sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum interact with immune genes of rainbow trout

Outer membrane vesicles (OMVs) released by gram-negative bacteria during host-pathogen interactions harbor cargos, such as DNA, RNA, toxins, and virulence factors. We hypothesized that sRNAs carried within OMVs of Flavobacterium psychrophilum interact with host immune genes and affect their expression. OMVs were isolated from F. psychrophilum and visualized using transmission electron microscopy (TEM). RNA-Seq datasets generated from whole-cell F. psychrophilum and their OMVs indicated enrichment of specific sRNAs in the OMVs compared to the parent cell. Fluorescent in situ hybridization (FISH) and confocal microscopy confirmed the expression of a randomly chosen sRNA. Integrated RNA-Seq analyses of host transcriptome and bacterial sRNAs on day 5 post-infection of F. psychrophilum -resistant and -susceptible rainbow trout genetic lines revealed 516 protein-coding, 595 lncRNA, and 116 bacterial sRNA differentially expressed (DE) transcripts. Integrated and network analyses of these DE transcripts revealed immune genes targeted by bacterial sRNAs. On the top of these genes, an isoform encoding anaphase-promoting complex subunit 13 (ANAPC13_1) was highly upregulated and exhibited interaction and reciprocal expression with 21 DE sRNAs enriched in OMVs and/or located in pathogenicity islands (PAIs). In vitro treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cell autolysis accompanied by dynamic changes in expression of host genes when profiled 24h following treatment. The OMV-enriched sRNAs, soFE013584 and soFE002123, showed high interactions with the protection of telomeres 1 gene (POT1); essential for chromosome stability and cellular viability. Modulation of the host gene expression following OMV-treatment, which favors elements from the phagocytic, endocytic, and antigen presentation pathways in addition to HSP70, HSP90, and cochaperone proteins, provided evidence for a potential role of OMVs in boosting the host immune response. In conclusion, our work identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.

Challenges for the Development of Extracellular Vesicle-Based Nucleic Acid Medicines

Nucleic acid drugs, such as siRNAs, antisense oligonucleotides, and miRNAs, exert their therapeutic effects by causing genetic changes in cells. However, there are various limitations in their delivery to target organs and cells, making their application to cancer treatment difficult. Extracellular vesicles (EVs) are lipid bilayer particles that are released from most cells, are stable in the blood, and have low immunogenicity. Methods using EVs to deliver nucleic acid drugs to target organs are rapidly being developed that take advantage of these properties. There are two main methods for loading nucleic acid drugs into EVs. One is to genetically engineer the parent cell and load the target gene into the EV, and the other is to isolate EVs and then load them with the nucleic acid drug. Target organ delivery methods include passive targeting using the enhanced permeation and retention effect of EVs and active targeting in which EVs are modified with antibodies, peptides, or aptamers to enhance their accumulation in tumors. In this review, we summarize the advantages of EVs as a drug delivery system for nucleic acid drugs, the methods of loading nucleic acid drugs into EVs, and the targeting of EVs to target organs.

Long noncoding RNA CRART16 confers 5-FU resistance in colorectal cancer cells by sponging miR-193b-5p

Background The emergence of chemoresistance to 5-fluorouracil (5-FU)-based chemotherapy is the main cause of treatment failure in advanced and metastatic colorectal cancer (CRC) patients. Long noncoding RNAs (lncRNAs) have been reported to be involved in 5-FU resistance. Previously, we first detected that lncRNA cetuximab resistance-associated RNA transcript 16 (CRART16) could contribute to cetuximab resistance by upregulating V-Erb-B2 erythroblastic leukemia viral oncogene homologue 3 (ERBB3) expression by sponging miR-371a-5p in CRC cells. The current study aimed to explore the role of CRART16 in acquired 5-FU resistance in CRC cells and its possible mechanism. Methods Quantitative real-time PCR (RT-qPCR) was used to measure the expression levels of CRART16 in a 5-FU-resistant CRC cell subline (SW620/5-FU) and the parent cell line. Lentivirus transduction was performed to establish SW620 and Caco-2 cells stably overexpressing CRART16. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were applied to measure cell chemosensitivity to 5-FU. Flow cytometric and immunofluorescence staining were adopted to assess cell apoptosis induced by 5-FU. The dual-luciferase reporter assay was used to validate the direct interactions between CRART16 and miR-193b-5p and between miR-193b-5p and high-mobility group AT-hook-2 (HMGA2). The expression levels of HMGA2, apoptosis-associated proteins and p-ERK were examined by western blotting. The statistical differences within any two groups were used Student’s t test. Results CRART16 was upregulated in SW620/5-FU cells. Overexpression of CRART16 reduced the sensitivity of CRC cells to 5-FU by attenuating apoptosis. In addition, CRART16 promoted 5-FU resistance by suppressing the expression of miR-193b-5p. Furthermore, CRART16 modulated the expression of HMGA2 by inhibiting miR-193b-5p and activated the MAPK signaling pathway. Conclusions CRART16 confers 5-FU resistance in CRC cells through the CRART16/miR-193b-5p/HMGA2/MAPK pathway.

