scholarly journals Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Svetlana Sokovic Bajic ◽  
Maria-Alexandra Cañas ◽  
Maja Tolinacki ◽  
Josefa Badia ◽  
Borja Sánchez ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Time Course ◽  
Intestinal Epithelial ◽  
Amino Acid Peptide ◽  
Gram Positive Bacteria ◽  
Ht29 Cells ◽  
Membrane Components ◽  
First Time ◽  
Putative Mechanism

AbstractIn recent years the role of extracellular vesicles (EVs) of Gram-positive bacteria in host-microbe cross-talk has become increasingly appreciated, although the knowledge of their biogenesis, release and host-uptake is still limited. The aim of this study was to characterize the EVs released by the dairy isolate Lactiplantibacillus plantarum BGAN8 and to gain an insight into the putative mechanism of EVs uptake by intestinal epithelial cells. The cryo-TEM observation undoubtedly demonstrated the release of EVs (20 to 140 nm) from the surface of BGAN8, with exopolysaccharides seems to be part of EVs surface. The proteomic analysis revealed that the EVs are enriched in enzymes involved in central metabolic pathways, such as glycolysis, and in membrane components with the most abundant proteins belonging to amino acid/peptide ABC transporters. Putative internalization pathways were evaluated in time-course internalization experiments with non-polarized HT29 cells in the presence of inhibitors of endocytic pathways: chlorpromazine and dynasore (inhibitors of clathrin-mediated endocytosis—CME) and filipin III and nystatin (disrupting lipid rafts). For the first time, our results revealed that the internalization was specifically inhibited by dynasore and chlorpromazine but not by filipin III and nystatin implying that one of the entries of L. plantarum vesicles was through CME pathway.

scholarly journals Modulation of RAB7A Protein Expression Determines Resistance to Cisplatin through Late Endocytic Pathway Impairment and Extracellular Vesicular Secretion

Cancers ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 52 ◽  
Author(s):  
Flora Guerra ◽  
Aurora Paiano ◽  
Danilo Migoni ◽  
Giulia Girolimetti ◽  
Anna Myriam Perrone ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Therapy Response ◽  
Endocytic Pathway ◽  
Endocytic Trafficking ◽  
Chemotherapy Response ◽  
Intracellular Accumulation ◽  
Human Patient ◽  
First Time ◽  
Vesicular Secretion

Background: Cisplatin (CDDP) is widely used in treatment of cancer, yet patients often develop resistance with consequent therapeutical failure. In CDDP-resistant cells alterations of endocytosis and lysosomal functionality have been revealed, although their causes and contribution to therapy response are unclear. Methods: We investigated the role of RAB7A, a key regulator of late endocytic trafficking, in CDDP-resistance by comparing resistant and sensitive cells using western blotting, confocal microscopy and real time PCR. Modulation of RAB7A expression was performed by transfection and RNA interference, while CDDP sensitivity and intracellular accumulation were evaluated by viability assays and chemical approaches, respectively. Also extracellular vesicles were purified and analyzed. Finally, correlations between RAB7A and chemotherapy response was investigated in human patient samples. Results: We demonstrated that down-regulation of RAB7A characterizes the chemoresistant phenotype, and that RAB7A depletion increases CDDP-resistance while RAB7A overexpression decreases it. In addition, increased production of extracellular vesicles is modulated by RAB7A expression levels and correlates with reduction of CDDP intracellular accumulation. Conclusions: We demonstrated, for the first time, that RAB7A regulates CDDP resistance determining alterations in late endocytic trafficking and drug efflux through extracellular vesicles.

scholarly journals Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences

2020 ◽  
Vol 21 (18) ◽  
pp. 6730
Author(s):  
Francesc Ibáñez ◽  
Juan R. Ureña-Peralta ◽  
Pilar Costa-Alba ◽  
Jorge-Luis Torres ◽  
Francisco-Javier Laso ◽  
...  
Keyword(s):  
Gender Differences ◽  
Extracellular Vesicles ◽  
Target Genes ◽  
Alcohol Intoxication ◽  
Peripheral Circulation ◽  
Female Adolescents ◽  
Circulating Micrornas ◽  
Male Adolescents ◽  
First Time

