scholarly journals Three Method-Combination Protocol for Improving Purity of Extracellular Vesicles

2020 ◽  
Vol 21 (9) ◽  
pp. 3071 ◽  
Author(s):  
Thomas Simon ◽  
Anish Kumaran ◽  
Diana-Florentina Veselu ◽  
Georgios Giamas
Keyword(s):  
Extracellular Vesicles ◽  
Protein Complexes ◽  
Genetic Material ◽  
Research Field ◽  
Functional Studies ◽  
Protein Contamination ◽  
Final Yield ◽  
High Yields

Extracellular vesicles (EVs) are nanosized structures able to carry proteins, lipids and genetic material from one cell to another with critical implications in intercellular communication mechanisms. Even though the rapidly growing EVs research field has sparked great interest in the last 20 years, many biological and technical aspects still remain challenging. One of the main issues that the field is facing is the absence of consensus regarding methods for EVs concentration from biofluids and tissue culture medium. Yet, not only can classic methods be time consuming, commercialized kits are also often quite expensive, especially when research requires analyzing numerous samples or concentrating EVs from large sample volumes. In addition, EV concentration often results in either low final yield or significant contamination of the vesicle sample with proteins and protein complexes of similar densities and sizes. Eventually, low vesicle yields highly limit any further application and data reproducibility while contamination greatly impacts extensive functional studies. Hence, there is a need for accessible and sustainable methods for improved vesicle concentration as this is a critical step in any EVs-related research study. In this brief report, we describe a novel combination of three well-known methods in order to obtain moderate-to-high yields of EVs with reduced protein contamination. We believe that such methods could be of high benefits for in vitro and in vivo functional studies.

Review: Gp-340/DMBT1 in mucosal innate immunity

2010 ◽  
Vol 16 (3) ◽  
pp. 160-167 ◽  
Author(s):  
Jens Madsen ◽  
Jan Mollenhauer ◽  
Uffe Holmskov
Keyword(s):  
Innate Immunity ◽  
Influenza A ◽  
Research Field ◽  
Secretory Iga ◽  
Functional Studies ◽  
Gram Positive Bacteria ◽  
Alternatively Spliced ◽  
Trefoil Factor 2

Deleted in Malignant Brain Tumour 1 (DMBT1) is a gene that encodes alternatively spliced proteins involved in mucosal innate immunity. It also encodes a glycoprotein with a molecular mass of 340 kDa, and is referred to as gp-340 (DMBT1gp340) and salivary agglutinin (DMBT1SAG). DMBT1gp340 is secreted into broncho-alveolar surface lining fluid whereas DMBTSAG is present in the saliva. The two molecules were shown to be identical and both interact with and agglutinate several Gram-negative and Gram-positive bacteria including Streptococcus mutans, a bacterium responsible for caries in the oral cavity. DMBT1gp340 interacts with surfactant proteins A and D (SP-D). DMBT1gp340 and SP-D can individually and together interact and agglutinate influenza A virus. DMBT1gp340 also binds to HIV-1 and facilitates transcytosis of the virus into epithelial cells. DMBT1 binds to a variety of other host proteins, including serum and secretory IgA, C1q, lactoferrin, MUC5B and trefoil factor 2 (TFF2), all molecules with involvement in innate immunity and/or wound-healing processes. Recent generation of Dmbt1-deficient mice has provided the research field of DMBT1 with a model that allows research to progress from in vitro studies to in vivo functional studies of the multifunctional proteins encoded by the DMBT1 gene.

scholarly journals Self-Assembling Metabolon Enables the Cell Free Conversion of Glycerol to 1,3-Propanediol

2021 ◽  
Vol 9 ◽  
Author(s):  
Qi Xu ◽  
Markus Alahuhta ◽  
Patrick Hewitt ◽  
Nicholas S. Sarai ◽  
Hui Wei ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Product Formation ◽  
Glycerol Concentration ◽  
Protein Scaffolds ◽  
Cellulolytic Bacteria ◽  
Glycerol Conversion ◽  
Self Assembling ◽  
High Yields

