Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc.

Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.

Dual-Wave Acoustofluidic Centrifuge for Ultrafast Concentration of Nanoparticles and Extracellular Vesicles.

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs.

Advances in Platelet Rich Plasma-Derived Extracellular Vesicles for Regenerative Medicine: A Systematic-Narrative Review.

The use of platelet-rich plasma (PRP) has gained increasing interest in recent decades. The platelet secretome contains a multitude of growth factors, cytokines, chemokines, and other biological biomolecules. In recent years, developments in the field of platelets have led to new insights, and attention has been focused on the platelets' released extracellular vesicles (EVs) and their role in intercellular communication. In this context, the aim of this review was to compile the current evidence on PRP-derived extracellular vesicles to identify the advantages and limitations fortheir use in the upcoming clinical applications. A total of 172 articles were identified during the systematic literature search through two databases (PubMed and Web of Science). Twenty publications met the inclusion criteria and were included in this review. According to the results, the use of PRP-EVs in the clinic is an emerging field of great interest that represents a promising therapeutic option, as their efficacy has been demonstrated in the majority of fields of applications included in this review. However, the lack of standardization along the procedures in both the field of PRP and the EVs makes it extremely challenging to compare results among studies. Establishing standardized conditions to ensure optimized and detailed protocols and define parameters such as the dose or the EV origin is therefore urgent. Further studies to elucidate the real contribution of EVs to PRP in terms of composition and functionality should also be performed. Nevertheless, research on the field provides promising results and a novel basis to deal with the regenerative medicine and drug delivery fields in the future.

microRNA-based signatures obtained from endometrial fluid identify implantative endometrium.

STUDY QUESTION: Is it possible to use free and extracellular vesicle-associated microRNAs (miRNAs) from human endometrial fluid (EF) samples as non-invasive biomarkers for implantative endometrium? SUMMARY ANSWER: The free and extracellular vesicle-associated miRNAs can be used to detect implantative endometrium in a non-invasive manner. WHAT IS KNOWN ALREADY: miRNAs and extracellular vesicles (EVs) from EF have been described as mediators of the embryo-endometrium crosstalk. Therefore, the analysis of miRNA from this fluid could become a non-invasive technique for recognizing implantative endometrium. This analysis could potentially help improve the implantation rates in ART. STUDY DESIGN, SIZE, DURATION: In this prospective study, we first optimized different protocols for EVs and miRNA analyses using the EF of a setup cohort (n = 72). Then, we examined differentially expressed miRNAs in the EF of women with successful embryo implantation (discovery cohort n = 15/validation cohort n = 30) in comparison with those for whom the implantation had failed (discovery cohort n = 15/validation cohort n = 30). Successful embryo implantation was considered when pregnancy was confirmed by vaginal ultrasound showing a gestational sac 4 weeks after embryo transfer (ET). PARTICIPANTS/MATERIALS, SETTING, METHODS: The EF of the setup cohort was obtained before starting fertility treatment during the natural cycle, 16-21 days after the beginning of menstruation. For the discovery and validation cohorts, the EF was collected from women undergoing frozen ET on Day 5, and the samples were collected immediately before ET. In this study, we compared five different methods; two of them based on direct extraction of RNA and the other three with an EV enrichment step before the RNA extraction. Small RNA sequencing was performed to determine the most efficient method and find a predictive model differentiating between implantative and non-implantative endometrium. The models were confirmed using quantitative PCR in two sets of samples (discovery and validation cohorts) with different implantation outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The protocols using EV enrichment detected more miRNAs than the methods based on direct RNA extraction. The two most efficient protocols (using polymer-based precipitation (PBP): PBP-M and PBP-N) were used to obtain two predictive models (based on three miRNAs) allowing us to distinguish between an implantative and non-implantative endometrium. The first Model 1 (PBP-M) (discovery: AUC = 0.93; P-value = 0.003; validation: AUC = 0.69; P-value = 0.019) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-148b-3p. Model 2 (PBP-N) (discovery: AUC = 0.92; P-value = 0.0002; validation: AUC = 0.78; P-value = 0.0002) used hsa-miR-200b-3p, hsa-miR-24-3p and hsa-miR-99b-5p. Functional analysis of these miRNAs showed strong association with key implantation processes such as in utero embryonic development or transforming growth factor-beta signaling. LARGE SCALE DATA: The FASTQ data are available in the GEO database (access number GSE178917). LIMITATIONS, REASONS FOR CAUTION: One important factor to consider is the inherent variability among the women involved in the trial and among the transferred embryos. The embryos were pre-selected based on morphology, but neither genetic nor molecular studies were conducted, which would have improved the accuracy of our tests. In addition, a limitation in miRNA library construction is the low amount of input RNA. WIDER IMPLICATIONS OF THE FINDINGS: We describe new non-invasive protocols to analyze miRNAs from small volumes of EF. These protocols could be implemented in clinical practice to assess the status of the endometrium before attempting ET. Such evaluation could help to avoid the loss of embryos transferred to a non-implantative endometrium. STUDY FUNDING/COMPETING INTEREST(S): J.I.-P. was supported by a predoctoral grant from the Basque Government (PRE_2017_0204). This study was partially funded by the Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). It was also supported by the Spanish Ministry of Economy and Competitiveness MINECO within the National Plan RTI2018-094969-B-I00, the European Union's Horizon 2020 research and innovation program (860303), the Severo Ochoa Centre of Excellence Innovative Research Grant (SEV-2016-0644) and the Instituto de Salud Carlos III (PI20/01131). The funding entities did not play any role in the study design, collection, analysis and interpretation of data, writing of the report or the decision to submit the article for publication. The authors declare no competing interests.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