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood–brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

scholarly journals Inhibition of  a v b 3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

2020 ◽  
Author(s):  
Wanessa Altei ◽  
Bianca Pachane ◽  
Patty K. Santos ◽  
Ligia Ribeiro ◽  
Bong Hwan Sung ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Organ Specific ◽  
Breast Cells ◽  
Mediate Cell ◽  
First Time ◽  
Cd63 Expression ◽  
Cell Cell

Abstract Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells.Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for avb3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments.Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry avb3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of avb3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content.Conclusion: In summary, our findings demonstrate for the first time a key role of avb3 integrin in cell-cell communication through SEVs.

scholarly journals Inhibition of  a v b 3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

2020 ◽  
Author(s):  
Wanessa Altei ◽  
Bianca Pachane ◽  
Patty K. Santos ◽  
Ligia Ribeiro ◽  
Bong Hwan Sung ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Αvβ3 Integrin ◽  
Organ Specific ◽  
Breast Cells ◽  
Mediate Cell ◽  
First Time ◽  
Cell Cell

Abstract Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs.

scholarly journals Extracellular Vesicles: An Overlooked Secretion System in Cyanobacteria

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 129 ◽  
Author(s):  
Steeve Lima ◽  
Jorge Matinha-Cardoso ◽  
Paula Tamagnini ◽  
Paulo Oliveira
Keyword(s):  
Extracellular Vesicles ◽  
Bacterial Cell ◽  
Cell Biology ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Formation Processes ◽  
Gram Negative ◽  
Nanoscale Structures ◽  
Membrane Components

In bacteria, the active transport of material from the interior to the exterior of the cell, or secretion, represents a very important mechanism of adaptation to the surrounding environment. The secretion of various types of biomolecules is mediated by a series of multiprotein complexes that cross the bacterial membrane(s), each complex dedicated to the secretion of specific substrates. In addition, biological material may also be released from the bacterial cell in the form of vesicles. Extracellular vesicles (EVs) are bilayered, nanoscale structures, derived from the bacterial cell envelope, which contain membrane components as well as soluble products. In cyanobacteria, the knowledge regarding EVs is lagging far behind compared to what is known about, for example, other Gram-negative bacteria. Here, we present a summary of the most important findings regarding EVs in Gram-negative bacteria, discussing aspects of their composition, formation processes and biological roles, and highlighting a number of technological applications tested. This lays the groundwork to raise awareness that the release of EVs by cyanobacteria likely represents an important, and yet highly disregarded, survival strategy. Furthermore, we hope to motivate future studies that can further elucidate the role of EVs in cyanobacterial cell biology and physiology.

scholarly journals Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

1985 ◽  
Vol 100 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T C Keller ◽  
K A Conzelman ◽  
R Chasan ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Intestinal Epithelium ◽  
Brush Border ◽  
Time Course ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.

scholarly journals Inhibition of αvβ3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Wanessa F. Altei ◽  
Bianca C. Pachane ◽  
Patty K. dos Santos ◽  
Lígia N. M. Ribeiro ◽  
Bong Hwan Sung ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Cell Communication ◽  
Αvβ3 Integrin ◽  
Organ Specific ◽  
Breast Cells ◽  
Mediate Cell ◽  
First Time ◽  
Cell Cell

Abstract Background Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. Graphical abstract

scholarly journals Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3

2017 ◽  
Vol 313 (6) ◽  
pp. G589-G598 ◽  
Author(s):  
Ram Lakhan ◽  
Veedamali S. Subramanian ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cells ◽  
Posttranscriptional Regulation ◽  
Intestinal Epithelial Cells ◽  
Significant Inhibition ◽  
Luciferase Reporter ◽  
Intestinal Epithelial ◽  
Functional Consequences ◽  
Uptake Process ◽  
First Time

Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the posttranscriptional level. We address this issue by focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3-3′-UTR, and showed (using pmirGLO-hRFVT3-3′-UTR) that the hRFVT3-3′-UTR is, indeed, a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3′-UTR construct, and also led to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Furthermore, cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in the level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for posttranscriptional regulation by miRNAs in intestinal epithelial cells and that this regulation has functional consequences on intestinal RF uptake. NEW & NOTEWORTHY Our findings show for the first time that RFVT3 is a target for posttranscriptional regulation by miR-423-5p in intestinal epithelial cells, and this regulation has functional consequences on intestinal riboflavin (RF) uptake process.