Cell free biocatalysis is showing promise as a replacement or complement to conventional microbial biocatalysts due to the potential for achieving high yields, titers, and productivities. However, there exist several challenges that need to be addressed before its broader industrial adoption is achieved. New paradigms and innovative solutions are needed to overcome these challenges. In this study we demonstrate high levels of glycerol conversion to 1,3-propanediol using a self-assembling metabolic pathway leveraging the arraying strategy (protein scaffolds) used by thermophilic cellulolytic bacteria to assemble their biomass degrading enzymes. These synthetic metabolons were capable of producing 1,3-PDO at a yield more than 95% at lower glycerol concentration and close to 70% at higher concentrations at a higher productivity rate than the equivalent microbial strain. One of the benefits of our approach is the fact that no enzyme purification is required, and that the assembly of the complex is accomplished in vivo before immobilization, while product formation is conducted in vitro. We also report the recovery of enzymatic activity upon fusion enzymes binding to these protein scaffolds, which could have broader applications when assembling arrayed protein complexes.

scholarly journals Extracellular Vesicles, the Road toward the Improvement of ART Outcomes

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2171
Author(s):  
Maria G. Gervasi ◽  
Ana J. Soler ◽  
Lauro González-Fernández ◽  
Marco G. Alves ◽  
Pedro F. Oliveira ◽  
...  
Keyword(s):  
Embryo Development ◽  
Extracellular Vesicles ◽  
Reproductive Tract ◽  
Assisted Reproductive Technologies ◽  
Genetic Material ◽  
Reproductive Technologies ◽  
Female Reproductive Tract ◽  
Vitro Fertilization

Nowadays, farm animal industries use assisted reproductive technologies (ART) as a tool to manage herds’ reproductive outcomes, for a fast dissemination of genetic improvement as well as to bypass subfertility issues. ART comprise at least one of the following procedures: collection and handling of oocytes, sperm, and embryos in in vitro conditions. Therefore, in these conditions, the interaction with the oviductal environment of gametes and early embryos during fertilization and the first stages of embryo development is lost. As a result, embryos obtained in in vitro fertilization (IVF) have less quality in comparison with those obtained in vivo, and have lower chances to implant and develop into viable offspring. In addition, media currently used for IVF are very similar to those empirically developed more than five decades ago. Recently, the importance of extracellular vesicles (EVs) in the fertility process has flourished. EVs are recognized as effective intercellular vehicles for communication as they deliver their cargo of proteins, lipids, and genetic material. Thus, during their transit through the female reproductive tract both gametes, oocyte and spermatozoa (that previously encountered EVs produced by male reproductive tract) interact with EVs produced by the female reproductive tract, passing them important information that contributes to a successful fertilization and embryo development. This fact highlights that the reproductive tract EVs cargo has an important role in reproductive events, which is missing in current ART media. This review aims to recapitulate recent advances in EVs functions on the fertilization process, highlighting the latest proposals with an applied approach to enhance ART outcome through EV utilization as an additive to the media of current ART procedures.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

A Systematic Review on Extracellular Vesicles-Enriched Fat Grafting: A Shifting Paradigm

2020 ◽  
Author(s):  
Mohammad Ghiasloo ◽  
Laura De Wilde ◽  
Kashika Singh ◽  
Patrick Tonnard ◽  
Alexis Verpaele ◽  
...  
Keyword(s):  
Systematic Review ◽  
Graft Survival ◽  
Extracellular Vesicles ◽  
Retention Rate ◽  
Fat Grafting ◽  
Retention Rates ◽  
Fat Graft ◽  
Cochrane Central Register

