing the internal ribosomal-entry-site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation-binding experiments, the gonadoliberin antagonist Cetrorelix showed high-affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I-Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten-times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK- cells coupled to the inositol phosphate signal-transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5-trisphosphate in a dose-dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth-arrested cells, a transient expression of the c-fos protooncogene was induced by gonadoliberin or [D-Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal-transduction pathway in this heterologous cell system.

Amino Acid Sequence↗

PubMed1977

Single-stranded DNA from oncornavirus-infected cells enriched in virus-specific DNA sequences.

We previously found that a minor fraction of single-stranded DNA (ss-DNA) isolated from native nuclear DNA of normal chicken embryonic cells and cells of other species hybridized with bulk nuclear DNA or cellular RNA in great excess. At least one-third of ss-DNA belonging to the nonrepetitious part of the cell genome could be hybridized to homologous RNAs. In the present work, similar results were obtained with ss-DNA from cells of chickens infected by avian myeloblastosis virus (AMV). To investigate whether this enrichment of ss-DNA in transcribed DNA sequences involves provirus DNA, radioactive AMV RNA and cDNA copies of AMV RNA were used. Most of the 70S AMV RNA hybridized much faster to ss-DNA from productively infected leukemic cells than to bulk DNA. cDNA, either double-stranded or single-stranded, made in the presence of actinomycin D hybridized to total nuclear DNA with similar kinetics. In contrast, about half of the double-stranded cDNA molecules hybridized 40-50 times faster to ss-DNA than to total DNA, indicating that only one of the provirus DNA strands seems to be present in ss-DNA. This was confirmed by the fact that relatively insignificant amounts of the ss-cDNA molecules made in the presence of actinomycin D could be annealed to ss-DNA as compared with bulk DNA. These results indicate that actively transcribed DNA sequences can be selectively distributed in the ss-DNA fraction, probably because of single strand breaks in the vicinity of transcription sites.

Animals↗

PubMed2026

Elevated intron retention implicates neuroinflammation in brains of individuals with alcohol use disorder.

Intron retention, a form of alternative RNA splicing, can occur as part of normal gene regulation or result from disruption of the splicing machinery. Retained introns can potentially form double-stranded RNA, activating innate immune sensors and inflammation. This mechanism has been implicated in cancer but has not been studied in neuropsychiatric diseases like alcohol use disorder. We systematically analysed transcriptome-wide intron retention events in post-mortem brain tissue from 142 individuals (66 with alcohol use disorder and 76 controls), encompassing 320 region-specific samples from the superior frontal cortex, nucleus accumbens, central nucleus and basolateral amygdala. Analyses were adjusted for demographic, technical and biological covariates. Validation was performed in alcohol-preferring (P) rats using long-read sequencing. In complementary experiments, immunofluorescent staining was used to detect double-stranded RNA in rat brain tissue, while single-cell RNA-sequencing was performed to test activation of double-stranded RNA-sensing pathways in human brains. Brains from individuals with alcohol use disorder showed significantly higher total intron retention compared with controls, independent of age, with females showing greater increases than males. A total of 368 introns were positively associated with alcohol use disorder, and these introns were significantly longer and had weaker splice acceptor sites compared with non-associated introns. Genes harbouring these intron retention events were enriched in Purkinje neurons, visual cortex neurons and oligodendrocytes. Computational predictions indicated these long introns could form duplex RNA structures. Increased double-stranded RNA was confirmed experimentally in multiple brain regions of alcohol-consuming rats, where it co-localized primarily with neuronal nuclei and dendrites. In individuals with alcohol use disorder, we found that multiple pathways including double-stranded RNA responses, neuroinflammation, interferon and NF-κB signalling, adaptive immunity and apoptosis were activated. In addition, NeuN-positive neuronal counts significantly decreased in both the prefrontal and visual cortices. Furthermore, single-cell analysis demonstrated upregulation of TICAM1, the target of double-stranded RNA sensor TLR3, in oligodendrocytes, as well as widespread activation of downstream inflammatory pathways across glial and neuronal cell types. These findings provide the first evidence that chronic alcohol consumption promotes an overall increase of intron retention in the brain and is associated with the presence of double-stranded RNA. Furthermore, the double-stranded RNA may contribute to neuronal loss and brain pathology by activating a neuroinflammatory response.

alcohol use disorder↗

PubMed2025

Single-cell mutational burden distributions in birth-death processes.

