Isolation and Characterization of Exosomes from Mouse Feces.
Ontology highlight
ABSTRACT:
SUBMITTER: Yang C
PROVIDER: S-EPMC7241525 | biostudies-literature | 2020 Apr
REPOSITORIES: biostudies-literature
Publications
Isolation and Characterization of Exosomes from Mouse Feces.
Yang Chunhua C Zhang Mingzhen M Sung Junsik J Wang Lixin L Jung Yunjin Y Merlin Didier D
Bio-protocol 20200401 8
Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, through which we can obtain a relatively more significant amount of exosomes from feces. This protocol is designed to effectively isolate a large number of ...[more]
Similar Datasets
Project description:Exosomes are a subset of extracellular vesicles that carry specific combinations of proteins, nucleic acids, metabolites, and lipids. Mounting evidence suggests that exosomes participate in intercellular communication and act as important molecular vehicles in the regulation of numerous physiological and pathological processes, including cancer development. Exosomes are released by various cell types under both normal and pathological conditions, and they can be found in multiple bodily fluids. Moreover, exosomes carrying a wide variety of important macromolecules provide a window into altered cellular or tissue states. Their presence in biological fluids renders them an attractive, minimally invasive approach for liquid biopsies with potential biomarkers for cancer diagnosis, prediction, and surveillance. Due to their biocompatibility and low immunogenicity and cytotoxicity, exosomes have potential clinical applications in the development of innovative therapeutic approaches. Here, we summarize recent advances in various technologies for exosome isolation for cancer research. We outline the functions of exosomes in regulating tumor metastasis, drug resistance, and immune modulation in the context of cancer development. Finally, we discuss prospects and challenges for the clinical development of exosome-based liquid biopsies and therapeutics.
| S-EPMC7652679 | biostudies-literature
Project description:Escherichia coli O104:H4, an hybrid pathotype of Shiga toxigenic and enteroaggregative E. coli, involved in a major foodborne outbreak in Germany in 2011, has not been detected in cattle feces. Serogroup O104 with H type other than H4 has been reported to cause human illnesses, but their prevalence and characteristics in cattle have not been reported. Our objectives were to determine the prevalence of E. coli O104 in feces of feedlot cattle, by culture and PCR detection methods, and characterize the isolated strains. Rectal fecal samples from a total of 757 cattle originating from 29 feedlots were collected at a Midwest commercial slaughter plant. Fecal samples, enriched in E. coli broth, were subjected to culture and PCR methods of detection. The culture method involved immunomagnetic separation with O104-specific beads and plating on a selective chromogenic medium, followed by serogroup confirmation of pooled colonies by PCR. If pooled colonies were positive for the wzxO104 gene, then colonies were tested individually to identify wzxO104-positive serogroup and associated genes of the hybrid strains. Extracted DNA from feces were also tested by a multiplex PCR to detect wzxO104-positive serogroup and associated major genes of the O104 hybrid pathotype. Because wzxO104 has been shown to be present in E. coli O8/O9/O9a, wzxO104-positive isolates and extracted DNA from fecal samples were also tested by a PCR targeting wbdDO8/O9/O9a, a gene specific for E. coli O8/O9/O9a serogroups. Model-adjusted prevalence estimates of E. coli O104 (positive for wzxO104 and negative for wbdDO8/O9/O9a) at the feedlot level were 5.7% and 21.2%, and at the sample level were 0.5% and 25.9% by culture and PCR, respectively. The McNemar's test indicated that there was a significant difference (P < 0.01) between the proportions of samples that tested positive for wzxO104 and samples that were positive for wzxO104, but negative for wbdDO8/O9/O9a by PCR and culture methods. A total of 143 isolates, positive for the wzxO104, were obtained in pure culture from 146 positive fecal samples. Ninety-two of the 143 isolates (64.3%) also tested positive for the wbdDO8/O9/O9a, indicating that only 51 (35.7%) isolates truly belonged to the O104 serogroup (positive for wzxO104 and negative for wbdDO8/O9/O9a). All 51 isolates tested negative for eae, and 16 tested positive for stx1 gene of the subtype 1c. Thirteen of the 16 stx1-positive O104 isolates were from one feedlot. The predominant serotype was O104:H7. Pulsed-field gel electrophoresis analysis indicated that stx1-positive O104:H7 isolates had 62.4% homology to the German outbreak strain and 67.9% to 77.5% homology to human diarrheagenic O104:H7 strains. The 13 isolates obtained from the same feedlot were of the same PFGE subtype with 100% Dice similarity. Although cattle do not harbor the O104:H4 pathotype, they do harbor and shed Shiga toxigenic O104 in the feces and the predominant serotype was O104:H7.
