A new cell labelling strategy to study the biogenesis of exosomes and their role in human melanoma progression driven by microenvironmental acidic pH

Gli esosomi sono nanovescicole di origine endosomiale, che rappresentano un mezzo importante di comunicazione cellula-cellula. Essendo coinvolti in diversi tipi di processi, sia fisiologici che patologici, sono diventati oggetto di sperimentazione clinica, nonostante molti meccanismi alla base della loro biogenesi rimangono tuttora sconosciuti. Quindi nel nostro laboratorio è stata messa a punto una metodica atta a produrre esosomi fluorescenti, in modo da poterne seguire la biogenesi nei diversi compartimenti intracellulari, il rilascio nell’ambiente extracellulare e la fusione con la membrana delle cellule riceventi. È stato quindi studiato, in un modello cellulare di melanoma umano, come questo precursore lipidico fluorescente è assorbito dalla cellula, accumulato all’interno del reticolo endoplasmatico, per poi diventare parte integrante della membrana degli esosomi. Queste nanovescicole fluorescenti sono state caratterizzate per dimensione, densità e presenza di marcatori proteici e successivamente è stata studiata con esperimenti di cinetica (5min – 24h) la secrezione. In seguito tale metodica è stata applicata allo studio della progressione del melanoma in condizioni di acidità microambientale. Nel melanoma è già noto che il pH microambientale è in grado di promuovere invasione e migrazione cellulare, e in questa tesi abbiamo analizzato il ruolo degli esosomi in tali processi. Quindi abbiamo studiato come cellule di melanoma umano, corrispondente a uno stadio intermedio, coltivate in ambiente acido producano una maggior quantità di esosomi, con un’aumentata capacità di trasferimento in cellule bersaglio. Inoltre queste vescicole presentano un profilo proteico che permette alle cellule riceventi di acquisire capacità migratorie e invasive. Infine, attraverso studi di meta-analisi ed ex vivo su biopsie di pazienti, è stato confermato che le molecole arricchite negli esosomi secreti in condizioni di acidità possano rappresentare dei marcatori della progressione del melanoma, convalidando quindi il valore diagnostico e prognostico degli esosomi.Exosomes, nanosized vesicles of endosomal origin, are worldwide recognized for their ability to transfer biological molecules, from cell to cell, crucial for both physiological and pathological processes. Hundred studies have been focused on exosome application also to clinics although biogenesis modalities are under investigation. Therefore, we set up a new effective fluorescent labelling strategy to trace exosome biogenesis and release with the aim to seek in human melanoma cell lines the impact that tumor progression may exert on their secretion and composition. Cells exposed to a fluorescent analogue of palmitic acid (Bodipy FL C16) were able to promptly synthetize fluorescent phospholipids, constituents of exosome membrane bilayer. Afterwards, we were able to follow exosome biogenesis from the intracellular sites of origin to cell secretion, chasing over time by direct cytofluorimetric analysis. To get insight into their function, we focused our studies on exosomes derived from melanoma cells maintained at low pH, which is a microenvironmental leverage for primary tumor to be transformed into widespread metastasis. When melanoma cells at specific intermediate stage were subjected to an acidic microenvironment, showed an increase in exosome release and transfer capability. Most importantly, when control melanoma cells were incubated with exosomes secreted in acidic medium acquired migratory and invasive capacities, demonstrating that exosomes carrying molecular payload can modify recipient cell program. Finally, meta-analysis and ex vivo studies confirmed the importance of acidic exosomes molecule content as marker of melanoma progression and so exosomes prognostic and diagnostic value

Effect of Water Stress on Photosynthetic Assimilation and Biomass Accumulation in Olive Tree

The climate change that we are attending in recent years requires a revision of the water resources management in order to en sure a sustainable supply. For this reason, in order to test the plants ability to face water stress, three water stress intensities (100% (T0), 50 (T1) and 25% (T2) field capacity (fc)), for 42 days in a greenhouse under semi-controlled conditions, were applied on 18 olive plants (Olea europaea L. cv 'Meski') of one year old. An evaluation of the response of the olive tree at different water supplies was conducted by monitoring the photosynthetic assimilation, chlorophyll index measured by SPAD, plant growth and dry matter accumulation. The results showed that photosynthesis has increased after 42 days for T0 plants while it increased during the first two weeks before dropping to the 42th day of the application of water restriction (AWR) for T1 plants; a decrease of 26% compared to control treatment. The photosynthetic assimilation of T2 plants has increased at a rate slower until the 28th day AWR then it has dropped after 42 days a decrease of 46% from the witness thus increasing the irrigation dose results in improved photosynthesis. The chlorophyll index fell 2, and 7% compared to the control treatment for T1 and T2, respectively, after 42 days AWR showing that fluid restriction affects the integrity of chlorophylls. Plant growth was shown by the development of a longer root for T2 plants ex plained by the search for minimum quantities of water available. The results showed that the control plants have accumulated more dry matter than stressed plants in the roots and the stem while the underground part/aerial part report seems indifferent to water stress. Water restriction has affected thereby biomass of roots and stems in favor of the leaves, the first operator of the photosyn thetic activit

ANTHRAQUINONE DYESTUFF AND GROWTH OF GALIUM ODORATUM ((L.) SCOP.) RHIZOMES IN RELATION TO ECOPHYSIOLOGICAL AND ONTOGENIC CONDITIONS

