- PEG-grafted phospholipids in vesicles: Effect of PEG chain length and concentration on mechanical properties. [Journal Article]
- CPChem Phys Lipids 2018 Dec 03
- Incorporation of low molecular weight poly-ethylene glycol (PEG) - grafted phospholipids in vesicle bilayers is known to increase the circulation time of liposomal drug delivery vehicles. Mechanical ...
Incorporation of low molecular weight poly-ethylene glycol (PEG) - grafted phospholipids in vesicle bilayers is known to increase the circulation time of liposomal drug delivery vehicles. Mechanical properties of giant unilamellar DPPC vesicles containing varying concentrations of DSPE-PEG (PEG MW: 550, 1000 and 2000) were measured by micropipette aspiration assay or osmotic swelling. While the area compressibility modulus did not change significantly, the bending modulus and water permeability of the bilayer was found to increase with increasing mole fraction of DSPE-PEG. This increase was more pronounced for higher molecular weight PEG. The measured bending modulus agreed with that predicted by scaling theory only at low mole fractions of DSPE-PEG. The water permeability was also measured as a function of the increase in area per lipid (due to steric repulsion between PEG chains), and for the same area per lipid, the PEG chain with MW 550 provided a greater resistance to water transport across the vesicle membrane compared to PEG 1000 and 2000. Lysis tension of the membrane, determined by osmotic lysis method at different loading rates showed a decrease in membrane strength on inclusion of the polymer lipid. These results suggest that liposome lifetime in the circulation and the rate of drug delivery are affected by the molecular weight and concentration of PEG in the bilayer.
- A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time. [Journal Article]
- PlosPLoS One 2018; 13(12):e0208508
- This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without usin...
This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively.
- Human bone marrow contains high levels of extracellular vesicles with a tissue-specific subtype distribution. [Journal Article]
- PlosPLoS One 2018; 13(12):e0207950
- Extracellular vesicles (EV) are shed from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. They are usu...
Extracellular vesicles (EV) are shed from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. They are usually measured as circulating EV in peripheral blood (PB) and other body fluids. However, little is known about the distribution, presence and impact of EV and their subpopulations in bone marrow (BM). In our study, we focused on the analysis of different EV subtypes in human BM as compared to EV subsets in PB.
- Roles of CD133 in microvesicle formation and oncoprotein trafficking in colon cancer. [Journal Article]
- FJFASEB J 2018 Dec 06; :fj201802018R
- Extracellular vesicles contain various cellular components that are involved in tumor growth, metastasis, and immune escape. Extracellular vesicles are classified into 2 groups, namely, exosomes and ...
Extracellular vesicles contain various cellular components that are involved in tumor growth, metastasis, and immune escape. Extracellular vesicles are classified into 2 groups, namely, exosomes and microvesicles (MV). Although the formation and roles of exosomes have been studied, the exact functions of MVs and mechanisms underlying MV release are not fully understood. We found that epidermal growth factor accelerates the release of MVs from the plasma membrane by inducing NF-κB activation and CD133 expression. The amount and sizes of budding MVs were found to be dependent on the expression level of CD133, which regulates the activities of the small guanosine 5'-triphosphatases RhoA and Rac1. CD133-containing MVs released from KRAS mutant colon cancer cells delivered KRAS mutant to adjacent nontumorigenic cells and activated KRAS downstream signaling. CD133-containing MVs were found to promote the migration and invasion of adjacent cells. CD133-containing MVs induced the development of chemoresistance by abolishing the inhibitory effects of anti-epidermal growth factor receptor (EGFR) drugs on cell proliferation and motility in colon cancer. These results suggest that CD133 acts as a novel modulator in MV release and in oncoprotein trafficking. CD133 can serve as a therapeutic target for treatment of anti-EGFR drug-resistant colon cancer.-Kang, M., Kim, S., Ko, J. Roles of CD133 in microvesicle formation and oncoprotein trafficking in colon cancer.
- Proteins regulating microvesicle biogenesis and multidrug resistance in cancer. [Review]
- PProteomics 2018 Dec 06; :e1800165
- Microvesicles (MV) are emerging as important mediators of intercellular communication. These signalling vectors are implicated in physiological processes ranging from vascular homeostasis to inflamma...