| S-EPMC4807062 | biostudies-literature
Project description:Lactic acid bacteria (LAB) are commonly used as probiotics; however, not all LAB strains have the same beneficial effects. To successfully use LAB as probiotics in canines, LAB species should originate from the canine intestinal tract as they display host specificity. The objective of this study was to investigate the phenotypic and genomic traits of potential probiotic LAB isolated from canine fecal samples. Twenty LAB samples were evaluated for their potential probiotic characteristics including resistance to low pH, bile salts, hydrophobicity, auto-aggregation, co-aggregation, adhesion to epithelia or mucosa, and production of inhibitory compounds. Additionally, we evaluated their safety and other beneficial effects on canine health, such as DPPH free radical scavenging, and β-galactosidase. Four strains demonstrated potential probiotic characteristics and were selected: Enterococcus hirae Pom4, Limosilactobacillus fermentum Pom5, Pediococcus pentosaceus Chi8, and Ligilactobacillus animalis FB2. Safety evaluations showed that all strains lacked hemolytic activity, could not produce biogenic amines, and did not carry any pathogenic genes. In addition, L. fermentum Pom5 and P. pentosaceus Chi8 displayed susceptibility to all antibiotics and concordant with the absence of antibiotic resistance genes. Based on their phenotypic and genomic characteristics, L. fermentum Pom5 and P. pentosaceus Chi8 were identified as potential probiotic candidates for canines.
| S-EPMC10359844 | biostudies-literature
Project description:Anaerobic fungi are prime candidates for the conversion of agricultural waste products to biofuels. Despite the increasing interest in these organisms, their growth requirements and metabolism remain largely unknown. The isolation of five strains of anaerobic fungi and their identification as Neocallimastix cameroonii, Caecomyces spec., Orpinomyces joyonii, Pecoramyces ruminantium, and Khoyollomyces ramosus, is described. The phylogeny supports the reassignment of Neocallimastix californiae and Neocallimastix lanati to Neocallimastix cameroonii and points towards the redesignation of Cyllamyces as a species of Caecomyces. All isolated strains including strain A252, which was described previously as Aestipascuomyces dubliciliberans, were further grown on different carbon sources and the produced metabolites were analyzed; hydrogen, acetate, formate, lactate, and succinate were the main products. Orpinomyces joyonii was lacking succinate production and Khoyollomyces ramosus was not able to produce lactate under the studied conditions. The results further suggested a sequential production of metabolites with a preference for hydrogen, acetate, and formate. By comparing fungal growth on monosaccharides or on the straw, a higher hydrogen production was noticed on the latter. Possible reactions to elevated sugar concentrations by anaerobic fungi are discussed.
| S-EPMC8399178 | biostudies-literature
Project description:BackgroundMost lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces.ResultsWe applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species.ConclusionsThese results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.
| S-EPMC7268542 | biostudies-literature
Project description:Membrane bound vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular vesicles (cirEVs) and exosomes in particular are recognized as a potential source of disease biomarkers. However, to exploit the use of circulatory exosomes as a biomarker, a rapid, high-throughput and reproducible method is required for their isolation and molecular analysis. We have developed a simple, low cost microfluidic-based platform to isolate cirEVs enriched in exosomes directly from blood serum allowing simultaneous capture and quantification of exosomes in a single device. To capture specific exosomes, we employed "ExoChip", a microfluidic device fabricated in polydimethylsiloxane (PDMS) and functionalized with antibodies against CD63, an antigen commonly overexpressed in exosomes. Subsequent staining with a fluorescent carbocyanine dye (DiO) that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34 ± 0.31 fold, p < 0.001) in exosomes captured in cancer patients when compared to healthy individuals. Exosomal origins of ExoChip immobilized vesicles were further confirmed using immuno-electron-microscopy and Western blotting. In addition, we demonstrate the ability of ExoChip to recover exosomes with intact RNA enabling profiling of exosomal-microRNAs through openarray analysis, which has potential applications in biomarker discovery. Based on our findings, ExoChip is a well suited platform to be used as an exosome-based diagnostic and research tool for molecular screening of human cancers.
