scholarly journals Unfolding and identification of membrane proteins from native cell membranes

2019 ◽  
Author(s):  
Nicola Galvanetto ◽  
Sourav Maity ◽  
Nina Ilieva ◽  
Zhongjie Ye ◽  
Alessandro Laio ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Single Cells ◽  
Total Content ◽  
Cell Types ◽  
Single Cell Level ◽  
Cell Level ◽  
Single Molecule Force Spectroscopy ◽  
Native Cell ◽  
Different Cell Types

AbstractIs the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or can it provide useful information even for real biological samples? Here, we describe a pipeline for analyzing native membranes based on high throughput single-molecule force spectroscopy. The protocol includes a technique for the isolation of the plasma membrane of single cells. Afterwards, one harvests hundreds of thousands SMSF traces from the sample. Finally, one characterizes and identifies the embedded membrane proteins. This latter step is the cornerstone of our approach and involves combining, within a Bayesian framework, the information of the shape of the SMFS Force-distance which are observed more frequently, with the information from Mass Spectrometry and from proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost single-cell level of resolution.

scholarly journals scAPAdb: a comprehensive database of alternative polyadenylation at single-cell resolution

2021 ◽  
Author(s):  
Sheng Zhu ◽  
Qiwei Lian ◽  
Wenbin Ye ◽  
Wei Qin ◽  
Zhe Wu ◽  
...  
Keyword(s):  
Single Cell ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Single Cell Level ◽  
Cell Heterogeneity ◽  
Rna Seq ◽  
Cell Level ◽  
Eukaryotic Gene ◽  
User Friendly ◽  
Different Cell Types

Abstract Alternative polyadenylation (APA) is a widespread regulatory mechanism of transcript diversification in eukaryotes, which is increasingly recognized as an important layer for eukaryotic gene expression. Recent studies based on single-cell RNA-seq (scRNA-seq) have revealed cell-to-cell heterogeneity in APA usage and APA dynamics across different cell types in various tissues, biological processes and diseases. However, currently available APA databases were all collected from bulk 3′-seq and/or RNA-seq data, and no existing database has provided APA information at single-cell resolution. Here, we present a user-friendly database called scAPAdb (http://www.bmibig.cn/scAPAdb), which provides a comprehensive and manually curated atlas of poly(A) sites, APA events and poly(A) signals at the single-cell level. Currently, scAPAdb collects APA information from > 360 scRNA-seq experiments, covering six species including human, mouse and several other plant species. scAPAdb also provides batch download of data, and users can query the database through a variety of keywords such as gene identifier, gene function and accession number. scAPAdb would be a valuable and extendable resource for the study of cell-to-cell heterogeneity in APA isoform usages and APA-mediated gene regulation at the single-cell level under diverse cell types, tissues and species.

scholarly journals Endothelial cells express the interleukin-1 receptor type I

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1262-1267
Author(s):  
D Boraschi ◽  
A Rambaldi ◽  
A Sica ◽  
P Ghiara ◽  
F Colotta ◽  
...  
Keyword(s):  
Binding Sites ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cross Linking ◽  
Type I ◽  
Single Cell Level ◽  
Vascular Cells ◽  
Cell Level ◽  
Myelomonocytic Cells ◽  
Different Cell Types

Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL- 1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.

scholarly journals Endothelial cells express the interleukin-1 receptor type I

Blood ◽  
1991 ◽  
Vol 78 (5) ◽  
pp. 1262-1267 ◽  
Author(s):  
D Boraschi ◽  
A Rambaldi ◽  
A Sica ◽  
P Ghiara ◽  
F Colotta ◽  
...  
Keyword(s):  
Binding Sites ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cross Linking ◽  
Type I ◽  
Single Cell Level ◽  
Vascular Cells ◽  
Cell Level ◽  
Myelomonocytic Cells ◽  
Different Cell Types

Abstract Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL- 1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.