The unpredictable carbon nanotube biocorona and a functionalization method to prevent protein biofouling.

BACKGROUND: The intrinsic physicochemical properties of carbon nanotubes (CNTs) make them unique tools in nanotechnology. Their elemental composition, resilience, thermal properties, and surface reactivity make CNTs also of undisputed interest in biotechnology. In particular, their extraordinary ability to capture biomolecules on their surface makes them essential in this field. The proteins adsorbed on the CNTs create a biological coating that endows them the ability to interact with some cell receptors, penetrate membranes or interfere with cell biomechanics, thus behaving as an active bio-camouflage. But some of these proteins unfold, triggering an immune response that unpredictably changes the biological activity of CNTs. For this reason, the control of the biocorona is fundamental in the nanobiotechnology of CNTs. RESULTS: Using TEM and AFM here we demonstrate a significant increase in CNTs diameter after protein functionalization. A quantitative analysis using TGA revealed that between 20 and 60% of the mass of functionalized nanotubes corresponds to protein, with single-walled CNTs capturing the highest amounts. To qualitatively/quantitatively characterize these biocoatings, we studied the biochemical "landscape" of the proteins captured by the different nanotubes after functionalization under various conditions. This study revealed a significant variability of the proteins in the corona as a function of the type of nanotube, the functionalization temperature, or the time after exposure to serum. Remarkably, the functionalization of a single type of CNT with sera from various human donors also resulted in different protein landscapes. Given the unpredictable assortment of proteins captured by the corona and the biological implications of this biocoating, we finally designed a method to genetically engineer and produce proteins to functionalize nanotubes in a controlled and customizable way. CONCLUSIONS: We demonstrate the high unpredictability of the spontaneous protein corona on CNTs and propose a versatile functionalization technique that prevents the binding of nonspecific proteins to the nanotube to improve the use of CNTs in biomedical applications.

Extracellular Vesicles in the Fungi Kingdom.

Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.

Hereditary tyrosinemia type I-associated mutations in fumarylacetoacetate hydrolase reduce the enzyme stability and increase its aggregation rate.

More than 100 mutations in the gene encoding fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1), a metabolic disorder characterized by elevated blood levels of tyrosine. Some of these mutations are known to decrease FAH catalytic activity, but the mechanisms of FAH mutation-induced pathogenicity remain poorly understood. Here, using diffusion ordered NMR spectroscopy, cryo-EM, and CD analyses, along with site-directed mutagenesis, enzymatic assays, and molecular dynamics simulations, we investigated the putative role of thermodynamic and kinetic stability in WT FAH and a representative set of 19 missense mutations identified in individuals with HT1. We found that at physiological temperatures and concentrations, WT FAH is in equilibrium between a catalytically active dimer and a monomeric species, with the latter being inactive and prone to oligomerization and aggregation. We also found that the majority of the deleterious mutations reduce the kinetic stability of the enzyme and always accelerate the FAH aggregation pathway. Depending mainly on the position of the amino acid in the structure, pathogenic mutations either reduced the dimer population or decreased the energy barrier that separates the monomer from the aggregate. The mechanistic insights reported here pave the way for the development of pharmacological chaperones that target FAH to tackle the severe disease HT1.