| S-EPMC4134440 | biostudies-literature
Project description:In the post-natal mammalian brain perivascular astrocytes (PAs) ensheath blood vessels to regulate their unique permeability properties known as the blood-brain barrier (BBB). Very little is known about PA-expressed genes and signaling pathways that mediate contact and communication with endothelial cells (ECs) to regulate BBB physiology. This is due, in part, to lack of suitable models to distinguish PAs from other astrocyte sub-populations in the brain. To decipher the unique biology of PAs, we used in vivo gene knock-in technology to fluorescently label these cells in the adult mouse brain followed by fractionation and quantitative single cell RNA sequencing. In addition, PAs and non-PAs were also distinguished with transgenic fluorescent reporters followed by gene expression comparisons using bulk RNA sequencing. These efforts have identified several genes and pathways in PAs with potential roles in contact and communication with brain ECs. These genes encode various extracellular matrix (ECM) proteins and adhesion receptors, secreted growth factors, and intracellular signaling enzymes. Collectively, our experimental data reveal a set of genes that are expressed in PAs with putative roles in BBB physiology.
| S-EPMC7544046 | biostudies-literature
Project description:Exosomes are nanoscale extracellular vesicles which regulate intercellular communication. They have great potential for application in nanomedicine. However, techniques for their isolation are limited by requirements for advanced instruments and costly reagents. In this study, we developed a lyophilization-based method for isolating exosomes from cultured cells. The isolated exosomes were characterized for protein content using Bradford assay, and for size distribution and shape using scanning electron microscopy (SEM) and nanoparticles tracking analysis (NTA). In addition, CD63, CD9, CD81, HSP70 and TSG101 were evaluated as essential exosomal surface markers using Western blot. Drug loading and release studies were performed to confirm their drug delivery properties using an in vitro model. Exosomes were also loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. All these characterizations confirmed successful exosome isolation with measurements of less than 150 nm, having a typical shape, and by expressing the known exosome surface protein markers. Finally, tyrosine kinase inhibitors (dasatinib and ponatinib) were loaded on the exosomes to evaluate their anticancer effects on leukemia cells (K562 and engineered Ba/F3-BCR-ABL) using MTT and Annexin-PI assays. The expression of MUC1 protein on the exosomes isolated from MCF-7 cells also indicated that their potential diagnostic properties were intact. In conclusion, we developed a new method for exosome isolation from cultured cells. These exosomes met all the essential requirements in terms of characterization, drug loading and release ability, and inhibition of proliferation and apoptosis induction in Ph+ leukemia cells. Based on these results, we are confident in presenting the lyophilization-based exosome isolation method as an alternative to traditional techniques for exosome isolation from cultured cells.
| S-EPMC10341925 | biostudies-literature
Project description:A new versatile actinobacterium designated as strain NJES-13 was isolated from the feces of the Antarctic emperor penguin. This new isolate was found to produce two active gephyromycin analogues and bioflocculanting exopolysaccharides (EPS) metabolites. Phylogenetic analysis based on pairwise comparison of 16S rRNA gene sequences showed that strain NJES-13 was closely related to Mobilicoccus pelagius Aji5-31T with a gene similarity of 95.9%, which was lower than the threshold value (98.65%) for novel species delineation. Additional phylogenomic calculations of the average nucleotide identity (ANI, 75.9-79.1%), average amino acid identity (AAI, 52.4-66.9%) and digital DNA-DNA hybridization (dDDH, 18.6-21.9%), along with the constructed phylogenomic tree based on the up-to-date bacterial core gene (UBCG) set from the bacterial genomes, unequivocally separated strain NJES-13 from its close relatives within the family Dermatophilaceae. Hence, it clearly indicated that strain NJES-13 represented a putative new actinobacterial species isolated from the gut microbiota of mammals inhabiting the Antarctic. The obtained complete genome of strain NJES-13 consisted of a circular 3.45 Mb chromosome with a DNA G+C content of 67.0 mol%. Furthering genome mining of strain NJES-13 showed the presence of five biosynthetic gene clusters (BGCs) including one type III PKS responsible for the biosynthesis of the core of gephyromycins, and a series of genes encoding for bacterial EPS biosynthesis. Thus, based on the combined phylogenetic and active metabolites characterization presented in this study, we confidently conclude that strain NJES-13 is a novel, fresh actinobacterial candidate to produce active gephyromycins and microbial bioflocculanting EPS, with potential pharmaceutical, environmental and biotechnological implications.
| S-EPMC8400323 | biostudies-literature
Project description: Not available
| S-EPMC3945919 | biostudies-literature