scholarly journals No evidence for DNA N6-methyladenine in mammals

2020 ◽  
Vol 6 (12) ◽  
pp. eaay3335 ◽  
Author(s):  
Karolos Douvlataniotis ◽  
Maike Bensberg ◽  
Antonio Lentini ◽  
Björn Gylemo ◽  
Colm E. Nestor
Keyword(s):  
Mammalian Cell ◽  
Bacterial Contamination ◽  
Cell Types ◽  
Published Data ◽  
Dna Modification ◽  
Bioinformatics Analyses ◽  
Experimental Approaches ◽  
Technological Limitations ◽  
Rna Contamination

N6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals.

scholarly journals Northstar enables automatic classification of known and novel cell types from tumor samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Fabio Zanini ◽  
Bojk A. Berghuis ◽  
Robert C. Jones ◽  
Benedetta Nicolis di Robilant ◽  
Rachel Yuan Nong ◽  
...  
Keyword(s):  
Cell Types ◽  
Neoplastic Cell ◽  
Computational Approach ◽  
Pancreatic Tumors ◽  
Published Data ◽  
Cell Type ◽  
Disease Heterogeneity ◽  
Manual Task ◽  
Insight Into

Abstract Single cell transcriptomics is revolutionising our understanding of tissue and disease heterogeneity, yet cell type identification remains a partially manual task. Published algorithms for automatic cell annotation are limited to known cell types and fail to capture novel populations, especially cancer cells. We developed northstar, a computational approach to classify thousands of cells based on published data within seconds while simultaneously identifying and highlighting new cell states such as malignancies. We tested northstar on data from glioblastoma, melanoma, and seven different healthy tissues and obtained high accuracy and robustness. We collected eleven pancreatic tumors and identified three shared and five private neoplastic cell populations, offering insight into the origins of neuroendocrine and exocrine tumors. Northstar is a useful tool to assign known and novel cell type and states in the age of cell atlases.

scholarly journals Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death.

1996 ◽  
Vol 133 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
M D Jacobsen ◽  
M Weil ◽  
M C Raff
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Apoptotic Cell ◽  
Interleukin 1 ◽  
Cell Types ◽  
Cysteine Proteases ◽  
A Cell ◽  
Bona Fide

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.

scholarly journals Ensemble learning for classifying single-cell data and projection across reference atlases

2020 ◽  
Vol 36 (11) ◽  
pp. 3585-3587
Author(s):  
Lin Wang ◽  
Francisca Catalan ◽  
Karin Shamardani ◽  
Husam Babikir ◽  
Aaron Diaz
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Status Quo ◽  
Supplementary Information ◽  
Published Data ◽  
Supplementary Data ◽  
Cell Type ◽  
Low Sensitivity ◽  
Project Data ◽  
Cell Data

Abstract Summary Single-cell data are being generated at an accelerating pace. How best to project data across single-cell atlases is an open problem. We developed a boosted learner that overcomes the greatest challenge with status quo classifiers: low sensitivity, especially when dealing with rare cell types. By comparing novel and published data from distinct scRNA-seq modalities that were acquired from the same tissues, we show that this approach preserves cell-type labels when mapping across diverse platforms. Availability and implementation https://github.com/diazlab/ELSA Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

Platelet Microparticle Production Is Regulated By STIM1 Dependent Entry Of Extracellular Ca2+ Through Orai1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1059-1059
Author(s):  
Matthew T Duvernay ◽  
Heidi Hamm
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Receptor Agonist ◽  
Conflicts Of Interest ◽  
Lipid Vesicles ◽  
Cell Types ◽  
Published Data ◽  
Receptor Stimulation ◽  
Intense Staining ◽  
Collagen Receptor

