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1.
Methods Mol Biol ; 2778: 43-52, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478270

RESUMO

Numerous bioinformatics tools allow predicting the localization of membrane proteins in the outer or inner membrane of Escherichia coli with high precision. Nevertheless, it might be desirable to experimentally verify such predictions or to assay the correct localization of recombinant or mutated variants of membrane proteins. Here we describe two methods (preferential detergent solubilization and sucrose-gradient fractionation) that allow to fractionate Gram-negative bacterial membranes and subsequently to enrich inner or outer membrane proteins.


Assuntos
Escherichia coli , Proteínas de Membrana , Membrana Celular , Escherichia coli/genética , Bactérias Gram-Negativas , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias , Fracionamento Celular/métodos
2.
Anal Biochem ; 687: 115445, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38135241

RESUMO

REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.


Assuntos
Núcleo Celular , Mitocôndrias , Humanos , Camundongos , Animais , Fracionamento Celular/métodos , Mitocôndrias/metabolismo , Citoplasma/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Frações Subcelulares/metabolismo
3.
Methods Mol Biol ; 2718: 253-269, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37665464

RESUMO

The identification of the molecular composition of extracellular vesicles (EV) by omics approaches, including proteomics, requires the separation of EV from non-EV confounding factors present in the source biofluid. In this protocol, we present the sequential implementation of density gradient ultracentrifugation and size-exclusion chromatography to prepare EV from cell-conditioned medium with high specificity and repeatability. This approach enables the recovery of intact purified EV suited for downstream functional assays and biomarker discovery by omics approaches.


Assuntos
Técnicas Citológicas , Vesículas Extracelulares , Vesículas Extracelulares/química , Fracionamento Celular , Meios de Cultivo Condicionados , Humanos , Técnicas Citológicas/métodos , Proteômica , Centrifugação com Gradiente de Concentração , Cromatografia em Gel
4.
Nat Commun ; 14(1): 5252, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37644046

RESUMO

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.


Assuntos
Endossomos , Humanos , Células HeLa , Reprodutibilidade dos Testes , Fracionamento Celular , Espectrometria de Massas
5.
Methods Mol Biol ; 2654: 159-167, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37106182

RESUMO

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.


Assuntos
Proteínas , Proteína 2 Associada à Membrana da Vesícula , Camundongos , Animais , Fracionamento Celular/métodos , Proteína 2 Associada à Membrana da Vesícula/análise , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas/metabolismo , Técnicas Citológicas , Organelas , Centrifugação com Gradiente de Concentração/métodos , Grânulos Citoplasmáticos , Frações Subcelulares/metabolismo
6.
J Cell Biol ; 222(6)2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-36920247

RESUMO

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.


Assuntos
Fracionamento Celular , Núcleo Celular , Proteoma , Animais , Fracionamento Celular/métodos , Linhagem Celular , Núcleo Celular/química , Mamíferos , Proteoma/análise , Proteômica/métodos , Citoplasma/química
7.
Cell Rep ; 42(3): 112211, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36884350

RESUMO

Stress granules (SGs) and processing bodies (PBs) are membraneless cytoplasmic assemblies regulating mRNAs under environmental stress such as viral infections, neurological disorders, or cancer. Upon antigen stimulation, T lymphocytes mediate their immune functions under regulatory mechanisms involving SGs and PBs. However, the impact of T cell activation on such complexes in terms of formation, constitution, and relationship remains unknown. Here, by combining proteomic, transcriptomic, and immunofluorescence approaches, we simultaneously characterized the SGs and PBs from primary human T lymphocytes pre and post stimulation. The identification of the proteomes and transcriptomes of SGs and PBs indicate an unanticipated molecular and functional complementarity. Notwithstanding, these granules keep distinct spatial organizations and abilities to interact with mRNAs. This comprehensive characterization of the RNP granule proteomic and transcriptomic landscapes provides a unique resource for future investigations on SGs and PBs in T lymphocytes.


