Diversité des vésicules extracellulaires dans le lait bovin et leurs activités dans les maladies inflammatoires de l'intestin

Les vésicules extracellulaires (VE) sont des « fragments » de cellules activement libérés dans tous les fluides biologiques. Elles transitent dans la circulation corporelle et transmettent leur contenu bioactif à d’autres cellules. Le lait est le fluide qui contient le plus de VE et celles-ci encapsulent plusieurs éléments bioactifs qui ont des effets anticancéreux, anti-inflammatoire et diminuent notamment les symptômes de la polyarthrite rhumatoïde in vivo. Durant ma thèse j’ai exploré la diversité des VE présentes dans le lait bovin commercial et j’ai étudié leurs activités biologiques dans le cadre des maladies inflammatoires chroniques de l’intestin (MICI). Les résultats que j’ai obtenus démontrent l’existence de plusieurs populations de VE dans le lait bovin commercial que j’ai pu discriminer grâce à l’utilisation du citrate de sodium pour leur isolation. J’ai découvert qu’elles étaient capables de survivre lors de la digestion in vitro durant laquelle elles protègent leur contenu bioactif, notamment des microARN. Après avoir décrit en détail les différents microARN et protéines encapsulées dans ces VE, j’ai pu trouver des marqueurs spécifiques pour certains sous-ensembles de VE du lait. J’ai aussi montré le transfert de miR-223 bovin à des cellules humaines in vitro et son activité biologique sur l’expression d’un gène rapporteur. J’ai alors exploré l’activité biologique des VE du lait dans un modèle murin de colite induite par le Dextran sulfate sodium (DSS). La prise orale de VE du lait a diminué les symptômes de la maladie, restauré en partie le microbiote intestinal et rétabli la barrière digestive et les niveaux de mucines. J’ai aussi découvert que différentes populations de VE du lait ont différents effets sur l’inflammation du côlon, notamment en modulant le niveau de certains microARN impliqués dans le développement des MICI. Le lait contient donc différentes VE avec des activités biologiques différentes capables de moduler l’inflammation et le développement de pathologies digestives. L’étude des mécanismes qui sous-tendent leur bioactivité pourrait impacter la prise en charge des maladies inflammatoires, permettrait une amélioration des formulations de lait pour les nouveau-nés et serait d’importance pour la santé publique et le traitement industriel du lait commercial.Extracellular vesicles (EVs) are cellular “fragments” actively released in all biological fluids. They are transported through body circulation and transmit their bioactive content to remote recipient cells. Milk is the biological fluid most enriched in EVs and these encapsulate several bioactive elements with anti-cancer and anti-inflammatory effects and reduce the symptoms of rheumatoid arthritis in vivo. During my thesis, I explored the diversity of EVs present in commercial bovine milk and studied their biological activities in the context of inflammatory bowel diseases (IBD). The results I obtained demonstrate the existence of several EV subsets in commercial bovine milk that I could discriminate using sodium citrate for their isolation. I found these EVs can survive during in-vitro digestion and protect their bioactive content, including microRNAs. After detailing the different microRNAs and proteins encapsulated in these EVs, I found specific markers for certain populations of milk EVs. I also reported the transfer of vesicular bovine miR-223 to human cells in vitro and its biological activity on the expression of a reporter gene. I then explored the biological activities of milk EVs in a mouse model of DSS-induced colitis. The oral intake of milk EVs decreased the symptoms of the disease, restored part of the intestinal microbiota, restored the intestinal barrier and replenished mucin levels. Also, different populations of milk EVs differentially modulated inflammation in the colon, notably by regulating the level of certain IBD-associated microRNAs. Milk therefore contains different EV subsets with different biological activities capable of modulating inflammation and the development of digestive pathologies. Studying the mechanisms underlying their bioactivity could impact the management of inflammatory diseases, improve milk formulations for newborns, and be of importance to public health and industrial milk processing

Recent technical innovations around HF radar technology and steps towards integrated national HF radar networks