scholarly journals IL-17 Affects the Progression, Metastasis, and Recurrence of Laryngeal Cancer via the Inhibition of Apoptosis through Activation of the PI3K/AKT/FAS/FASL Pathways

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Yang Song ◽  
Ming Yang ◽  
Hongjian Zhang ◽  
Yan Sun ◽  
Ye Tao ◽  
...  
Keyword(s):  
Laryngeal Cancer ◽  
Time Course ◽  
Clinical Stage ◽  
Immune Surveillance ◽  
Tumor Promoter ◽  
Interleukin 17 ◽  
Normal Tissues ◽  
First Time

Background. Cytokines play important roles in the development and prognosis of laryngeal cancer (LC). Interleukin-17 (IL-17) from a distinct subset of CD4+ T cells may significantly induce cancer-elicited inflammation to prevent tumor immune surveillance. Methods. The expression levels of IL-17 were examined among 60 patients with LC. Immunofluorescence colocalization experiments were performed to verify the localization of IL-17 and FAS/FASL in Hep-2 and Tu212 cells. The role of IL-17 was determined using siRNA techniques in the LC cell line. Results. In the LC patients, cytokines were dysregulated in LC tissues compared with normal tissues. It was found that IL-17 was overexpressed in a cohort of 60 LC tumors paired with nontumor tissues. Moreover, high IL-17 expression was significantly associated with the advanced T category, the late clinical stage, differentiation, lymph node metastasis, and recurrence. In addition, the time course expression of FAS and FASL was observed after stimulation and treatment with the IL-17 stimulator. Finally, in vitro experiments demonstrated that IL-17 functioned as an oncogene by inhibiting the apoptosis of LC cells via the PI3K/AKT/FAS/FASL pathways. Conclusions. In summary, these findings demonstrated for the first time the role of IL-17 as a tumor promoter and a prometastatic factor in LC and indicated that IL-17 may have an oncogenic role and serve as a potential prognostic biomarker and therapeutic target in LC.

scholarly journals Staphylococcus aureus Extracellular Vesicles Carry Biologically Active β-Lactamase

2013 ◽  
Vol 57 (6) ◽  
pp. 2589-2595 ◽  
Author(s):  
Jaewook Lee ◽  
Eun-Young Lee ◽  
Si-Hyun Kim ◽  
Dae-Kyum Kim ◽  
Kyong-Su Park ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Extracellular Vesicles ◽  
Extracellular Vesicle ◽  
Biologically Active ◽  
Gram Positive ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Heat Treated ◽  
Protease Digestion

ABSTRACTGram-positive bacteria naturally produce extracellular vesicles. However, little is known regarding the functions of Gram-positive bacterial extracellular vesicles, especially in the bacterial community. Here, we investigated the role ofStaphylococcus aureusextracellular vesicles in interbacterial communication to cope with antibiotic stress. We found thatS. aureusliberated BlaZ, a β-lactamase protein, via extracellular vesicles. These extracellular vesicles enabled other ampicillin-susceptible Gram-negative and Gram-positive bacteria to survive in the presence of ampicillin. However,S. aureusextracellular vesicles did not mediate the survival of tetracycline-, chloramphenicol-, or kanamycin-susceptible bacteria. Moreover,S. aureusextracellular vesicles did not contain theblaZgene. In addition, the heat-treatedS. aureusextracellular vesicles did not mediate the survival of ampicillin-susceptible bacteria. The β-lactamase activities ofS. aureussoluble and extracellular vesicle-associated BlaZ were similar, but only the extracellular vesicle-associated BlaZ was resistant to protease digestion, which suggests that the enzymatic activity of BlaZ in extracellular vesicles is largely protected by the vesicle structure. Our observations provide evidence of the important role ofS. aureusextracellular vesicles in antibiotic resistance, which allows the polymicrobial community to continue to evolve and prosper against antibiotics.

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