Assuntos
Ativação Linfocitária , Corpos de Processamento , Proteoma , Grânulos de Estresse , Linfócitos T , Transcriptoma , Grânulos de Estresse/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Corpos de Processamento/metabolismo , Proteoma/metabolismo , Transcriptoma/genética , Proteômica , Perfilação da Expressão Gênica , Humanos , Masculino , Feminino , Adulto , Células Cultivadas , RNA/análise , Biossíntese de Proteínas , Transcrição Gênica , Fracionamento Celular
8.
Methods Mol Biol ; 2643: 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36952174

RESUMO

Sophisticated organelle fractionation strategies were the workhorse of early peroxisome research and led to the characterization of the principal functions of the organelle. However, even in the era of molecular biology and "omics" technologies, they are still of importance to unravel peroxisome-specific proteomes, confirm the localization of still uncharacterized proteins, analyze peroxisome metabolism or lipid composition, or study their protein import mechanism. To isolate and analyze peroxisomes for these purposes, density gradient centrifugation still represents a highly reliable and reproducible technique. This article describes two protocols to purify peroxisomes from either liver tissue or the HepG2 hepatoma cell line. The protocol for liver enables purification of peroxisome fractions with high purity (95%) and is therefore suitable to study low-abundant peroxisomal proteins or analyze their lipid composition, for example. The protocol presented for HepG2 cells is not suitable to gain highly pure peroxisomal fractions but is intended to be used for gradient profiling experiments and allows easier manipulation of the peroxisomal compartment, e.g., by gene knockdown or protein overexpression for functional studies. Both purification methods therefore represent complementary tools to be used to analyze different aspects of peroxisome physiology. Please note that this is an updated version of a protocol, which has been published in a former volume of Methods in Molecular Biology.


Assuntos
Fígado , Peroxissomos , Animais , Peroxissomos/metabolismo , Fracionamento Celular/métodos , Fígado/metabolismo , Mamíferos , Centrifugação com Gradiente de Concentração/métodos , Lipídeos
9.
Methods Mol Biol ; 2643: 13-31, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36952175

RESUMO

Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species, and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions, and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.


Assuntos
Peroxissomos , Saccharomyces cerevisiae , Peroxissomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Fracionamento Celular/métodos
10.
Methods Mol Biol ; 2643: 321-331, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36952195

RESUMO

Subcellular fractionation approaches have allowed for the identification of various functionally distinct organelles including peroxisomes. The methods enable enrichment of organelles and combined with downstream assays allow for the identification of biochemical functions, composition, and structural characteristics of these compartments. In this chapter, we describe the methods for differential centrifugation and Nycodenz gradients in the yeast Saccharomyces cerevisiae and describe assays for fatty acid ß-oxidation in intact cells and in peroxisomal fractions.


Assuntos
Peroxissomos , Proteínas de Saccharomyces cerevisiae , Peroxissomos/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Fracionamento Celular/métodos , Centrifugação , Proteínas de Saccharomyces cerevisiae/metabolismo , Frações Subcelulares , Oxirredução
11.
Methods Mol Biol ; 2615: 3-16, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36807780

RESUMO

Detailed analysis of mitochondrial function cannot be achieved without good quality preparations of isolated mitochondria. Ideally, the isolation protocol should be quick, while producing a reasonably pure pool of mitochondria that are still intact and coupled. Here, we describe a fast and simple method for the purification of mammalian mitochondria relying on isopycnic density gradient centrifugation. We describe specific steps that should be taken into consideration when functional mitochondria from different tissues should be isolated. This protocol is suitable for the analysis of many aspects of the organelle's structure and function.


Assuntos
DNA Mitocondrial , Mitocôndrias , Camundongos , Animais , Mitocôndrias/genética , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Mamíferos/genética
12.
Methods Mol Biol ; 2615: 41-55, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36807783

RESUMO

The isolation of organelles devoid of other cellular compartments is crucial for studying organellar proteomes and the localization of newly identified proteins, as well as for assessing specific organellar functions. Here, we describe a protocol for the isolation of crude and highly pure mitochondria from Saccharomyces cerevisiae and provide methods for testing the functional integrity of the isolated organelles.


Assuntos
Mitocôndrias , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fracionamento Celular/métodos , Mitocôndrias/metabolismo , Organelas/metabolismo , Controle de Qualidade
13.
Methods Mol Biol ; 2625: 7-15, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653629

RESUMO

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).


Assuntos
Sacarose , Membranas Sinápticas , Membranas Sinápticas/metabolismo , Sacarose/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Membrana Celular , Centrifugação , Lipídeos , Fracionamento Celular/métodos
14.
FEBS Lett ; 597(2): 246-261, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36217875

RESUMO

The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 s stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable the assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.


Assuntos
Coração , Mitocôndrias , Camundongos , Animais , Citosol/metabolismo , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Cardíacas/metabolismo , Fracionamento Celular/métodos
15.
J Vis Exp ; (187)2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36190269

RESUMO

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.


Assuntos
Membranas Sinápticas , Sinaptossomos , Animais , Encéfalo/metabolismo , Fracionamento Celular/métodos , Camundongos , Frações Subcelulares , Vesículas Sinápticas/metabolismo , Sinaptossomos/metabolismo
16.
Methods Cell Biol ; 170: 47-58, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35811103

RESUMO

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.