Abstract Microparticles are submicron lipid vesicles packed with protein and nucleic acid. They are found in abundance circulating in the vasculature and can be generated from a number of cell types including endothelial cells, leukocytes, and platelets. The microparticles generated from platelets far out number those generated from other cell types and their incidence is correlated with a myriad of cardiovascular diseases. We recently demonstrated that stimulation of gel filtered human platelets through Protease activated receptor (PAR) 4 leads to the generation of 4-5 times more platelet microparticles (PMP) than PAR1 stimulation. Platelet microparticle (PMP) production was demonstrated to be downstream of a Rho kinase-dependent signaling pathway. Consistently, more myosinIIa phosphorylation was observed downstream of PAR4 stimulation. PMP generation was quantified by flow cytometry using polystyrene microbeads of standardized size for appropriate size gating, and CD41a (aIIb) and CD62p (P-selectin) staining for positive identification of platelet derived membranes. Submicron particles positive for both CD41a and CD62p were classified as true PMP. Recently, we have expanded these observations to platelet stimulation with thrombin, convulxin (a GPVI collagen receptor agonist), and dual PAR/collagen receptor stimulation. Thrombin and PAR4-activating peptide (AP) stimulation leads to equivalent levels of PMP production, confirming that PAR4 is the major thrombin receptor responsible for PMP generation, with PAR1 playing only a minor role. Collagen receptor stimulation with convulxin lead to a comparable level of PMP generation as thrombin stimulation. However, co-stimulation with convulxin and thrombin or convulxin and PAR1-AP or PAR4-AP lead to PMP production exceeding the sum of PMP by convulxin and PAR agonist alone by as much as 350%, suggesting a synergistic response. It is well documented that PMP generation does not occur in the absence of extracellular Ca2+. Recently published data collected with Orai1 knockout mice indicate that the majority of extracellular Ca2+ entry into the platelet is mediated by the plasma membrane Ca2+ channel Orai1. STIM1 is an ER calcium sensor that migrates near the cell surface upon depletion of intracellular Ca2+ stores to oligomerize and activate Orai1. In an attempt to further elucidate the signaling pathways responsible for PMP generation we treated platelets with the STIM1 inhibitor SKF 96365. Preincubation with SKF 96365 nearly abolished PMP production induced by PAR4-AP, thrombin, convulxin, or the combination of PAR and collagen receptor agonist. Scanning Electron Microscopy of PAR4-AP stimulated platelets in suspension revealed extended filipodia and bag-like structures protruding from the platelet core which correlated with the size of PMPs as analyzed by flow cytometry. Pretreatment with the SKF 96365 or exclusion of extracellular Ca2+ prevented the formation of the microparticle-like extensions and blunted filipodia extension. Finally, confocal analysis of Orai1 staining on platelets spread on a collagen matrix and co-stimulated with PAR4-AP revealed Orai1 throughout the plasma membrane with intense staining of the microparticle-like structures. These data suggest that PMP generation is nucleated by STIM1 dependent Orai1 Ca2+ entry. Current efforts are focused on elucidating the mechanism by which PAR and collagen receptor agonists differentially regulate STIM1 or Orai1 activity to mediate PMP generation. Disclosures: No relevant conflicts of interest to declare.

An X-ray microanalysis survey of the concentration of elements in the cytoplasm of different mammalian cell types

1979 ◽  
Vol 101 (3) ◽  
pp. 493-501 ◽  
Author(s):  
Ivan L. Cameron ◽  
Thomas B. Pool ◽  
Nancy K. R. Smith
Keyword(s):  
Mammalian Cell ◽  
Cell Types ◽  
X Ray

scholarly journals Cell-specific and region-specific transcriptomics in the multiple sclerosis model: Focus on astrocytes

2017 ◽  
Vol 115 (2) ◽  
pp. E302-E309 ◽  
Author(s):  
Noriko Itoh ◽  
Yuichiro Itoh ◽  
Alessia Tassoni ◽  
Emily Ren ◽  
Max Kaito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Sclerosis ◽  
Spinal Cord ◽  
Cholesterol Synthesis ◽  
Neurological Diseases ◽  
Cell Types ◽  
Specific Gene ◽  
Bioinformatics Analyses ◽  
Pathway Gene ◽  
A Cell

Changes in gene expression that occur across the central nervous system (CNS) during neurological diseases do not address the heterogeneity of cell types from one CNS region to another and are complicated by alterations in cellular composition during disease. Multiple sclerosis (MS) is multifocal by definition. Here, a cell-specific and region-specific transcriptomics approach was used to determine gene expression changes in astrocytes in the most widely used MS model, experimental autoimmune encephalomyelitis (EAE). Astrocyte-specific RNAs from various neuroanatomic regions were attained using RiboTag technology. Sequencing and bioinformatics analyses showed that EAE-induced gene expression changes differed between neuroanatomic regions when comparing astrocytes from spinal cord, cerebellum, cerebral cortex, and hippocampus. The top gene pathways that were changed in astrocytes from spinal cord during chronic EAE involved decreases in expression of cholesterol synthesis genes while immune pathway gene expression in astrocytes was increased. Optic nerve from EAE and optic chiasm from MS also showed decreased cholesterol synthesis gene expression. The potential role of cholesterol synthesized by astrocytes during EAE and MS is discussed. Together, this provides proof-of-concept that a cell-specific and region-specific gene expression approach can provide potential treatment targets in distinct neuroanatomic regions during multifocal neurological diseases.