Microvesicles (MV) are emerging as important mediators of intercellular communication. These signalling vectors are implicated in physiological processes ranging from vascular homeostasis to inflammation. They also play an important role in the cancer pathology and progression. MVs can be formed in response to stimuli, or in the case of cancer, spontaneously and constitutively. Cellular activation is perhaps the most widely reported initiator of MV biogenesis, however, the precise mechanism remains undefined. What is clear, is cellular activation causes an increase in intracellular calcium and this modulates a number of membrane bound and cytosolic proteins that untether the membrane from the underlying cytoskeleton. This results in outward budding and ultimately in the vesiculation of the plasma membrane. Uncovering the proteins involved in regulating MV biogenesis is of interest given their role in the dissemination of deleterious cancer traits. Our laboratory was the first to report that MVs shed from drug-resistant cancer cells transfer multidrug resistance (MDR) proteins to drug-sensitive cells, conferring the MDR phenotype in a matter of hours. MDR is attributed to the overexpression of ABC transporters, primarily P-glycoprotein and MRP1. These ATP-dependent efflux transporters can efflux a broad range of substrates out of the cytoplasm. Their expression and functionality is dependent on a number of proteins that are involved in their translation, trafficking from the Golgi, as well as their subcellular localisation. In particular, FERM domains proteins have been implicated in supporting the functionality of these proteins in drug-resistant cells and in recipient cells during intercellular transfer by vesicles. Herein, we have compiled the most recent research on the proteins involved in MV biogenesis and in the dissemination of MV-mediated MDR. We also draw attention to unanswered questions in the literature that may prove to be of benefit in ongoing efforts to improve clinical response to chemotherapy and circumventing MDR. This article is protected by copyright. All rights reserved.
- Two ancient neuropeptides, PACAP and AVP, modulate motivated behavior at synapses in the extrahypothalamic brain: a study in contrast. [Review]
- CTCell Tissue Res 2018 Dec 05
- We examine evolutionary aspects of two primordial neuropeptides, arginine vasopressin (AVP) and pituitary adenylate cyclase-activating polypeptide (PACAP); the distribution of AVP and PACAP and their...
We examine evolutionary aspects of two primordial neuropeptides, arginine vasopressin (AVP) and pituitary adenylate cyclase-activating polypeptide (PACAP); the distribution of AVP and PACAP and their receptors in mammals; AVP and PACAP release patterns relevant to their roles in neuroendocrine control in brain and periphery; and finally the intricate interlocking of homeostatic and allostatic regulation created by extrahypothalamic AVP and PACAP projections to brain circuit nodes important in controlling appetitive, avoidance and aggressive motor responses. A cardinal feature of peptide neurotransmission important in regulatory control of organismic responses and emphasized in this review, is that neuropeptides are released from large dense-core vesicles docked not only within axonal varicosities and dendrites but also at presynaptic nerve terminal sites, along with small clear synaptic vesicles, at active zones. Peptide transmitter nerve terminals, from hypothalamic and other projections, are distributed widely to multiple brain areas important in integrative control of behavior. They converge with heterologous inputs that release other transmitters, including other peptides, in the same areas. The concept of a quasi-hormonal effect of peptide neurotransmission through coordinated release at multiple synapses throughout the brain echoes earlier conceptualizations of "action-at-a-distance" by diffusion following peptide release at non-synaptic sites. Yet, it recognizes that peptide delivery occurs with neuroanatomical precision, from discrete peptide-containing brain nuclei, via highly distributed projections to multiple extrahypothalamic nodes, registering multiple homeostatic, hedonistic, aversive and reproductive drives that modulate real-time motor decisions. There is paradigmatic value in the discussion of these two particular ancient neuropeptides, for peptide-centric translational neuroendocrinology and peptide GPCR-based neurotherapeutics.
- Extracellular vesicles from mature dendritic cells (DC) differentiate monocytes into immature DC. [Journal Article]
- LSLife Sci Alliance 2018; 1(6):e201800093
- During inflammation, murine and human monocytes can develop into dendritic cells (DC), but this process is not entirely understood. Here, we demonstrate that extracellular vesicles (EV) secreted by m...
During inflammation, murine and human monocytes can develop into dendritic cells (DC), but this process is not entirely understood. Here, we demonstrate that extracellular vesicles (EV) secreted by mature human DC (maDC) differentiate peripheral monocytes into immature DC, expressing a unique marker pattern, including 6-sulfo LacNAc (slan), Zbtb46, CD64, and CD14. While EV from both maDC and immature DC differentiated monocytes similar to GM-CSF/IL-4 stimulation, only maDC-EV produced precursors, which upon maturation stimulus developed into T-cell-activating and IL-12p70-secreting maDC. Mechanistically, maDC-EV induced cell signaling through GM-CSF, which was abundant in EV as were IL-4 and other cytokines and chemokines. When injected into the mouse skin, murine maDC-EV attracted immune cells including monocytes that developed activation markers typical for inflammatory cells. Skin-injected EV also reached lymph nodes, causing a similar immune cell infiltration. We conclude that DC-derived EV likely serve to perpetuate an immune reaction and may contribute to chronic inflammation.
- Archaea Symbiont of T. cruzi Infection May Explain Heart Failure in Chagas Disease. [Journal Article]
- FCFront Cell Infect Microbiol 2018; 8:412
- Background: Archaeal genes present in Trypanosoma cruzi may represent symbionts that would explain development of heart failure in 30% of Chagas disease patients. Extracellular vesicles in periphera...