Assuntos
Células-Tronco Neoplásicas , Proteômica , Animais , Encéfalo/metabolismo , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Mamíferos/metabolismo , Células-Tronco Neoplásicas/patologia , Proteoma/metabolismo , Proteômica/métodos , Frações Subcelulares/metabolismo
17.
Methods Mol Biol ; 2442: 713-726, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320554

RESUMO

Fractionation of HeLa cell nuclear extracts by glycerol gradient centrifugation separates endogenous uracil-rich small nuclear ribonucleoprotein complexes (U snRNP) into numerous particles sedimenting from 7S to greater than 60S. Complexes sedimenting at 10S contain a single U snRNP (U1 snRNP) and galectin-3. Addition of antibodies specific for galectin-3 to fractions containing these 10S complexes coprecipitates U1 snRNP, indicating that a fraction of the U1 snRNP is associated with this galectin. Galectin-3 has been shown by depletion-reconstitution studies to be an integral splicing component involved both in spliceosome assembly and splicing activity. The first step in initiation of spliceosome assembly is binding of U1 snRNP to the 5' splice site of the premessenger RNA substrate. The finding that U1 snRNP and galectin-3 are associated in splicing extracts hints that this complex affords a potential entry point for galectin-3 into the splicing pathway. Addition of U1 snRNP-galectin-3 complexes immunoselected from the 10S region of glycerol gradients to a U1-depleted nuclear extract initiates splicing activity with the formation of splicing intermediates and mature mRNA. This chapter describes the materials and methods for these experiments that document galectin-3-U1 snRNP complexes initiate the splicing reaction in a U1-depleted nuclear extract.


Assuntos
Galectina 3 , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1 , Spliceossomos , Fracionamento Celular , Galectina 3/genética , Galectina 3/metabolismo , Células HeLa/metabolismo , Humanos , Espaço Intranuclear/química , Espaço Intranuclear/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Spliceossomos/metabolismo , Uracila/análise , Uracila/metabolismo
18.
Physiol Rep ; 10(4): e15195, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35179318

RESUMO

Mobilization of glycogen, the short-term storage form of glucose, is the body's first defense against hypoglycemia and is critical for energy provision during high intensity exercise. Therefore, to advance metabolic research, it is critical to be able to accurately measure glycogen concentrations, including during a prolonged fast and other glycogen-modulating interventions. Unfortunately, prior enzymatic methods of glycogen detection have been plagued by poor detection in the lower range, high sample mass requirements, and complicated and/or expensive protocols which introduce substantial technical variability into the measured glycogen concentrations. To address these limitations, here we report a streamlined and versatile glycogen extraction protocol coupled with an optimized phenol-sulfuric acid quantification protocol. With this method, we demonstrate how glycogen can be extracted from only 20 mg of tissue with one centrifugation step and quantified with a highly precise (Intra-assay %CV ranges from 5-10%) and sensitive (proportionality constant for glycogen = 0.07279 A.U./µg) assay. The cost of all materials equates to ~10 cents per sample. Therefore, this method represents an attractive means of assessing ex vivo tissue glycogen content including at the extremes of glycogen concentrations.


Assuntos
Glicogênio/análise , Animais , Fracionamento Celular/métodos , Fracionamento Químico/métodos , Glicogênio/metabolismo , Fígado/química , Fígado/metabolismo , Camundongos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Fenol/química , Ácidos Sulfúricos/química
19.
PLoS One ; 17(2): e0264003, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35157741

RESUMO

The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 µg/g dry weight, eight had copper concentrations between 100 and 180 µg/g, 14 had copper concentrations between 181 and 700 µg/g, and two had copper concentrations >700 µg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.


Assuntos
Cobre/análise , Fígado/química , Espectrofotometria Atômica/veterinária , Animais , Gatos , Fracionamento Celular , Núcleo Celular/química , Citosol/química , Feminino , Humanos , Masculino , Microssomos Hepáticos/química
20.
PLoS One ; 17(1): e0260222, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35085240

RESUMO

BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.


Assuntos
Plaquetas/metabolismo , Sepse/genética , Transcriptoma/genética , Idoso , Sequência de Bases/genética , Plaquetas/patologia , Fracionamento Celular/métodos , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Ativação Plaquetária/genética , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Testes de Função Plaquetária , RNA Circular/análise , RNA Circular/genética , Sepse/sangue , Análise de Sequência de RNA/métodos
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