HF Radar is a proven technology for ocean observing that has at present more than 600 references in the world and which is since April 2016 in application on all continents for the purpose of 2D surface currents and waves monitoring. This has only been possible 44 years after its first implementation in 1972 in San Clemente Island in California, thanks to the permanent evolution of the radar technology with key achievements such as the present unique system compactness and its capability to make a very efficient use of the radio spectrum. This paper firstly presents some of the latest technical innovations around HF radar technology that are making the technology even more reliable, accurate and useful such as the new Automatic Antenna Pattern Generation solution based on AIS vessel positions and the implementation of new added value trajectory models forced by HF Radar surface currents able to accurately predict oil spill transport and movement of particles on the sea or persons adrift. Secondly, we present current plans existing in the Iberian Peninsula HF Radar network to integrate HF Radar technology as an operational component of a national tsunami early warning system. Finally, the advanced HF Radar system implemented by the Direction de la Météorologie Nationale du Royaume du Maroc is presented; one of the latest deployed systems and the first permanent system operating in Africa.Peer Reviewe

Identification of protein markers for extracellular vesicle (EV) subsets in cow’s milk

Extracellular vesicles (EVs), like exosomes, are small membrane vesicles involved in cell-to-cell communications that modulate numerous biological processes. We previously discovered a new EV subset in milk (sedimenting at 35,000 g; 35 K) that protected its cargo (RNAs and proteins) during simulated digestion and was more enriched in microRNAs than exosomes (sedimenting at 100 K). Here, we used LC-MS/MS to push further the comparison between these two pellets. Commonly used EV markers were not differentially enriched between the pellets, questioning their use with cow's milk EVs. Similarly, the majority of the quantified proteins were equally enriched between the two pellets. Nevertheless, 20 proteins were specific to 35 K, while 41 were specifically enriched in 100 K (p < 0.05), suggesting their potential use as specific markers. Loaded with these proteins, the EVs in these pellets might regulate translation, proliferation and cell survival for 35 K, and metabolism, extracellular matrix turnover and immunity for 100 K. This approach also brought new insights into milk EV-associated integrins and their possible role in specifically targeting recipient cell types. These findings may help better discriminate between milk EVs, improve our understanding of milk EV-associated protein function and their possible use as therapeutic tools for the management of immunity- and metabolism-associated disorders

Extracellular vesicles isolated from milk can improve gut barrier dysfunction induced by malnutrition

Malnutrition impacts approximately 50 million children worldwide and is linked to 45% of global mortality in children below the age of five. Severe acute malnutrition (SAM) is associated with intestinal barrier breakdown and epithelial atrophy. Extracellular vesicles including exosomes (EVs; 30–150 nm) can travel to distant target cells through biofluids including milk. Since milk-derived EVs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. Mice were fed either a control (18%) or a low protein (1%) diet for 14 days to induce malnutrition. From day 10 to 14, they received either bovine milk EVs or control gavage and were sacrificed on day 15, 4 h after a Fluorescein Isothiocyanate (FITC) dose. Tissue and blood were collected for histological and epithelial barrier function analyses. Mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. Despite continued low protein diet feeding, milk EV treatment improved intestinal permeability, intestinal architecture and cellular proliferation. Our results suggest that EVs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Milk microRNAs in human health and disease

MicroRNAs are small noncoding RNAs responsible for regulating 40% to 60% of gene expression at the posttranscriptional level. The discovery of circulating microRNAs in several biological fluids opened the path for their study as biomarkers and long‐range cell‐to‐cell communication mediators. Their transfer between individuals in the case of blood transfusion, for example, and their high enrichment in milk have sparked the interest for microRNA transfer through diet, especially from mothers to infants during breastfeeding. The extension of such paradigm led to the study of milk microRNAs in the case of cow or goat milk consumption in adults. Here we provide a comprehensive critical review of the key findings surrounding milk microRNAs in human, cow, and goat milk among other species. We discuss the data on their biological properties, their use as disease biomarkers, their transfer between individuals or species, and their putative or verified functions in health and disease of infants and adult consumers. This work is based on all the literature available and integrates all the results, theories, debates, and validation studies available so far on milk microRNAs and related areas of investigations. We critically discuss the limitations and outline future aspects and avenues to explore in this rapidly growing field of research that could impact public health through infant milk formulations or new therapies. We hope that this comprehensive review of the literature will provide insight for all teams investigating milk RNAs’ biological activities and help ensure the quality of future reports