Background: Archaeal genes present in Trypanosoma cruzi may represent symbionts that would explain development of heart failure in 30% of Chagas disease patients. Extracellular vesicles in peripheral blood, called exosomes (< 0.1 μm) or microvesicles (>0.1 μm), present in larger numbers in heart failure, were analyzed to determine whether they are derived from archaea in heart failure Chagas disease. Methods: Exosomes and microvesicles in serum supernatant from 3 groups were analyzed: heart failure Chagas disease (N = 26), asymptomatic indeterminate form (N = 21) and healthy non-chagasic control (N = 16). Samples were quantified with transmission electron microscopy, flow cytometer immunolabeled with anti-archaemetzincin-1 antibody (AMZ 1, archaea collagenase) and probe anti-archaeal DNA and zymography to determine AMZ1 (Archaeal metalloproteinase) activity. Results: Indeterminate form patients had higher median numbers of exosomes/case vs. heart failure patients (58.5 vs. 25.5, P < 0.001), higher exosome content of AMZ1 antigens (2.0 vs. 0.0; P < 0.001), and lower archaeal DNA content (0.2 vs. 1.5, P = 0.02). A positive correlation between exosomes and AMZ1 content was seen in indeterminate form (r = 0.5, P < 0.001), but not in heart failure patients (r = 0.002, P = 0.98). Higher free archaeal DNA (63.0 vs. 11.1, P < 0.001) in correlation with exosome numbers (r = 0.66, P = 0.01) was seen in heart failure but not in indeterminate form (r = 0.29, P = 0.10). Flow cytometer showed higher numbers of AMZ1 microvesicles in indeterminate form (64 vs. 36, P = 0.02) and higher archaeal DNA microvesicles in heart failure (8.1 vs. 0.9, P < 0.001). Zymography showed strong% collagenase activity in HF group, mild activity in IF compared to non-chagasic healthy group (121 ± 14, 106 ± 13 and 100; P < 0.001). Conclusions: Numerous exosomes, possibly removing and degrading abnormal AMZ1 collagenase, are associated with indeterminate form. Archaeal microvesicles and their exosomes, possibly associated with release of archaeal AMZ1 in heart failure, are future candidates of heart failure biomarkers if confirmed in larger series, and the therapeutic focus in the treatment of Chagas disease.
- Exosomes and miRNA-Loaded Biomimetic Nanovehicles, a Focus on Their Potentials Preventing Type-2 Diabetes Linked to Metabolic Syndrome. [Review]
- FIFront Immunol 2018; 9:2711
- Exosomes are small membrane vesicles of 30-150 nm, members of the extracellular vesicle family and secreted by various cell types. Different studies describe specific microRNA (miRNA) with altered ex...
Exosomes are small membrane vesicles of 30-150 nm, members of the extracellular vesicle family and secreted by various cell types. Different studies describe specific microRNA (miRNA) with altered expression in serum and/or plasma of patients suffering from diabetes or metabolic syndrome. Diabetic cardiomyocyte-derived exosomes loaded with miRNAs like miR-320-3p (or 320a) have been shown regulating angiogenesis on endothelial cell cultures. Insufficient myocardial angiogenesis is the major manifestation of diabetes-caused ischemic cardiovascular disease. Studies on transfer of functional microRNAs between mouse dendritic cells via exosomes have shown that some miRNAs (miR-320-3p, 29b-3p, 7a-5p) are distributed in immature and mature exosomes. Among these miRNAs, miR-320-3p is better known in epigenetics for silencing polr3d gene by binding to its promoter in Human Embryonic Kidney-293 cells. Moreover, quantitative and stoichiometric analysis of the microRNA content of exosomes highlights the lack of reliable natural source of such particles loaded with miRNA opening the need for tailoring exosomes or nanoparticles delivering efficiently miRNA intimately linked to immunity, metabolism and epigenetics in target cells. However, loading of extracellular mature miRNA into recipient cells comes with a cost by at least impeding dynamic localization of miRNAs in nucleoli or inefficient miRNA delivery due to rapid recycling by exonucleases. All these works are calling for the design of new biomimetic vehicles and in vivo assessment of miRNA functionality when delivered by natural or biomimetic nanoparticles in order to control metabolic diseases from infancy to adulthood.
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- The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells. [Journal Article]
- FMFront Microbiol 2018; 9:2765
- Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the comp...
Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of Aeromonas hydrophila OMVs is missing. In this study we analyzed the composition of purified OMVs from A. hydrophila ATCC® 7966TM by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90-170 nm. Moreover, 211 unique proteins were found in OMVs from A. hydrophila; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from A. hydrophila ATCC® 7966TM induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of A. hydrophila ATCC® 7966TM and their interaction with the host cell.