scholarly journals Single-molecule long-read sequencing reveals the chromatin basis of gene expression

2019 ◽  
Author(s):  
Yunhao Wang ◽  
Anqi Wang ◽  
Zujun Liu ◽  
Andrew Thurman ◽  
Linda S. Powers ◽  
...  
Keyword(s):  
Single Cell ◽  
Long Range ◽  
Single Molecule ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Single Cell Level ◽  
Dna Molecules ◽  
Cell Level ◽  
Single Dna Molecules ◽  
Long Read

ABSTRACTGenome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remains poorly studied at the single-cell level. Here we present a new experimental approach MeSMLR-seq (methyltransferase treatment followed by single-molecule long-read sequencing) for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules, and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding transcription start site for silently- and actively-transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin statuses spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly-heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming.

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 transcriptomic analysis elucidates APOE genotype specific changes across cell types in two brain regions in Alzheimer’s disease

2021 ◽  
Author(s):  
Stella Belonwu ◽  
Yaqiao Li ◽  
Daniel Bunis ◽  
Arjun Arkal Rao ◽  
Caroline Warly Solsberg ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Apoe Genotype ◽  
Cell Types ◽  
Brain Regions ◽  
Single Cell Level ◽  
Cell Level ◽  
Single Nucleus

Abstract Alzheimer’s Disease (AD) is a complex neurodegenerative disease that gravely affects patients and imposes an immense burden on caregivers. Apolipoprotein E4 (APOE4) has been identified as the most common genetic risk factor for AD, yet the molecular mechanisms connecting APOE4 to AD are not well understood. Past transcriptomic analyses in AD have revealed APOE genotype-specific transcriptomic differences; however, these differences have not been explored at a single-cell level. Here, we leverage the first two single-nucleus RNA sequencing AD datasets from human brain samples, including nearly 55,000 cells from the prefrontal and entorhinal cortices. We observed more global transcriptomic changes in APOE4 positive AD cells and identified differences across APOE genotypes primarily in glial cell types. Our findings highlight the differential transcriptomic perturbations of APOE isoforms at a single-cell level in AD pathogenesis and have implications for precision medicine development in the diagnosis and treatment of AD.

scholarly journals Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

2020 ◽  
Vol 11 (25) ◽  
pp. 6352-6361 ◽  
Author(s):  
Kaixiang Zhang ◽  
Shangshang Qin ◽  
Sixuan Wu ◽  
Yan Liang ◽  
Jinghong Li
Keyword(s):  
Single Cell ◽  
Antibiotic Treatment ◽  
Single Molecule ◽  
Antibiotic Susceptibility ◽  
Single Cell Level ◽  
Microfluidic Systems ◽  
Cell Level ◽  
Single Molecule Level ◽  
Recent Developments ◽  
Susceptibility Tests

Recent developments of microfluidics-based antibiotic susceptibility tests (ASTs) at the single-cell or single-molecule level are summarized for guiding antibiotic treatment.

scholarly journals Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

2009 ◽  
Vol 75 (13) ◽  
pp. 4550-4556 ◽  
Author(s):  
Vicky G. Kastbjerg ◽  
Dennis S. Nielsen ◽  
Nils Arneborg ◽  
Lone Gram
Keyword(s):  
Listeria Monocytogenes ◽  
Single Cell ◽  
Intracellular Ph ◽  
Bacterial Population ◽  
Single Cells ◽  
Viable Cell ◽  
Population Subdivision ◽  
Single Cell Level ◽  
Cell Level ◽  
Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

scholarly journals Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification

2011 ◽  
Vol 57 (7) ◽  
pp. 1032-1041 ◽  
Author(s):  
Thomas Kroneis ◽  
Jochen B Geigl ◽  
Amin El-Heliebi ◽  
Martina Auer ◽  
Peter Ulz ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Molecular Genetic ◽  
Target Cells ◽  
Whole Genome ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Amplification

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

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