Genetic mutations are footprints of cancer evolution and reveal critical dynamic parameters of tumour growth, which otherwise are hard to measure in vivo. The mutation accumulation in tumour cell populations has been described by various statistics, such as site frequency spectra (SFS), single-cell division distributions (DD) and mutational burden distributions (MBD). While DD and SFS have been intensively studied in phylogenetics especially after the development of whole genome sequencing technology of bulk samples, MBD has drawn attention more recently with the single-cell sequencing data. Although those statistics all arise from the same somatic evolutionary process, an integrated understanding of these distributions is missing and requires novel mathematical tools to better inform the ecological and evolutionary dynamics of tumours. Here we introduce dynamical matrices to analyse and unite the SFS, DD and MBD and derive recurrence relations for the expectations of these three distributions. While we successfully recover classic exact results in pure-birth cases for the SFS and the DD through our new framework, we derive a new expression for the MBD and approximate all three distributions when death is introduced. We demonstrate a natural link between the SFS and the single-cell MBD, and show that the MBD can be regenerated through the DD. Counter-intuitively, the single-cell MBD is mainly driven by the stochasticity arising in the DD, rather than the extra stochasticity in the number of mutations at each cell division.

Single-Cell Analysis↗

PubMed2006

Sequence- and concentration-dependent effects of acute and long-term exposure to the bisphosphonate ibandronate in combination with single and multiple fractions of ionising radiation doses in human breast cancer cell lines.

Both bisphosphonates and radiotherapy are highly effective for the management of bone metastases. Our in vitro study examined the cytotoxic effects resulting from combinations of ibandronate and ionising radiations (RX) in various sequences on breast cancer cells. Single radiation doses were given before, at halftime of, or after acute ibandronate incubation (48 h). Single or fractionated radiation doses were applied at the end of chronic ibandronate incubation (5 weeks). Combination of acute ibandronate exposure and single radiation doses led to synergistic cytotoxic effects in MDA-MB-231 cell line, but only with low ibandronate concentrations in MCF-7 cell line. In both cell lines, synergy was more marked when ibandronate followed RX. After long-term ibandronate exposure, only high single radiation doses induced synergistic effects in MDA-MB-231 cell line. Synergy was only detected with low ibandronate concentrations in MCF-7 cell line. In both cell lines, fractionated radiation doses exerted similar effects. The combination of ibandronate with radiation can exert synergistic effects on the inhibition of breast cancer cells growth, depending on cell line, drug sequence and dosage. Our data might provide a rationale for associating bisphosphonates and radiotherapy for the treatment of bone metastases from breast cancer.

Antineoplastic Agents↗

PubMed2002

CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis.

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.

Actins↗

PubMed2003

Single cell lineage analysis in human focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a developmental malformation of the human cerebral cortex that is closely associated with epilepsy. Dysplastic, 'balloon' and heterotopic neurons that comprise FCD are heterogeneous cell populations that exhibit abnormal morphologies. A pivotal unanswered question is how these cell types are generated during cortical development. As a strategy to define the lineage relationships between cells in FCD, the size of a heterozygous trinucleotide (CAG) repeat sequence within the X-chromosome encoded androgen receptor (XAR) mRNA was determined by RT-PCR and direct sequencing in single, microdissected cells from six female patients with FCD compared with control cortex. As a consequence of X-chromosome inactivation, only one of the two XAR alleles is expressed in all somatic cells, and cell types derived from distinct progenitors will express XAR CAG repeat lengths of differing size. Disparate XAR CAG repeat lengths were detected with equal frequency in single dysplastic, 'balloon' and heterotopic neurons in FCD whereas the XAR CAG repeats lengths in control cortex were identical in 70-80% of closely apposed neurons. These results support a random X-inactivation pattern in FCD. We propose that dysplastic, 'balloon' and heterotopic neurons in FCD derive from a population of progenitor cells or post-mitotic neurons during cortical development.

Adult↗