On the Role of Electrostatic Repulsion in Topological Defect-Driven Membrane Fission

Within a modified Langevin Poisson–Boltzmann model of electric double layers, we derived an analytical expression for osmotic pressure between two charged surfaces. The orientational ordering of the water dipoles as well as the space dependencies of electric potentials, electric fields, and osmotic pressure between two charged spheres were taken into account in the model. Thus, we were able to capture the interaction between the parent cell and connected daughter vesicle or the interactions between neighbouring beads in necklace-like membrane protrusions. The predicted repulsion between them can facilitate the topological antidefect-driven fission of membrane daughter vesicles and the fission of beads of undulated membrane protrusions.

Engineering Cardiac Small Extracellular Vesicle-Derived Vehicles with Thin-Film Hydration for Customized microRNA Loading

Cell therapies for myocardial infarction, including cardiac ckit+ progenitor cell (CPC) therapies, have been promising, with clinical trials underway. Recently, paracrine signaling, specifically through small extracellular vesicle (sEV) release, was implicated in cell-based cardiac repair. sEVs carry cardioprotective cargo, including microRNA (miRNA), within a complex membrane and improve cardiac outcomes similar to that of their parent cells. However, miRNA loading efficiency is low, and sEV yield and cargo composition vary with parent cell conditions, minimizing sEV potency. Synthetic mimics allow for cargo-loading control but consist of much simpler membranes, often suffering from high immunogenicity and poor stability. Here, we aim to combine the benefits of sEVs and synthetic mimics to develop sEV-like vesicles (ELVs) with customized cargo loading. We developed a modified thin-film hydration (TFH) mechanism to engineer ELVs from CPC-derived sEVs with pro-angiogenic miR-126 encapsulated. Characterization shows miR-126+ ELVs are similar in size and structure to sEVs. Upon administration to cardiac endothelial cells (CECs), ELV uptake is similar to sEVs too. Further, when functionally validated with a CEC tube formation assay, ELVs significantly improve tube formation parameters compared to sEVs. This study shows TFH-ELVs synthesized from sEVs allow for select miRNA loading and can improve in vitro cardiac outcomes.

Assembly of tetraspanins, galectin-3, and distinct N-glycans defines the solubilization signature of seminal prostasomes from normozoospermic and oligozoospermic men

Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

Extracellular Vesicles in Innate Immune Cell Programming

Extracellular vesicles (EV) are a heterogeneous group of bilipid-enclosed envelopes that carry proteins, metabolites, RNA, DNA and lipids from their parent cell of origin. They mediate cellular communication to other cells in local tissue microenvironments and across organ systems. EV size, number and their biologically active cargo are often altered in response to pathological processes, including infection, cancer, cardiovascular diseases and in response to metabolic perturbations such as obesity and diabetes, which also have a strong inflammatory component. Here, we discuss the broad repertoire of EV produced by neutrophils, monocytes, macrophages, their precursor hematopoietic stem cells and discuss their effects on the innate immune system. We seek to understand the immunomodulatory properties of EV in cellular programming, which impacts innate immune cell differentiation and function. We further explore the possibilities of using EV as immune targeting vectors, for the modulation of the innate immune response, e.g., for tissue preservation during sterile injury such as myocardial infarction or to promote tissue resolution of inflammation and potentially tissue regeneration and repair.