Small non-coding RNAs derived from eukaryotic ribosomal RNA

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will present a nonexhaustive list of referenced small non-coding RNAs (ncRNAs) derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs). We will focus on the rRFs that are experimentally verified, and discuss their origin, length, structure, biogenesis, association with known regulatory proteins, and potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because no one could believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places

Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation

Platelet Pathogen Reduction Technologies Alter the MicroRNA Profile of Platelet-Derived Microparticles

Despite improvements in donor screening and increasing efforts to avoid contamination and the spread of pathogens in clinical platelet concentrates (PCs), the risks of transfusion-transmitted infections remain important. Relying on an ultraviolet photo activation system, pathogen reduction technologies (PRTs), such as Intercept and Mirasol, utilize amotosalen, and riboflavin (vitamin B2), respectively, to mediate inactivation of pathogen nucleic acids. Although they are expected to increase the safety and prolong the shelf life of clinical PCs, these PRTs might affect the quality and function of platelets, as recently reported. Upon activation, platelets release microparticles (MPs), which are involved in intercellular communications and regulation of gene expression, thereby mediating critical cellular functions. Here, we have used small RNA sequencing (RNA-Seq) to document the effect of PRT treatment on the microRNA profiles of platelets and derived MPs. PRT treatment did not affect the microRNA profile of platelets. However, we observed a specific loading of certain microRNAs into platelet MPs, which was impaired by treatment with Intercept or its Additive solution (SSP+). Whereas, Intercept had an impact on the microRNA profile of platelet-derived MPs, Mirasol did not impact the microRNA profile of platelets and derived MPs, compared to non-treated control. Considering that platelet MPs are able to transfer their microRNA content to recipient cells, and that this content may exert biological activities, those findings suggest that PRT treatment of clinical PCs may modify the bioactivity of the platelets and MPs to be transfused and argue for further investigations into PRT-induced changes in clinical PC content and function.Funding Agencies|Fonds de Recherche du Quebec-Sante (FRQ-S) [262093]; Canadian Blood Services (CBS)/Canadian Institutes of Health Research (CIHR) Partnership-Blood Utilization and Conservation Initiative, via Health Canada [286,777, 327,364]</p

An Expanded Landscape of Unusually Short RNAs in 11 Samples from Six Eukaryotic Organisms

Small RNA sequencing (sRNA-Seq) approaches unveiled sequences derived from longer non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA) fragments, known as tRFs and rRFs, respectively. However, rRNAs and RNAs shorter than 16 nt are often depleted from library preparations/sequencing analyses, although they may be functional. Here, we sought to obtain a complete repertoire of small RNAs by sequencing the total RNA from 11 samples of 6 different eukaryotic organisms, from yeasts to human, in an extended 8- to 30-nt window of RNA length. The 8- to 15-nt window essentially contained fragments of longer non-coding RNAs, such as microRNAs, PIWI-associated RNAs (piRNAs), small nucleolar RNAs (snoRNAs), tRNAs and rRNAs. Notably, unusually short RNAs 16 nt in bilaterian organisms. A new RT-qPCR method confirmed that two unusually short rRFs of 12 and 13 nt were more overly abundant (~3-log difference) than two microRNAs. We propose to not deplete rRNA and to reduce the lower threshold of RNA length to include unusually short RNAs in sRNA-Seq analyses and datasets, as their abundance and diversity support their potential role and importance as biomarkers of disease and/or mediators of cellular function