Single-cell analyses of nitrergic neurons in simple nervous systems

1999 ◽  
Vol 202 (4) ◽  
pp. 333-341 ◽  
Author(s):  
L.L. Moroz ◽  
R. Gillette ◽  
J.V. Sweedler
Keyword(s):  
Single Cell ◽  
Cell Model ◽  
Single Cells ◽  
Chemical Species ◽  
No Oxidation ◽  
Cell Level ◽  
Pharmacological Manipulations ◽  
Physiological And Biochemical

Understanding the role of the gaseous messenger nitric oxide (NO) in the nervous system is complicated by the heterogeneity of its nerve cells; analyses carried out at the single cell level are therefore important, if not critical. Some invertebrate preparations, most especially those from the gastropod molluscs, provide large, hardy and identified neurons that are useful both for the development of analytical methodologies and for cellular analyses of NO metabolism and its actions. Recent modifications of capillary electrophoresis (CE) allow the use of a small fraction of an individual neuron to perform direct, quantitative and simultaneous assays of the major metabolites of the NO-citrulline cycle and associated biochemical pathways. These chemical species include the products of NO oxidation (NO2-/NO3-), l-arginine, l-citrulline, l-ornithine, l-argininosuccinate, as well as selected NO synthase inhibitors and cofactors such as NADPH, biopterin, FMN and FAD. Diverse cotransmitters can also be identified in the same nitrergic neuron. The sensitivity of CE methods is in the femtomole to attomole range, depending on the species analysed and on the specific detector used. CE analysis can be combined with prior in vivo electrophysiological and pharmacological manipulations and measurements to yield multiple physiological and biochemical values from single cells. The methodologies and instrumentation developed and tested using the convenient molluscan cell model can be adapted to the smaller and more delicate neurons of other invertebrates and chordates.

scholarly journals Single-cell RNA-seq revealed the role of Alkbh5 in reproduction

2021 ◽  
Author(s):  
Xiaozhong Shen ◽  
Gangcai Xie
Keyword(s):  
Single Cell ◽  
Messenger Rna ◽  
Single Cells ◽  
Rna Seq ◽  
Single Cell Sequencing ◽  
Differential Gene ◽  
Higher Eukaryotes

AbstractN(6)-methyladenosine (m(6)a) is the most common internal modification of messenger RNA (mRNA) in higher eukaryotes. According to previous literature reports, alkbh5, as another demethylase in mammals, can reverse the expression of m(6)a gene in vivo and in vitro. In order to reveal the effect of Alkbh5 deletion on the level of single cells in the testis during spermatogenesis in mice, the data were compared using single-cell sequencing. In this article, we discussed the transcription profile and cell type identification of mouse testis, the expression of mitochondrial and ribosomal genes in mice, the analysis of differential gene expression, and the effects of Alkbh5 deletion, and try to explain the role and influence of Alkbh5 on reproduction at the level of single-cell sequencing.

scholarly journals SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

scholarly journals PapRIV, a BV-2 microglial cell activating quorum sensing peptide

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yorick Janssens ◽  
Nathan Debunne ◽  
Anton De Spiegeleer ◽  
Evelien Wynendaele ◽  
Marta Planas ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Model ◽  
Dependent Manner ◽  
Communication Signals ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

Abstract 257: Clonal Analysis Of Multipotent Cardiovascular Progenitors That Generate Cardiomyocytes During Development

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Konstantina Ioanna Sereti ◽  
Paniz Kamran Rashani ◽  
Peng Zhao ◽  
Reza Ardehali
Keyword(s):  
Single Cell ◽  
Heart Development ◽  
Cardiac Development ◽  
Clonal Analysis ◽  
Cardiac Cells ◽  
Single Cell Level ◽  
Reporter System ◽  
Cell Level ◽  
Cardiac Progenitors

It has been proposed that cardiac development in lower vertebrates is driven by the proliferation of cardiomyocytes. Similarly, cycling myocytes have been suggested to direct cardiac regeneration in neonatal mice after injury. Although, the role of cardiomyocyte proliferation in cardiac tissue generation during development has been well documented, the extent of this contribution as well as the role of other cell types, such as progenitor cells, still remains controversial. Here we used a novel stochastic four-color Cre-dependent reporter system (Rainbow) that allows labeling at a single cell level and retrospective analysis of the progeny. Cardiac progenitors expressing Mesp1 or Nkx2.5 were shown to be a source of cardiomyocytes during embryonic development while the onset of αMHC expression marked the developmental stage where the capacity of cardiac cells to proliferate diminishes significantly. Through direct clonal analysis we provide strong evidence supporting that cardiac progenitors, as opposed to mature cardiomyocytes, are the main source of cardiomyocytes during cardiac development. Moreover, we have identified quadri-, tri-, bi, and uni-potent progenitors that at a single cell level can generate cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Although existing cardiomyocytes undergo limited proliferation, our data indicates that it is mainly the progenitors that contribute to heart development. Furthermore, we show that the limited proliferation capacity of cardiomyocytes observed during normal development was enhanced following neonatal cardiac injury allowing almost complete regeneration of the scared tissue. However, this ability was largely absent in adult injured hearts. Detailed characterization of dividing cardiomyocytes and proliferating progenitors would greatly benefit the development of novel therapeutic options for cardiovascular diseases.