scholarly journals Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing

Mutagenesis ◽  
2010 ◽  
Vol 25 (6) ◽  
pp. 531-538 ◽  
Author(s):  
James M. Parry ◽  
Elizabeth Parry ◽  
Pascal Phrakonkham ◽  
Raffaella Corvi
Keyword(s):  
Mammalian Cell ◽  
Published Data ◽  
Genotoxicity Testing

scholarly journals Microglia and monocytes in inflammatory CNS disease: integrating phenotype and function

2021 ◽  
Author(s):  
Alanna G. Spiteri ◽  
Claire L. Wishart ◽  
Roger Pamphlett ◽  
Giuseppe Locatelli ◽  
Nicholas J. C. King
Keyword(s):  
Viral Encephalitis ◽  
Neurological Diseases ◽  
Cell Types ◽  
Cell Type ◽  
Neuroinflammatory Disease ◽  
Experimental Systems ◽  
Immune Mediated ◽  
Experimental Approaches ◽  
And Function ◽  
Disease Specific

AbstractIn neurological diseases, the actions of microglia, the resident myeloid cells of the CNS parenchyma, may diverge from, or intersect with, those of recruited monocytes to drive immune-mediated pathology. However, defining the precise roles of each cell type has historically been impeded by the lack of discriminating markers and experimental systems capable of accurately identifying them. Our ability to distinguish microglia from monocytes in neuroinflammation has advanced with single-cell technologies, new markers and drugs that identify and deplete them, respectively. Nevertheless, the focus of individual studies on particular cell types, diseases or experimental approaches has limited our ability to connect phenotype and function more widely and across diverse CNS pathologies. Here, we critically review, tabulate and integrate the disease-specific functions and immune profiles of microglia and monocytes to provide a comprehensive atlas of myeloid responses in viral encephalitis, demyelination, neurodegeneration and ischemic injury. In emphasizing the differential roles of microglia and monocytes in the severe neuroinflammatory disease of viral encephalitis, we connect inflammatory pathways common to equally incapacitating diseases with less severe inflammation. We examine these findings in the context of human studies and highlight the benefits and inherent limitations of animal models that may impede or facilitate clinical translation. This enables us to highlight common and contrasting, non-redundant and often opposing roles of microglia and monocytes in disease that could be targeted therapeutically.

scholarly journals Power Analysis of Single Cell RNA-Sequencing Experiments

2016 ◽  
Author(s):  
Valentine Svensson ◽  
Kedar Nath Natarajan ◽  
Lam-Ha Ly ◽  
Ricardo J Miragaia ◽  
Charlotte Labalette ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Developmental Process ◽  
Cell Types ◽  
Rna Degradation ◽  
Published Data ◽  
Minute Amount ◽  
Trade Offs ◽  
Single Cell Rna Sequencing

AbstractHigh-throughput single cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, and has revealed new cell types, and new insights into developmental process and stochasticity in gene expression. There are now several published scRNA-seq protocols, which all sequence transcriptomes from a minute amount of starting material. Therefore, a key question is how these methods compare in terms of sensitivity of detection of mRNA molecules, and accuracy of quantification of gene expression. Here, we assessed the sensitivity and accuracy of many published data sets based on standardized spike-ins with a uniform raw data processing pipeline. We developed a flexible and fast UMI counting tool (https://github.com/vals/umis) which is compatible with all UMI based protocols. This allowed us to relate these parameters to sequencing depth, and discuss the trade offs between the different methods. To confirm our results, we performed experiments on cells from the same population using three different protocols. We also investigated the effect of RNA degradation on spike-in molecules, and the average efficiency of scRNA-seq on spike-in molecules versus endogenous RNAs.

scholarly journals Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase

2021 ◽  
Vol 118 (4) ◽  
pp. e2016778118
Author(s):  
Zebulon G. Levine ◽  
Sarah C. Potter ◽  
Cassandra M. Joiner ◽  
George Q. Fei ◽  
Behnam Nabet ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Mammalian Cell ◽  
Control Cell ◽  
Cell Types ◽  
Cell Physiology ◽  
Enzymatic Reactions ◽  
A Cell ◽  
Glcnac Transferase ◽  
Catalytic Functions

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT’s myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT’s enzymatic activities for cell viability. Using genetic complementation, we found that OGT’s glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT’s catalytic and noncatalytic activities affect protein abundance, we found that OGT’s noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

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