Melting of Paraffin Waxes Embedded in a Porous Matrix Made by Additive Manufacturing

The recent advances in additive manufacturing technology have widened the choice of materials that can be printed, opening new frontiers in the field of heat transfer devices. This paper explores the use of a solid porous matrix in which paraffin waxes, having different melting temperatures (42, 55, and 64 °C), were embedded. The solid matrix is made by additive manufacturing. The parent cell of the porous matrix occupies the volume of a cube with an edge of 5 mm. The entire 3D printed matrix has a square base with an edge of 100 mm, and it has a height of 20 mm. The solid matrix was printed between two plates, each one with a thickness of 10 mm, where thermocouples were inserted, and it was tested in an upright position, laterally heated applying three different heat fluxes (10, 15, and 20 kW m−2). The experimental results are given in terms of the temperature of the heated side, as well as of the phase change material, during the heating process. The temperature reached by the heated side and the time needed to completely melt the paraffin waxes are compared at the different working conditions. Furthermore, the thermal conductivities and diffusivities of the three paraffins and of the parent material of the porous matrix were experimentally evaluated.

Tumor-Derived Exosomes (TEX) and Their Role in Immuno-Oncology

Extracellular vesicles (EVs) play a key role in health and disease, including cancer. Tumors produce a mix of EVs differing in size, cellular origin, biogenesis and molecular content. Small EVs (sEV) or exosomes are a subset of 30–150 nm (virus–size) vesicles originating from the multivesicular bodies (MVBs) and carrying a cargo that in its content and topography approximates that of a parent cell. Tumor-derived exosomes (TEX) present in all body fluids of cancer patients, are considered promising candidates for a liquid tumor biopsy. TEX also mediate immunoregulatory activities: they maintain a crosstalk between the tumor and various non-malignant cells, including immunocytes. Effects that EVs exert on immune cells may be immunosuppressive or immunostimulatory. Here, we review the available data for TEX interactions with immunocytes, focusing on strategies that allow isolation from plasma and separation of TEX from sEV produced by non-malignant cells. Immune effects mediated by either of the subsets can now be distinguished and measured. The approach has allowed for the comparison of molecular and functional profiles of the two sEV fractions in plasma of cancer patients. While TEX carried an excess of immunosuppressive proteins and inhibited immune cell functions in vitro and in vivo, the sEV derived from non-malignant cells, including CD3(+)T cells, were variably enriched in immunostimulatory proteins and could promote functions of immunocytes. Thus, sEV in plasma of cancer patients are heterogenous, representing a complex molecular network which is not evident in healthy donors’ plasma. Importantly, TEX appear to be able to reprogram functions of non-malignant CD3(+)T cells inducing them to produce CD3(+)sEV enriched in immunosuppressive proteins. Ratios of stimulatory/inhibitory proteins carried by TEX and by CD3(+)sEV derived from reprogrammed non-malignant cells vary broadly in patients and appear to negatively correlate with disease progression. Simultaneous capture from plasma and functional/molecular profiling of TEX and the CD3(+)sEV fractions allows for defining their role as cancer biomarkers and as monitors of cancer patients’ immune competence, respectively.

The Transformative Impact of Extracellular Vesicles on Developing Sperm

Objective To review the role of extracellular vesicles (EVs) released from the male reproductive tract and their impact on developing sperm. We discuss how sperm exiting the seminiferous tubules, although developmentally mature, require further modification. Acquisition of various functions including increased motility, transfer of cargoes and ability to undertake the acrosome reaction are mediated through the interaction between sperm and EVs. Methods A review of the literature identified that EVs are released from different portions of the male reproductive tract, notably the epididymis and prostate. These EVs interact with sperm as they pass from the seminiferous tubules to the epididymis and vas deferens prior to ejaculation. Results EVs are small lipid bound particles carrying bespoke RNA, protein and lipid cargoes. These cargoes are loaded based on the state of the parent cell and are used to communicate with recipient cells. In sperm, these cargoes are essential for post testicular modification. Sperm extracted from the proximal epididymis are poorly motile and unable to carry out the acrosome reaction. Conclusions Interaction between developing sperm and EVs is important for the subsequent function of sperm. Little is known however about EVs released from the seminiferous tubules to developing sperm or in the fetal and pre-pubertal testes. A greater understanding, especially in the formation and development of the spermatogonial stem cell niche may lead to new insights as to how damage to this niche may be prevented and preserve future fertility.