Abstract Background Recent evidence confirms that mesenchymal stem cells (MSCs) facilitate angiogenesis mainly through paracrine function. Extracellular vesicles (EVs) are regarded as key components of the cell secretome, possessing functional properties of their source cells. Subsequently, MSC-EVs have emerged as a novel cell-free approach to improve fat graft retention rate. Objectives To provide a systematic review of all studies reporting the use of MSC-EVs to improve graft retention rate. Methods A systematic search was undertaken using the Embase, PubMed and the Cochrane Central Register of Controlled Trials databases. Outcome measures included donor/receptor organism of the fat graft, study model, intervention groups, evaluation intervals, EV research data, in vitro and in vivo results. Results Of the total 1717 articles, 62 full-texts were screened. Seven studies reporting on 294mice were included. Overall, EV treated groups showed higher graft retention rates compared to untreated groups. Notably, retention rate was similar following EV- and MSC-treatment. In addition to reduced inflammation, graft enrichment with EVs resulted in early revascularization and better graft integrity. Interestingly, hypoxic preconditioning of MSCs improved their beneficial paracrine effects and led to a more proangiogenic EV population, as observed by both in vitro and in vivo results. Conclusions MSC-EVs appear to offer an interesting cell-free alternative to improve fat graft survival. While their clinical relevance remains to be determined, it is clear that not the cells, but their secretome is essential for graft survival. Thus, a paradigm shift from cell-assisted lipotransfer towards ‘secretome-assisted lipotransfer’ is well on its way.

scholarly journals Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

Membranes ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 411
Author(s):  
Nader Kameli ◽  
Anya Dragojlovic-Kerkache ◽  
Paul Savelkoul ◽  
Frank R. Stassen
Keyword(s):  
Human Health ◽  
Extracellular Vesicles ◽  
Preliminary Results ◽  
In Vivo Experiments ◽  
Health And Disease ◽  
Conducting Research ◽  
Protocol Standardization ◽  
Insight Into

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals The Therapeutic Effect of Extracellular Vesicles on Asthma in Pre-Clinical Models: A Systematic Review Protocol

2021 ◽  
Vol 1 (1) ◽  
pp. 84-95
Author(s):  
Patience O. Obi ◽  
Jennifer E. Kent ◽  
Maya M. Jeyaraman ◽  
Nicole Askin ◽  
Taiana M. Pierdoná ◽  
...  
Keyword(s):  
Systematic Review ◽  
Extracellular Vesicles ◽  
Current Knowledge ◽  
Grey Literature ◽  
Specific Treatment ◽  
Therapeutic Effects ◽  
Management Options ◽  
Paracrine Signalling

Asthma is the most common pediatric disease, characterized by chronic airway inflammation and airway hyperresponsiveness. There are several management options for asthma, but no specific treatment. Extracellular vesicles (EVs) are powerful cellular mediators of endocrine, autocrine and paracrine signalling, and can modulate biophysiological function in vitro and in vivo. A thorough investigation of therapeutic effects of EVs in asthma has not been conducted. Therefore, this systematic review is designed to synthesize recent literature on the therapeutic effects of EVs on physiological and biological outcomes of asthma in pre-clinical studies. An electronic search of Web of Science, EMBASE, MEDLINE, and Scopus will be conducted on manuscripts published in the last five years that adhere to standardized guidelines for EV research. Grey literature will also be included. Two reviewers will independently screen the selected studies for title and abstract, and full text based on the eligibility criteria. Data will be extracted, narratively synthesized and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This systematic review will summarize the current knowledge from preclinical studies investigating the therapeutic effects of EVs on asthma. The results will delineate whether EVs can mitigate biological hallmarks of asthma, and if so, describe the underlying mechanisms involved in the process. This insight is crucial for identifying key pathways that can be targeted to alleviate the burden of asthma. The data will also reveal the origin, dosage and biophysical characteristics of beneficial EVs. Overall, our results will provide a scaffold for future intervention and translational studies on asthma treatment.

scholarly journals POLRMT mutations impair mitochondrial transcription causing neurological disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Monika Oláhová ◽  
Bradley Peter ◽  
Zsolt Szilagyi ◽  
Hector Diaz-Maldonado ◽  
Meenakshi Singh ◽  
...  
Keyword(s):  
Progressive External Ophthalmoplegia ◽  
Global Developmental Delay ◽  
Disease Mechanism ◽  
Mitochondrial Transcription ◽  
Functional Studies ◽  
Mtdna Deletions ◽  
Molecular Nature ◽  
Important Disease

AbstractWhile >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.

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