scholarly journals Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

2018 ◽  
Author(s):  
Joshua C. Russell ◽  
Gennifer E. Merrihew ◽  
Julia E. Robbins ◽  
Nadia Postupna ◽  
Tyek-Kyun Kim ◽  
...  
Keyword(s):  
Cell Culture ◽  
Caenorhabditis Elegans ◽  
Extracellular Vesicles ◽  
Mammalian Cell Culture ◽  
Model Systems ◽  
C Elegans ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Invertebrate Model

AbstractCells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classesof molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsysamples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolateEVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on ascale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system.

scholarly journals ESPO/ BIOLOGICAL SCIENCES SECTION SYMPOSIUM: EMERGING INSIGHTS IN INTERACTIONS AND NETWORKS IN THE BIOLOGY OF AGING DEVELOPING C. ELEGANS TO STUDY AGE-RELATED EXTRACELLULAR VESICLE SIGNALS

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S424-S424
Author(s):  
Joshua Russell ◽  
Matt Kaeberlein
Keyword(s):  
Biological Sciences ◽  
Mammalian Cell Culture ◽  
Extracellular Vesicle ◽  
Model Systems ◽  
C Elegans ◽  
Physiological Processes ◽  
Age Related ◽  
Powerful Approach ◽  
Invertebrate Model ◽  
Biology Of Aging

Abstract All cells release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classes of molecules, including nucleic acids, proteins, and lipids. EV-signaling has been shown to be impacted by many age-related physiological processes such as inflammation, mitochondrial stress, and autophagy as well as directly mediate critical functions in cellular senescence and aging. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsy samples has become a powerful approach for uncovering the messages that are packaged into these organelles. Caenorhabditis elegans is a premier model for dissecting the genetics of aging however, EV analysis has not been tenable in invertebrate model systems due to lack of methods for obtaining sufficient amounts of pure EVs. We developed a method for isolating pure EVs from C. elegans with yields sufficient for mass spectrometry and RNAseq. Here we present the analysis of the genetic and protein cargos of EVs collected from wild type and long-lived mutants collected at different time points across their lifespans. As the first investigation of age-related EV signals in an invertebrate model system we believe these results will provide insights into cell non-autonomous mechanisms of aging.

Isolation and characterization of extracellular vesicles

2019 ◽  
Vol 1 (1) ◽  
pp. 18-26
Author(s):  
Fabia Fricke ◽  
Dominik Buschmann ◽  
Michael W. Pfaffl
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Body Fluids ◽  
Cell Culture Media ◽  
Advantages And Disadvantages ◽  
The Past ◽  
Isolation And Characterization ◽  
Basic Biology

Research into extracellular vesicles (EVs) gained significant traction in the past decade and EVs have been investigated in a wide variety of studies ranging from basic biology to diagnostic and therapeutic applications. Since EVs are secreted by most, if not all, eukaryotic and prokaryotic cells, they have been detected in body fluids as diverse as blood, urine and saliva as well as in cell culture media. In this chapter, we will provide an overview of EV isolation and characterization strategies and highlight their advantages and disadvantages.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

The size of extracellular vesicles secreted by different types of stem cells

2018 ◽  
pp. 38-42
Author(s):  
И.Б. Алчинова ◽  
М.В. Полякова ◽  
И.Н. Сабурина ◽  
М.Ю. Карганов
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Living Organism ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Study Results ◽  
Multipotent Mesenchymal Stem Cells ◽  
Rat Adipose Tissue ◽  
Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

2020 ◽  
Author(s):  
Dario Brambilla ◽  
Laura Sola ◽  
Elisa Chiodi ◽  
Natasa Zarovni ◽  
Diogo Fortunato ◽  
...  
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Biological Fluids ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Disease Diagnosis ◽  
Cell Culture Supernatant ◽  
Transmission Electron ◽  
Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

scholarly journals Isolation and Characterization of Strain Exiguobacterium sp. KRL4, a Producer of Bioactive Secondary Metabolites from a Tibetan Glacier

2021 ◽  
Vol 9 (5) ◽  
pp. 890
Author(s):  
Pietro Tedesco ◽  
Fortunato Palma Esposito ◽  
Antonio Masino ◽  
Giovanni Andrea Vitale ◽  
Emiliana Tortorella ◽  
...  
Keyword(s):  
Phenotypic Analysis ◽  
Biological Assays ◽  
C Elegans ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Protein Encoding ◽  
Nematocidal Activity ◽  
Extremophilic Microorganisms ◽  
Unique Source

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 °C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds.

scholarly journals 109 Isolation and characterization of myometrium and decidual cell-derived extracellular vesicles

2021 ◽  
Vol 224 (2) ◽  
pp. S75-S76
Author(s):  
Megan Shepherd ◽  
Enkhtuya Radnaa ◽  
Rheanna Urrabaz-Garza ◽  
Talar Kechichian ◽  
Ourlad Alzeus G. Tantengco ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Decidual Cell ◽  
Isolation And Characterization

scholarly journals Isolation and characterization of pathogenic Escherichia coli bacteriophages from chicken and beef offal

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Celosia Lukman ◽  
Christopher Yonathan ◽  
Stella Magdalena ◽  
Diana Elizabeth Waturangi
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
High Efficiency ◽  
Multiplicity Of Infection ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Pathogenic Escherichia Coli ◽  
Family Based

Abstract Objective This study was conducted to isolate and characterize lytic bacteriophages for pathogenic Escherichia coli from chicken and beef offal, and analyze their capability as biocontrol for several foodborne pathogens. Methods done in this research are bacteriophage isolation, purification, titer determination, application, determination of host range and minimum multiplicity of infection (miMOI), and bacteriophage morphology. Results Six bacteriophages successfully isolated from chicken and beef offal using EPEC and EHEC as host strain. Bacteriophage titers observed between 109 and 1010 PFU mL−1. CS EPEC and BL EHEC bacteriophage showed high efficiency in reduction of EPEC or EHEC contamination in meat about 99.20% and 99.04%. The lowest miMOI was 0.01 showed by CS EPEC bacteriophage. CI EPEC and BL EPEC bacteriophage suspected as Myoviridae family based on its micrograph from Transmission Electron Microscopy (TEM). Refers to their activity, bacteriophages isolated in this study have a great potential to be used as biocontrol against several foodborne pathogens.

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