A custom-made functionalization method to control the biological identity of nanomaterials.

Here we propose a one-step strategy to endow nanomaterials with a custom-designed bio-identity. This study designs a universal 'nanomaterial binding domain' that can be genetically attached to any protein ensuring precise and spontaneous protein orientation. We demonstrate how, despite the simplicity of the method, the bioconjugation achieved: (i) is highly efficient, even in the presence of competing proteins, (ii) is stable at extreme physiological conditions (pH ranges 5.2-9.0; NaCl concentrations 0-1 M); (iii) prevents unwanted protein biofouling days after incubation in biologically-relevant conditions; and finally, (iv) avoids nanoparticle interaction with promiscuous unspecific receptors. In summary, this protein biocoating technique, applicable to a wide array of nano-designs, integrates material science and molecular biology procedures to create hybrid nanodevices with well-defined surfaces and predictable biological behaviors, opening a chapter in precision nanodiagnostics, nanosensing or nanotherapeutic applications.

Design of Polymeric and Biocompatible Delivery Systems by Dissolving Mesoporous Silica Templates.

There are many nanoencapsulation systems available today. Among all these, mesoporous silica particles (MSPs) have received great attention in the last few years. Their large surface-to-volume ratio, biocompatibility, and versatility allow the encapsulation of a wide variety of drugs inside their pores. However, their chemical instability in biological fluids is a handicap to program the precise release of the therapeutic compounds. Taking advantage of the dissolving capacity of silica, in this study, we generate hollow capsules using MSPs as transitory sacrificial templates. We show how, upon MSP coating with different polyelectrolytes or proteins, fully customized hollow shells can be produced. These capsules are biocompatible, flexible, and biodegradable, and can be decorated with nanoparticles or carbon nanotubes to endow the systems with supplementary intrinsic properties. We also fill the capsules with a fluorescent dye to demonstrate intracellular compound release. Finally, we document how fluorescent polymeric capsules are engulfed by cells, releasing their encapsulated agent during the first 96 h. In summary, here, we describe how to assemble a highly versatile encapsulation structure based on silica mesoporous cores that are completely removed from the final polymeric capsule system. These drug encapsulation systems are highly customizable and have great versatility as they can be made using silica cores of different sizes and multiple coatings. This provides capsules with unique programmable attributes that are fully customizable according to the specific needs of each disease or target tissue for the development of nanocarriers in personalized medicine.

EV-associated miRNAs from peritoneal lavage as potential diagnostic biomarkers in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide. Current systematic methods for diagnosing have inherent limitations so development of a minimally-invasive diagnosis, based on the identification of sensitive biomarkers in liquid biopsies could therefore facilitate screening among population at risk. METHODS: In this study, we aim to develop a novel approach to identify highly sensitive and specific biomarkers by investigating the use of extracellular vesicles (EVs) isolated from the peritoneal lavage as a source of potential miRNA diagnostic biomarkers. We isolated EVs by ultracentrifugation from 25 ascitic fluids and 25 peritoneal lavages from non-cancer and CRC patients, respectively. Analysis of the expression of EV-associated miRNAs was performed using Taqman OpenArray technology through which we could detect 371 miRNAs. RESULTS: 210 miRNAs were significantly dysregulated (adjusted p value < 0.05 and abs(logFC) ≥ 1). The top-10 miRNAs, which had the AUC value higher than 0.95, were miRNA-199b-5p, miRNA-150-5p, miRNA-29c-5p, miRNA-218-5p, miRNA-99a-3p, miRNA-383-5p, miRNA-199a-3p, miRNA-193a-5p, miRNA-10b-5p and miRNA-181c-5p. CONCLUSIONS: This finding opens the avenue to the use of EV-associated miRNA of peritoneal lavages as an untapped source of biomarkers for CRC.

Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions, such as primary sclerosing cholangitis (PSC), are risk factors. Noninvasive differential diagnosis between intrahepatic CCA and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate noninvasive biomarkers for PSC, CCA, and HCC are not available. In the search for novel biomarkers, serum extracellular vesicles (EV) were isolated from CCA (n = 43), PSC (n = 30), or HCC (n = 29) patients and healthy individuals (control, n = 32); and their protein content was characterized. By using nanoparticle tracking analysis, serum EV concentration was found to be higher in HCC than in all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by nanoparticle tracking analysis), and markers (clusters of differentiation 9, 63, and 81 by immunoblot) indicated that most serum EV were exosomes. Proteome profiles (by mass spectrometry) revealed multiple differentially expressed proteins among groups. Several of these proteins showed high diagnostic values with maximum area under the receiver operating characteristic curve of 0.878 for CCA versus control, 0.905 for CCA stage I-II versus control, 0.789 for PSC versus control, 0.806 for noncirhottic PSC versus control, 0.796 for CCA versus PSC, 0.956 for CCA stage I-II versus PSC, 0.904 for HCC versus control, and 0.894 for intrahepatic CCA versus HCC. Proteomic analysis of EV derived from CCA human cells in vitro revealed higher abundance of oncogenic proteins compared to EV released by normal human cholangiocytes. Orthotopic implant of CCA human cells in the liver of immunodeficient mice resulted in the release to serum of EV containing some similar human oncogenic proteins. CONCLUSION: Proteomic signatures found in serum EV of CCA, PSC, and HCC patients show potential usefulness as diagnostic tools. (Hepatology 2017;66:1125-1143).

Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation.

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed "inflammaging" has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process.

Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation.

In Arabidopsis thaliana, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high-affinity phosphate transporters. They are transcriptionally induced by phosphate starvation and require PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1) to exit the endoplasmic reticulum (ER), indicating intracellular traffic as an additional level of regulation of PHT1 activity. Our study revealed that PHF1 acts on PHT1, upstream of vesicle coat protein COPII formation, and that additional regulatory events occur during PHT1 trafficking and determine its ER exit and plasma membrane stability. Phosphoproteomic and mutagenesis analyses revealed modulation of PHT1;1 ER export by Ser-514 phosphorylation status. Confocal microscopy analysis of root tip cells showed that PHT1;1 is localized to the plasma membrane and is present in intracellular endocytic compartments. More precisely, PHT1;1 was localized to sorting endosomes associated with prevacuolar compartments. Kinetic analysis of PHT1;1 stability and targeting suggested a modulation of PHT1 internalization from the plasma membrane to the endosomes, followed by either subsequent recycling (in low Pi) or vacuolar degradation (in high Pi). For the latter condition, we identified a rapid mechanism that reduces the pool of PHT1 proteins present at the plasma membrane. This mechanism is regulated by the Pi concentration in the medium and appears to be independent of degradation mechanisms potentially regulated by the PHO2 ubiquitin conjugase. We propose a model for differential trafficking of PHT1 to the plasma membrane or vacuole as a function of phosphate concentration.

The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques.

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions.

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.

Lipidomics and biodistribution of extracellular vesicles-secreted by hepatocytes from Zucker lean and fatty rats.

Extracellular vesicles (EVs) have been involved in metabolic syndrome, although their specific role in the development of the pathology is still unknown. To further study the role of EVs, we have analysed by Raman tweezers microspectroscopy and mass spectrometry-based lipidomics the small EVs population secreted by fatty (ZF) and lean (ZL) hepatocytes obtained from Zucker rats. We have also explored in vivo and ex vivo biodistribution of these EVs through fluorine-18-radiolabelling using a positron emission tomography imaging. Based on the proportion of proteins to lipids and the types of lipids, our results indicate that within the range of small EVs, primary hepatocytes secrete different subpopulations of particles. These differences were observed in the enrichment of triglyceride species in EVs secreted by ZF hepatocytes. Biodistribution experiments showed accumulation in the brain, heart, lungs, kidney and specially in bladder after intravenous administration. In summary, we show that EVs released by a fatty hepatocytes carry a different lipid signature compared to their lean counterpart. Biodistribution experiment has shown no difference in the distribution of EVs secreted by ZF and ZL hepatocytes but has given us a first view of possible target organs for these particles. Our results might open a door to both pathology studies and therapeutic interventions.

Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.