scholarly journals Direct observation of frequency modulated transcription in single cells using light activation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Daniel R Larson ◽  
Christoph Fritzsch ◽  
Liang Sun ◽  
Xiuhau Meng ◽  
David S Lawrence ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Cell Model ◽  
Single Cells ◽  
Single Gene ◽  
Gene Activation ◽  
Light Activation ◽  
Dynamic Synthesis ◽  
Single Cell Model

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.

scholarly journals High Throughput Immunophenotyping and Expression Profiling at Single Cell Level Reveal BCR-ABL1 Dependent Surface Markers of Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2920-2920
Author(s):  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Peter Taus ◽  
Jiri Mayer ◽  
Martin Culen
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Single Cells ◽  
Transcript Level ◽  
Surface Expression ◽  
Surface Markers ◽  
Cell Level ◽  
Tki Treatment

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

scholarly journals Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 285
Author(s):  
Eszter Széles ◽  
Krisztina Nagy ◽  
Ágnes Ábrahám ◽  
Sándor Kovács ◽  
Anna Podmaniczki ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Single Cell ◽  
Single Cells ◽  
Fluorescence Induction ◽  
Photochemical Quenching ◽  
Support Surface ◽  
Single Cell Level ◽  
Microfluidic Platforms ◽  
Cell Level ◽  
Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

scholarly journals Single-cell tumor-immune microenvironment of BRCA1/2 mutated high-grade serous ovarian cancer

2021 ◽  
Author(s):  
Inga-Maria Launonen ◽  
Nuppu Lyytikäinen ◽  
Julia Casado ◽  
Ella Anttila ◽  
Angéla Szabó ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Single Cells ◽  
High Grade ◽  
Prognostic Role ◽  
Immune Microenvironment ◽  
Proteomic Data ◽  
Tumor Immune Microenvironment ◽  
Cellular Phenotypes

Abstract The majority of high-grade serous ovarian cancers (HGSCs) are deficient in homologous recombination (HR) DNA repair, most commonly due to mutations or hypermethylation of the BRCA1/2 genes. We aimed to discover how BRCA1/2 mutations shape the cellular phenotypes and spatial interactions of the tumor microenvironment. Using a highly multiplex immunofluorescence and image analysis we generated spatial proteomic data for 21 markers in 124,623 single cells from 112 tumor cores originating from 31 tumors with BRCA1/2 mutation (BRCA1/2mut), and from 13 tumors without alterations in HR genes (HRwt). We identified a phenotypically distinct tumor microenvironment in the BRCA1/2mut tumors with evidence of increased immunosurveillance. Importantly, we found an opposing prognostic role of a proliferative tumor-cell phenotypic subpopulation in the HR-genotypes, which associated with enhanced spatial tumor-immune interactions by the CD8+ and CD4+T-cells in BRCA1/2mut tumors. The single-cell spatial landscapes indicate distinct patterns of spatial immunosurveillance with the premise to improve immunotherapeutic strategies and patient stratification in HGSC.

scholarly journals Performance Comparison of Reverse Transcriptases for Single-Cell Studies

2019 ◽  
Vol 66 (1) ◽  
pp. 217-228 ◽  
Author(s):  
Daniel Zucha ◽  
Peter Androvic ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Single Cell ◽  
Reverse Transcription ◽  
Cell Model ◽  
Single Cells ◽  
Performance Comparison ◽  
Reaction Yield ◽  
Reverse Transcriptases ◽  
Reverse Transcription Pcr ◽  
Cell Experiment ◽  
Almost All

Abstract BACKGROUND Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. METHODS We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. RESULTS All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%–90% reaction positivity rate) and in their absolute reaction yield (7.3%–137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H− and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H−) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). CONCLUSIONS Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.

scholarly journals Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

2017 ◽  
Vol 114 (18) ◽  
pp. E3659-E3668 ◽  
Author(s):  
Ann Wiegand ◽  
Jonathan Spindler ◽  
Feiyu F. Hong ◽  
Wei Shao ◽  
Joshua C. Cyktor ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Single Cells ◽  
Constitutive Expression ◽  
Multiple Time ◽  
Hiv Rna ◽  
Infected Cells ◽  
The Impact ◽  
Hiv 1

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

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