Galium odoratum, a member of the Rubiaceae family is known to accumulate anthraquinone dyestuff in the rhizome. The objective of our research was to document ecophysiological variables that modulate the anthraquinone dyestuff content and the growth rate of the underground part of this species. Several variables were taken into consideration : (1) the plant population phenology and the geographic origin of the plant population, for plant material collected in 4 natural stands identified in forests of Belgium, Grand-Duchy of Luxembourg and France, (2) the plant growth conditions, i.e. natural stands vs controlled environment and (3) the plant propagation path, i.e. clonal vs seed propagation. In natural stands, the anthraquinone dyestuff content varied with the plant phenology, being minimal at full flowering stage and the highest at leaf yellowing. It also varied depending on the geographic location of the plant population. Such a difference between plant populations was not observed when plants were grown in controlled environment. Typically, the anthraquinone dyestuff of the plant rhizome was higher in controlled environment than in natural stands, suggesting a strong impact of the growth conditions on anthraquinone biosynthesis. Additionnally, the plant propagation path does not influence the anthraquinone dyestuff content, but when newly formed rhizome was separated into fragments of increasing age –from 27 to 105 days-, a linear increase of the dyestuff with the rhizome ageing was observed

Effect of Aluminium and Aqueous extract of Rosmarinus officinalis on rat Brain: Impact on Neurobehavioral and Histological study

'Rosmarinus officilnalis' is a plant used in Mediterranean diet and traditional medicine, possessing various antioxidant and cytoprotective bioactivities. In this study, we investigated the potential neuroprotective efficacy of aqueous Rosemary extract (AER) against neurotoxicity induced by Aluminum (Al), in terms of behavioral, biochemical and histological aspects in young rats. an intraperitoneal injection of Al, at the weekly dose of 60mg/Kg was given to the animals. A treatment of 150mg/Kg/day of AER was administered by gavage over periods of 6 or 12weeks. Al caused intense changes over time in body and brain weight, increase in neurological disorders such as depression, anxiety, and deficiency in memory skills. Results show also disturbances in locomotors activity, with a significant inhibition of AchE and increase LDH activity compared to control. Additionally, Al induced structural damages in the cerebral cortex, and the CA1 region of hippocampus. However, treatment with AER resulted in improved depression and anxiety state, locomotors activity and restored memory skills. Results show that AER increase the AchE activity and decreased neuronal loss in the cerebral cortex and the CA1 region of hippocampus with the 6weeks treatment but induced disruption and structural modification of brain tissue after the 12 weeks treatment. The Aqueous extract of Rosemary possess a neuroprotector and corrective effect against neurological alterations induced by Aluminum, but when administered over a long period of time, the extract can cause a no beneficial effect and morphologic modifications in cerebral tissue and behavior test. Keywords: Rosmarinus officinalis, Aluminum, neuro-behavior, brain structure

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT's toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3',5'-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology

Ecophysiological Responses of Two Olive Tree Cultivars (Olea europaea L. CV Koroneiki and Chemlali) Under Three Water Treatments

In this paper, our objective is to assess and compare the effects of three levels of irrigation application on the ecophysiological behavior and plant dry matter accumulation of two young olive tree cultivars Koroneiki, a promising greek cultivar, and Chemlali, the best local cultivar. According to this assessment, we can determine the most efficient water treatment that could be suitable for Mediterranean environments subjected to water shortage conditions. Measurements were made in the green house of the Tunisian Olive tree Institute under normal day-light conditions from March, 16th to April, 21st 2015. Three water treatments were applied which are T100% (control treatment: Daily irrigation at 100% of Available Water Content (AWC)), T50% (Daily irrigation at 50% of AWC) and T0% (Without watering). The results showed that the two olive tree cultivars possess important mechanisms to overtake limited water resources. However, some striking variations existed between the two cultivars studied. Indeed, as water stress increased (T0%), Chemlali maintained longer a high midday leaf water potential compared (- 4.54 MPa) to Koroneiki (- 5.8 MPa). T50% treatment seems to be sufficient for both cultivars. Measurements of total Osmotic Adjustment (OA) showed that olive trees use this mechanism to create very negative leaf water potentials in order to be able to extract water from a dry root environment. The root/shoot ratio of Chemlali plants at T50% treatment was the highest (1.08) comparatively with the other two treatments (0.70 and 0.79 for T100% and T0% AWC water treatments, respectively). This result shows that Chemlali plants valorize low quantities of water (T50%) rather than high quantities (T100%) and Koroneiki plants behave better when it is irrigated at 100% AWC. To conclude, Chemlali plants irrigated at 50% AWC, compared to Koroneiki plants, are the most suitable to tolerate water restriction conditions

WATER USE IN OLIVE ORCHARDS ESTIMATED BY PHYSIOLOGIC AND CLIMATIC METHODS IN TUNISIA

This experimental study conducted on irrigated olive trees (cv. Meski) in arid climate in Enfidha-Tunisia. This study aims to estimate the water needs of the Olive Trees for a possible optimization of irrigation.To do this, a calculation of water requirements was performed by physiologic and climatic methods and compared to traditional irrigation practiced by farmer (T0). The physiologic method is based on the xylem sap flow (T1) and the climatic method is based on the reference evapotranspiration (T2).Results showed that the values of the highest transpiration are recorded under 60% ET0 (T2).Under this treatment, the average of transpiration was about 50% of the applied irrigation dose, with soil evaporation average about 15%. These results allowed us to conclude that the physiological method (T1) allows a better estimate of water needs. This method also improves the profitability of the olive tree with a better optimization of water use (save 35% of water)

The conditional inactivation of the β-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility

Using the Cre/loxP system we conditionally inactivated β-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell–cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of β-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression