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The branched-chain amino acids: leucine, isoleucine and valine occupy a special place among the essential amino acids because of their importance not only in the structure of proteins but also in general and cerebral metabolism. Among the first amino acids absorbed after food intake, they play a major role in the regulation of protein synthesis and insulin secretion. They are involved in the modulation of brain uptake of monoamine precursors with which they may compete for occupancy of a common transporter. In the brain, branched-chain amino acids are involved not only in protein synthesis but also in the metabolic cycles of GABA and Glutamate, and in energy metabolism. In particular, they can affect GABAergic neurons and the excitation/inhibition balance. Branched-chain amino acids are known for the 24-hour rhythmicity of their plasma concentrations, which is remarkably conserved in rodent models. This rhythmicity is partly circadian, independent of sleep and food. Moreover, their concentration increases when sleep is disturbed and in obesity and diabetes. The mechanisms regulating these rhythms and their physiological impact remain poorly understood. In this context, the Drosophila model has not yet been widely used, but it is highly relevant and the first results indicate that it can generate new concepts. The elucidation of the metabolism and fluxes of branched-chain amino acids is beginning to shed light on the mysterious connections between clock, sleep, and metabolism, opening the possibility of new therapies., Les acides aminés branchés : leucine, isoleucine et valine occupent une place particulière parmi les acides aminés essentiels de par leur importance non seulement dans la structure des protéines mais aussi dans le métabolisme général et cérébral. Ils sont parmi les premiers acides aminés absorbés après la prise alimentaire et jouent un rôle majeur dans la régulation de la synthèse protéique et de la sécrétion d’insuline. Ils participent à la modulation de l’import cérébral des précurseurs de monoamines avec lesquels ils peuvent entrer en compétition pour l’occupation d’un transporteur commun. Dans le cerveau, les acides aminés branchés interviennent également dans les cycles métaboliques du GABA et du Glutamate, et dans le métabolisme énergétique. Ils peuvent notamment affecter les neurones GABAergiques et la balance excitation/inhibition. Les acides aminés branchés sont connus pour la rythmicité sur 24h de leurs concentrations plasmatiques qui est remarquablement conservée chez les modèles rongeurs. Cette rythmicité est en partie circadienne, indépendante du sommeil et de l’alimentation. Par ailleurs leur concentration augmente lorsque le sommeil est perturbé ainsi que dans l’obésité et le diabète. Les mécanismes régulant ces rythmes et leur impact physiologique restent mal compris. Dans ce contexte, le modèle drosophile a encore été peu utilisé, mais il a toute sa pertinence et les premiers résultats indiquent qu’il peut générer de nouveaux concepts. L’élucidation du métabolisme et des flux des acides aminés branchés commence à éclairer les connections mystérieuses qui existent entre horloge, sommeil, et métabolisme, ouvrant la possibilité de nouvelles thérapies.

International audience; The presence of CD8+ T cells with a memory phenotype in nonimmunized mice has been noted for decades, but it was not until about 2 decades ago that they began to be studied in greater depth. Currently called virtual memory CD8+ T cells, they consist of a heterogeneous group of cells with memory characteristics, without any previous contact with their specific antigens. These cells were identified in mice, but a few years ago, a cell type with characteristics equivalent to the murine ones was described in healthy humans. In this review, we address the different aspects of its biology mainly developed in murine models and what is currently known about its cellular equivalent in humans.

International audience; Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. The non-receptor tyrosine kinase c-Abelson (cAbl) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is down-activated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from PAH patients (PAH-ECs). We found cAbl and phosphorylated cAbl levels to be lower in the endothelium of remodeled pulmonary vessels in the lungs of PAH patients than controls. Similar observations were obtained for the lungs of sugen+hypoxia (SuHx) and monocrotaline (MCT) rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA-interference in Control-ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with DPH reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of crosstalk between cAbl and bone morphogenetic protein receptor type II (BMPRII). This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

SummaryAlthough oncogene-induced senescence (OIS) is a potent tumor-suppressor mechanism, recent studies revealed that cells can escape from OIS with features of transformed cells. However, the mechanisms that promote OIS escape remain unclear, and evidence of post-senescent cells in human cancers is missing. Here, we unravel the regulatory mechanisms underlying OIS escape using dynamic multidimensional profiling. We demonstrate a critical role for AP1 and POU2F2 transcription factors for escape from OIS and identify ‘senescence-associated chromatin scars (SACS)’ as an epigenetic memory of OIS, detectable during colorectal cancer progression. POU2F2 levels are elevated already in precancerous lesions and as cells escape from OIS, and its expression and binding activity to cis-regulatory elements are associated with decreased patient survival. Our results support a model in which POU2F2 exploits a precoded enhancer landscape to promote senescence escape and reveal POU2F2 gene signatures and SACS as valuable biomarkers with diagnostic and prognostic potential.

International audience; Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

International audience; Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Background: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome (also known as hand–heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. Methods: We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome ( Tbx5 G125R ) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). Results: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Tbx5 G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5 G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Conclusions: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

International audience; Single cell multimodal analysis is at the frontier of single cell research: it defines the roles and functions of distinct cell types through simultaneous analysis to provide unprecedented insight into cellular processes. Current single cell approaches are rapidly moving toward multimodal characterizations. It replaces one-dimensional single cell analysis, for example by allowing for simultaneous measurement of transcription and post-transcriptional regulation, epigenetic modifications and/or surface protein expression. By providing deeper insights into single cell processes, multimodal single cell analyses paves the way to new understandings in various cellular processes such as cell fate decisions, physiological heterogeneity or genotype–phenotype linkages. At the forefront of this, microfluidics is key for high-throughput single cell analysis. Here, we present an overview of the recent multimodal microfluidic platforms having a potential in biomedical research, with a specific focus on their potential clinical applications.

One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity—by interfering with metaphase completion—resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency., Un certain nombre de facteurs de stress et de médiateurs inflammatoires (cytokines, protéases, médiateurs du stress oxydatif) libérés pendant l'inflammation ou l'ischémie stimulent et activent les cellules du sang, de la paroi vasculaire ou des tissus. Les réponses fonctionnelles et phénotypiques les plus connues des cellules activées sont (1) l'expression et/ou la libération immédiate de molécules bioactives stockées ou nouvellement synthétisées, et (2) le décollement de la membrane suivi de la libération de microvésicules. Une réponse ultime, à savoir la formation de pièges extracellulaires par les neutrophiles (NETs), sort du cadre de ce travail. L'objectif principal de cet article est de donner un aperçu du mécanisme de réception et d'activation du plasminogène à la surface des microvésicules dérivées des cellules, nouveaux acteurs de la fibrinolyse et de la protéolyse. Le rôle de la plasmine liée aux microvésicules dans des contextes pathologiques impliquant l'inflammation, l'athérosclérose, l'angiogenèse et la croissance tumorale, reste à étudier. D'autres études sont nécessaires pour déterminer si les microvésicules profibrinolytiques sont impliquées dans un équilibre finement régulé avec les microvésicules pro-coagulantes, qui assure une hémostase équilibrée, conduisant au maintien de la perméabilité vasculaire.

International audience; Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 μm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer’s disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

According to the commonly used metaphor of "genetic programs", cells adapt to environmental fluctuations through more or less complex signalling pathways pre-programmed in their genomes, leading to a compartmentalized description of cellular activities. However, the different cellular activities are intimately linked and interdependent. I analysed the response of a cell model, a melanoma-derived cell line, to cell growth inhibitors directed against BRAF and MEK proteins. The results obtained show that these inhibitors decrease the bioavailability of certain amino acids such as glutamate and aspartate. This leads to the accumulation of ribosomes on mRNAs coding for proteins enriched in these amino acids, and we show that this translation arrest leads to the degradation of these mRNAs whose composition is biased. However, proteins enriched in glutamate and aspartate are frequently involved in DNA metabolism, and in particular in its repair. Thus, through their effects on amino acid metabolism, cell growth inhibitors cause the arrest of the biosynthesis of glutamate and aspartate enriched proteins involved in DNA repair. This decrease in the biosynthesis of DNA repair proteins dependent on cell metabolism is discussed in the context of the emergence of cancer cells carrying mutations and being resistant, i.e. no longer responding to anti-cancer therapies such as the combination of BRAF and MEK inhibitors often used in the treatment of melanoma., Préprogrammées dans leurs génomes, conduisant à une description cloisonnée des activités cellulaires. Pourtant, les différentes activités cellulaires sont intimement liées et interdépendantes. J’ai analysé la réponse d’un modèle cellulaire, une lignée dérivée d’un mélanome, exposée à des inhibiteurs de la croissance cellulaire dirigés contre les protéines BRAF et MEK. Les résultats obtenus montrent que ces inhibiteurs diminuent la biodisponibilité de certains acides aminés comme le glutamate et l’aspartate. Ceci entraîne l’accumulation de ribosomes sur des ARNm codant pour des protéines enrichies en ces acides aminés, et nous montrons que cet arrêt de traduction entraîne la dégradation de ces ARNm dont la composition est biaisée. Or, les protéines enrichies en glutamate et aspartate sont fréquemment impliquées dans le métabolisme de l’ADN, et en particulier dans sa réparation. Ainsi, via leurs effets sur le métabolisme des acides aminés, des inhibiteurs de croissance cellulaire entraînent l’arrêt de la biosynthèse de protéines enrichies en glutamate et aspartate impliquées dans la réparation de l’ADN. Cette diminution de la biosynthèse des protéines de la réparation de l’ADN dépendante du métabolisme cellulaire est discutée dans le contexte de l’émergence de cellules cancéreuses porteuses de mutations et étant résistantes, c’est-à-dire ne répondant plus à des thérapies anti-cancéreuses comme la combinaison d’inhibiteurs BRAF et MEK souvent utilisée dans le traitement des mélanomes.

Introduction: The aim of this study was to investigate endogenous retrovirus (ERV) expression and type I interferon (IFN) activation in human pleural mesothelioma (PM) and their association with clinical outcome. Methods: The expression of ERV was determined from PM cohorts and mesothelial precursor RNA sequencing data. The expression of ERV was confirmed by quantitative polymerase chain reaction (qPCR). Methylation of genomic DNA was assessed by quantitative methylation-specific PCR. DNA demethylation was induced in cells by demethylating agent 5-Aza-2’-deoxycytidine (5-Aza-CdR) treatment. To block type I IFN signaling, the cells were treated with ruxolitinib or MAVS silencing. The expression of IFN-stimulated genes (ISGs) was determined by qPCR and Western blot. Circulating ERVs were detected by qPCR. Results: Long terminal repeats (LTRs) represent the most abundant transposable elements up-regulated in PM. Within the LTR, ERVmap_1248 and LTR7Y, which are specifically enriched in PM, were further analyzed. The 5-Aza-CdR treatment increased the levels of ERVmap_1248 expression and induced ERVmap_1248 promoter demethylation in mesothelial cells. In addition, ERVmap_1248 promoter was more demethylated in the mesothelioma tissue compared with nontumor tissue. The 5-Aza-CdR treatment of the mesothelial cells also increased the levels of ISGs. Basal ISG expression was higher in the mesothelioma cells compared with the mesothelial cells, and it was significantly decreased by ruxolitinib treatment or MAVS silencing. Furthermore, ISG expression was higher in the tumor tissue with high expression levels of ERVmap_1248. High expression of ERVmap_1248 was associated with longer overall survival and BAP1 mutations. ERVmap_1248 and LTR7Y can be detected in the PM plasma. Conclusions: We provide clues for patient stratification especially for immunotherapy where best clinical responses are associated with an activated basal immune response., JTO Clinical and Research Reports, 3 (12), ISSN:2666-3643

New procedures envisioned for dental pulp regeneration after pulpectomy include cell homing strategy. It involves host endogenous stem cell recruitment and activation. To meet this cell-free approach, we need to design a relevant scaffold to support cell migration from tissues surrounding the dental root canal. A composite membrane made of electrospun poly(lactic acid) nanofibers and electrosprayed polycaprolactone with tannic acid (TA) microparticles which mimics the architecture of the extracellular matrix was first fabricated. After rolling the membrane in the form of a 3D conical scaffold and subsequently coating it with gelatin, it can be directly inserted into the root canal. The porous morphology of the construct was characterized by SEM at different length scales. It was shown that TA was released from the 3D conical scaffold after 2 days in PBS at 37 °C. Biocompatibility studies were first assessed by seeding human dental pulp stem cells (DPSCs) on planar membranes coated or not coated with gelatin to compare the surfaces. After 24 h, the results highlighted that the gelatin-coating increased the membrane biocompatibility and cell viability. Similar DPSC morphology and proliferation on both membrane surfaces were observed. The culture of DPSCs on conical scaffolds showed cell colonization in the whole cone volume, proving that the architecture of the conical scaffold was suitable for cell migration. Keywords: cell homing; dental pulp regeneration; dental pulp stem cells; electrospinning; tissue engineering.

Polymeric nanoparticles (NPs) are highly attractive for biomedical applications due to their potential biodegradability and capacity to encapsulate different loads, notably drugs and contrast agents. For in vivo optical bioimaging, NPs should operate in the near-infrared region (NIR) and exhibit stealth properties. In the present work, we applied the approach of ionic dye insulation with bulky hydrophobic counterions for encapsulation of near-infrared cyanine dyes (Cy5.5 and Cy7 bearing two octadecyl chains) into biodegradable polymer (PLGA) NPs. We found that at high dye loading (20–50 mM with respect to the polymer), the bulkiest fluorinated tetraphenylborate counterion minimized best the aggregation-caused quenching and improved fluorescence quantum yields of both NIR dyes, especially of Cy5.5. In addition, bulky counterions also enabled formation of small 40 nm polymeric NPs in contrast to smaller counterions. To provide them stealth properties, we prepared 40 nm dye-loaded PEGylated NPs through nanoprecipitation of synthetic PLGA–PEG block copolymer with the dye/counterion salt. The obtained NIR NPs loaded with Cy5.5 dye salt allowed in vivo imaging of wild-type mice with a good contrast after IV injection. Compared to the bare PLGA NPs, PLGA–PEG NPs exhibited significantly slower accumulation in the liver. Biodistribution studies confirmed the preferential accumulation in the liver, although PLGA and PLGA–PEG NPs could also be distributed in other organs, with the following tendency: liver > spleen > lungs > kidney > heart > testis > brain. Overall, the present work validated the counterion approach for encapsulation of NIR cyanine dyes into biodegradable polymer NPs bearing covalently attached PEG shell. Thus, we propose a simple and robust methodology for preparation of NIR fluorescent biodegradable polymer NPs, which could further improve the existing optical imaging for biomedical applications., Bulky hydrophobic counterions were applied for encapsulation of near-infrared cyanine dyes into biodegradable polymer matrix with minimized self-quenching, yielding fluorescent nanoparticles with stealth PEG shell for in vivo imaging.

International audience; Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid–triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients’ glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients’ tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients’ tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients’ tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

International audience; Bladder cancer is the 10th most common cancer in the world and has a high risk of recurrence and metastasis. In order to sustain high energetic needs, cancer cells undergo complex metabolic adaptations, such as a switch toward aerobic glycolysis, that can be exploited therapeutically. Reactive oxygen species (ROS) act as key regulators of cancer metabolic reprogramming and tumorigenesis, but the sources of ROS remain unidentified. Monoamine oxidases (MAOs) are mitochondrial enzymes that generate H 2 O 2 during the breakdown of catecholamines and serotonin. These enzymes are particularly important in neurological disorders, but recently, a new link between MAOs and cancer has been uncovered, involving their production of ROS. At present, the putative role of MAOs in bladder cancer has never been evaluated. We observed that human urothelial tumor explants and the bladder cancer cell line AY27 expressed both MAO-A and MAO-B isoforms. Selective inhibition of MAO-A or MAO-B limited mitochondrial ROS accumulation, cell cycle progression and proliferation of bladder cancer cells, while only MAO-A inhibition prevented cell motility. To test whether ROS contributed to MAO-induced tumorigenesis, we used a mutated form of MAO-A which was unable to produce H 2 O 2. Adenoviral transduction of the WT MAO-A stimulated the proliferation and migration of AY27 cells while the Lys305Met MAO-A mutant was inactive. This was consistent with the fact that the antioxidant Trolox strongly impaired proliferation and cell cycle progression. Most interestingly, AY27 cells were highly dependent on glucose metabolism to sustain their growth, and MAO inhibitors potently reduced glycolysis and oxidative phosphorylation, due to pyruvate depletion. Accordingly, MAO inhibitors decreased the expression of proteins involved in glucose transport (GLUT1) and transformation (HK2). In conclusion, urothelial cancer cells are characterized by a metabolic shift toward glucose-dependent metabolism, which is important for cell growth and is under the regulation of MAO-dependent oxidative stress.

Adherent-invasive Escherichia coli (AIEC) were investigated for their involvement in the induction/chronicity of intestinal inflammation in Crohn's disease (CD). AIEC gut establishment is favoured by overexpression of the glycoprotein CEACAM6 in the ileal epithelium. We generated a transgenic mouse model, named ‘Vill-hCC6’, in which the human CEACAM6 gene was under the control of the villin promoter, conditioning expression in the small intestine. We demonstrated that CEACAM6 is strongly expressed in the small intestine mucosa and is correlated with numerous glycosylations displayed at the brush border of enterocytes. Ex vivo, the AIEC–enterocyte interaction was enhanced by CEACAM6 expression and necessitated the presence of the bacterial adhesive factor FimH. Finally, AIEC bacteria preferentially persisted in a FimH-dependent manner in the ileal mucosa of Vill-hCC6 mice compared to wild-type mice. This preclinical model opens new perspectives in the mechanistic study of the AIEC pathobiont and represents a valuable tool to evaluate the efficacy of new strategies to eliminate AIEC implanted in the ileal mucosa, such as phages, inhibitory and/or anti-virulence molecules, or CRISPR-based strategies targeting virulence or fitness factors of AIEC bacteria.

International audience; DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

International audience; Gene-transfer methods are useful to study the structural or functional roles of recombinant proteins in vitro. In particular, adenovirus-mediated gene transduction results in strong efficiency and high level of expression in primary cells such as cardiomyocytes, which are difficult to transfect with classical methods. Here, we describe a protocol that enables efficient expression of MAO A in both primary cells and cell lines. Following expression of recombinant MAO A, substrate-induced activation of the enzyme can be assessed by measuring production of reactive oxygen species and downstream signal transduction pathways in cells. This model allows to decipher the biological function of MAO A on metabolism, mitochondrial fitness, cell death/survival, and proliferation.

The mechanical, chemical, or infectious mediated tissue damages affect all epithelial barriers including the skin. The rapid resolution of theses damages is key to recover their integrity and function, it involves the joint action of several immune cells that infiltrate the barrier tissues. The interest surrounding the study of MAIT (mucosal-associated immune T) cells in repair stems from their high frequency in the skin (up to 10% of T cells) and their capacity to rapidly secrete various effector molecules. Recently, it has been shown both in vitro and in vivo that MAIT cells are involved in wound healing. However, the mechanisms that underly this pro-repair function remain elusive. Using the congenic B6-MAITCAST strain (10 times more MAIT cells than the C57BL/6 mice) and an full thickness excision model mimicking the human repair process (Dunn et al., 2013), we aimed at unravelling these mechanisms. With these tools, we first show that MAIT cells accelerates wound closure. Because MAIT cell numbers and frequency increased following injury without increased proliferation, we hypothesized that MAIT cells were recruited to the wound site. Using skin graft experiments we evidenced that, contrary to the resident gdT cells, MAIT cells are rapidly recruited into the inflamed site. The Kaedes mouse (in which local ultraviolet light induces a shift in fluorescence) strain and the use of FTY720 treatment demonstrated that recruited cells transited through the lymph nodes rather than from the surrounding skin. We next investigated whether TCR signaling, via MR1 the MAIT cell antigen presenting molecule, could be necessary both for the recruitment and the tissue repair function. To investigate MAIT cells reliance on TCR signaling, we performed parabiosis of MR1+ and MR1- animals and MR1- skin grafted onto MR1+ animals, and we showed that MAIT cell recruitment was MR1-independent. Instead, the recruitment relied on the CXCL16-CXCR6 signaling, as shown by blocking CXCL16 in vivo and deleting CXCR6 on MAIT cells by CRISPR-Cas9 genetic modification. Adoptive ransfer experiments further demonstrated that wound closure does not depend on a continuous TCR signaling.Concerning their effector function our scRNAseq analysis suggested that MAIT cell repair program was acquired already at steady state in the skin. Additionally, we observed an increase in epithelial tongue length and proliferation within these tongues in the presence of MAIT cells as compared to their absence. Thus, we presupposed that MAIT cells could play a direct effector function on keratinocyte growth. This theory was supported by scRNAseq that showed that signatures associated to amphiregulin-producing pro-repair cells were up-regulated by skin MAIT cells . We observed that amphiregulin produced by PLZF cells was necessary in our model and that MAIT cells increased its production at the protein level following wounding. Transfer experiments of Areg deficient MAIT cells formally demonstrated that Areg produced by MAIT cells was instrumental for promoting wound repair.Overall, our study describes the dynamics of MAIT cells within the skin, the signals they rely on for recruitment and how they promote tissue repair in an MR1 independent manner. It also provides evidence to an Areg mediated mechanism supporting this repair function. These additional insights on MAIT cell biology could provide key therapeutic axes that could help alleviate the pain, discomfort and complications caused by unhealed or poorly healed wounds., Les dommages tissulaires d'origine mécanique, chimique ou infectieuse affectent toutes les barrières épithéliales, y compris la peau. La résolution rapide de ces dommages est essentielle pour que ces barrières recouvrent leur intégrité et fonction, elle implique l'action conjointe de cellules immunitaires infiltrant ces tissus. L'intérêt entourant l'étude des cellules MAIT (cellules T immunitaires associées aux muqueuse) dans la réparation provient de leur fréquence élevée dans la peau (jusqu'à 10% des cellules T) et de leur capacité à sécréter rapidement diverses molécules effectrices. Récemment, il a été démontré à la fois in vitro et in vivo que les cellules MAIT sont impliquées dans la cicatrisation des plaies. Cependant, les mécanismes qui sous-tendent cette fonction pro-réparatrice demeurent inconnus. En utilisant la souche congénique B6-MAITCAST (10 fois plus de cellules MAIT que les souris C57BL/6) et un modèle d'excision mimant le processus de réparation humain (Dunn et al., 2013), nous avons cherché à élucider ces mécanismes. Grâce à ces outils, nous montrons que les cellules MAIT accélèrent la fermeture des plaies. Ayant observé l’augmentation du nombre et de la fréquence des cellules MAIT au sein des plaies sans augmentation de leur prolifération, nous avons émis l'hypothèse qu’elles étaient recrutées. À l’aide de greffe de peau, nous avons mis en évidence que, contrairement aux cellules gdT résidentes, les cellules MAIT sont recrutées au site inflammatoire. La souris Kaedes (où la lumière ultraviolette induit un changement de fluorescence) et le traitement par FTY720 démontrent que les cellules recrutées transitent par les ganglions lymphatiques et ne migrent pas depuis d'autres endroits de la peau. Nous avons ensuite cherché à savoir si la signalisation du TCR, via MR1, la molécule de présentation de l'antigène des cellules MAIT, était nécessaire pour leur recrutement et fonction réparatrice. Pour étudier la dépendance des cellules MAIT à la signalisation TCR, nous avons réalisé une parabiose d'animaux MR1+ et MR1- et une greffe de peau MR1- sur des animaux MR1+, ceci a montré que leur recrutement était MR1 indépendant. Les expériences de blocage de CXCL16 et de suppression génétique de CXCR6 sur les cellules MAIT par CRISPR-Cas9 montrent que le recrutement est dépendant de la signalisation CXCL16-CXCR6. Nos expériences de transfert adoptif démontrent également que la fermeture de la plaie est indépendante de la signalisation du TCR.Concernant leur fonction effectrice, notre analyse scRNAseq suggère que le programme de réparation des cellules MAIT acquis dans la peau à l’état basal. De plus, nous observons qu’en présence des cellules MAIT il y a une augmentation significative de la longueur des langues épithéliales et de la prolifération au sein de ces langues. Ainsi, nous avons présupposé que les cellules MAIT pouvaient agir directement sur la croissance des kératinocytes. Cette théorie est appuyée par le scRNAseq montrant une augmentation de l’expression de signatures associées à des cellules pro-réparatrices produisant de l'amphireguline (Areg) par les cellules MAIT cutanées. Conjointement, nous observons la nécessité de l’Areg produite par les cellules PLZF dans notre modèle et l’augmentation de sa production par les cellules MAIT. Des expériences de transfert de cellules MAIT déficientes en Areg démontrent que l'Areg produite par les cellules MAIT est déterminante pour la réparation des plaies.Dans l'ensemble, notre étude décrit la dynamique des cellules MAIT dans la peau, les signaux sur lesquels elles s'appuient pour leur recrutement et la manière dont elles promeuvent la réparation des tissus. Elle fournit également des preuves d'un mécanisme médié par l'Areg sous-tendant cette fonction. Ces connaissances sur la biologie des cellules MAIT fournissent de nouveaux axes thérapeutiques qui pourraient atténuer la douleur, l'inconfort et les complications causés par les plaies non ou mal cicatrisées.

International audience

International audience; Medullary and extra-medullary hematopoiesis has been shown to govern inflammatory cell infiltration and subsequently cardiac remodeling and function after acute myocardial infarction (MI). Emerging evidence positions adipose tissue (AT) as an alternative source of immune cell production. We, therefore, hypothesized that AT could act as a reservoir of inflammatory cells that participate in cardiac homeostasis after MI. To reveal the distinct role of inflammatory cells derived from AT or bone marrow (BM), chimeric mice were generated using standard repopulation assays. We showed that AMI increased the number of AT-derived macrophages in the cardiac tissue. These macrophages exhibit pro-inflammatory characteristics and their specific depletion improved cardiac function as well as decreased infarct size and interstitial fibrosis. We then reasoned that the alteration of AT-immune compartment in type 2 diabetes could, thus, contribute to defects in cardiac remodeling. However, in these conditions, myeloid cells recruited in the infarcted heart mainly originate from the BM, and AT was no longer used as a myeloid cell reservoir. Altogether, we showed here that a subpopulation of cardiac inflammatory macrophages emerges from myeloid cells of AT origin and plays a detrimental role in cardiac remodeling and function after MI. Diabetes abrogates the ability of AT-derived myeloid cells to populate the infarcted heart.

International audience; Immunological synapse formation results from a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, and the intracellular traffic of several vesicular organelles. T cell polarization is key for both T cell activation lead- ing to T cell proliferation and differentiation, and for T cell effector functions such as polarized secretion of cytokines by helper T cells, or polarized delivery of lytic granules by cytotoxic T cells. Efficient targeting of lytic granules by cytotoxic T cells is a crucial event for the con- trol and elimination of infected or tumor cells. Understanding how lytic granule delivery is regulated and quantifying its efficiency under physiological and pathological conditions may help to improve immune responses against infection and cancer.

International audience

Background: We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. Methods: We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. Results: We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. Conclusions: Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

International audience; The gut microbiota produces a wide variety of metabolites, which interact with intestinal cells and contribute to host physiology. The effect of gut commensal bacteria on host protein SUMOylation, an essential ubiquitin-like modification involved in various intestinal functions, remains, however, unknown. Here, we show that short chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) produced by the gut microbiota increase protein SUMOylation in intestinal cells in a pH-dependent manner. We demonstrate that these metabolites inactivate intestinal deSUMOylases and promote the hyperSUMOylation of nuclear matrix-associated proteins. We further show that BCFAs inhibit the NF-κB pathway, decrease pro-inflammatory cytokine expression, and promote intestinal epithelial integrity. Together, our results reveal that fatty acids produced by gut commensal bacteria regulate intestinal physiology by modulating SUMOylation and illustrate a new mechanism of dampening of host inflammatory responses triggered by the gut microbiota.

Automating the extraction of meaningful temporal information from sequences of microscopy images represents a major challenge to characterize dynamical biological processes. So far, strong limitations in the ability to quantitatively analyze single-cell trajectories have prevented large-scale investigations to assess the dynamics of entry into replicative senescence in yeast. Here, we have developed DetecDiv, a microfluidic-based image acquisition platform combined with deep learning-based software for high-throughput single-cell division tracking. We show that DetecDiv can automatically reconstruct cellular replicative lifespans with high accuracy and performs similarly with various imaging platforms and geometries of microfluidic traps. In addition, this methodology provides comprehensive temporal cellular metrics using time-series classification and image semantic segmentation. Last, we show that this method can be further applied to automatically quantify the dynamics of cellular adaptation and the real-time cell survival upon exposure to environmental stress. Hence, this methodology provides an all-in-one toolbox for high-throughput phenotyping for cell cycle, stress response, and replicative lifespan assays.

The antibiotic tetracycline demeclocycline (DMC) was recently reported to rescue α-synuclein (α-Syn) fibril-induced pathology. However, the antimicrobial activity of DMC precludes its po-tential use in long-term neuroprotective treatments. Here, we synthesized a DMC derivative with residual antibiotic activity and improved neuroprotective effects. The molecule, called de-rivative demeclocycline (DDMC), was obtained by the removal of both dimethylamino substitu-ents at position 4 and the reduction of the hydroxyl group at position 12a on ring A of DMC. The modifications strongly diminished its antibiotic activity against Gram-positive and Gram-negative bacteria. Moreover, this compound preserved the low toxicity of DMC in dopaminergic cell lines while improving its ability to interfere with α-Syn amyloid-like aggregation, showing the highest effectiveness of all tetracyclines tested. Likewise, DDMC demonstrated the ability to reduce seeding induced by the exogenous addition of α-Syn preformed fibrils (α-SynPFF ) in ex vitro models and in SH-SY5Y-α-Syn-tRFP cells. In addition, in the presence of DDMC, α-SynPFF were less inflammogenic, as they dampened the release of tumor necrosis factor α (TNF-α) and glutamate by microglial cells compared to control fibrils. Our results suggest that DDMC may be a promising drug candidate for hit-to-lead development and preclinical studies in PD and other synucleinopathies.

The Golgi apparatus (GA) is essential for intracellular sorting, trafficking and the targeting of proteins to specific cellular compartments. Anatomically, the GA spreads all over the cell but is also particularly enriched close to the base of the primary cilium. This peculiar organelle protrudes at the surface of almost all cells and fulfills many cellular functions, in particular during development, when a dysfunction of the primary cilium can lead to disorders called ciliopathies. While ciliopathies caused by loss of ciliated proteins have been extensively documented, several studies suggest that alterations of GA and GA-associated proteins can also affect ciliogenesis. Here, we aim to discuss how the loss-of-function of genes coding these proteins induces ciliary defects and results in ciliopathies.

Efficient food use and disease resistance are two major challenges in pig growth, and are linked to the physiology of the gut. Intestinal pig organoids are three-dimensional cell cultures used as in vitro models. However, because of their culture conditions and structure, transfection efficiency is often reduced. Recently, the development of mRNA synthesis and transfection has opened new opportunities. Because mRNA does not need to enter the nucleus to be expressed, as opposed to plasmid DNA, mRNA transfection efficiency is expected to be greater than with plasmid DNA. We compared mRNA transfection to plasmid transfection in intestinal organoids and cell cultures, to compare their delivery and expression, using fluorescence microscopy, target genes methylation and mRNA expression. From our findings, cell line transfection efficiency varies depending on the type of material used, RNA being more efficient than plasmid transfection, and the culture medium may occasionally make a difference. Transfection of cell lines with CrisprOff plasmid allowed us to modify both methylation and expression of GLUT2, our target gene. Finally, we increased survival rates of our organoids after transfection, and hope to try mRNA transfection of CrisprOff, a new plasmid, to be able to induce epigenetic editing in pig organoids. L'utilisation efficace des aliments et la résistance aux maladies sont deux défis majeurs de la croissance des porcs liés à la physiologie de l'intestin. Les organoïdes intestinaux de porc sont des cultures cellulaires tridimensionnelles utilisées comme modèles in vitro. Cependant, l'efficacité de leur transfection est faible, en raison de leurs conditions de culture. Récemment, le développement de la transfection par ARNm a ouvert de nouvelles perspectives car il est exprimé dans le cytoplasme, contrairement à l'ADN plasmidique; son efficacité de transfection devrait donc être supérieure à ce dernier. Nous avons comparé la transfection d'ARNm à celle de plasmides dans des organoïdes intestinaux et des cultures cellulaires, pour mesurer leur expression par microscopie à fluorescence, la méthylation des gènes cibles et l'expression d'ARNm. D'après nos résultats, l'efficacité de transfection des lignées est meilleure avec l'ARN que les plasmides, et le milieu de culture pourrait l'influencer également. La transfection des lignées cellulaires avec le plasmide CrisprOff nous a permis de modifier à la fois la méthylation et l'expression de GLUT2, notre gène cible. Nous avons augmenté le taux de survie de nos organoïdes après transfection, et espérons transfecter l'ARNm de CrisprOff, afin d'induire une édition épigénétique dans les organoïdes de porc.

International audience; The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.

Background: Radiotherapy (RT) is commonly used in the management of digestive tumors, including rectal cancer (RC) and pancreatic ductal adenocarcinoma (PDAC), but with distinct indications. RT is often combined with chemotherapy to increase efficiency but at the cost of important toxicity, leaving out fragile patients. Naturally occurring bioactive food components (BFCs), including resveratrol and capsaicin, may potentiate the antitumor effect of ionizing radiation. This radiosensitization may be of great interest if surrounding healthy tissues are spared. Moreover, the combination of BFCs may provide an improved anti-tumor effect. Objectives: This work evaluated resveratrol and capsaicin radiosensitization of PDAC and RC models and explored mechanisms involved in their tumor-specific action, at the cellular and molecular levels. Methods: Cell survival was evaluated in vitro in the presence of BFCs on different pancreatic and colorectal tumor and non-tumor cell lines. Resveratrol metabolization was evaluated by mass spectrometry, and correlated with the expression of UDP-glucosyltransferases 1A (UGT1A) genes by RT-qPCR. The expression of UGT1A genes in human colorectal tissues was studied with RNAseq databases available online. Functional validation of UGT1A enzymes implication in resveratrol sensitivity of colorectal cells established by UGT1A expression modulation. Radiosensitization was assessed by apoptosis induction and cell cycle impact exploration. Molecular pathways involved in radiosensitization were analyzed by western blotting. In vivo radiosensitization analysis was tested on subcutaneous human tumor cell xenografts. Results: Resveratrol (R) and capsaicin (C) were more cytotoxic toward tumor cells and this effect was enhanced in combination. Concomitantly, cell cycle was arrested and apoptosis was induced. In addition, resveratrol resistance was related to the expression of UGTs in intestinal tumor and non-tumor cell lines. UGT1A8 and UGT1A10 gene expression were downregulated in CRC tumor tissues in comparison to matched healthy tissues. This down expression was correlated to the prognostic of CRC patients. Downregulation of UGT1A10 gene expression by shRNA in an initial resveratrol resistant cell line reduced resveratrol metabolization capacity and increased resveratrol toxicity. Conversely, overexpression of the UGT1A10 gene in an initial resveratrol sensitive tumor cell line increased the metabolization accompanied by decreased cytotoxicity. Resveratrol affected intestinal sensitive tumor cell homeostasis with a cell-size increase, cell-cycle modulation, and decreased uridylate nucleotide levels, resulting in a global reduction of transcription and translation. This impact on global cell activity was restricted to tumor cells. Xenografted tumors were radiosensitized by resveratrol and capsaicin associated with a cell cycle arrest and an increase in apoptosis. Moreover, a better tumor growth control was obtained for mice irradiated and treated with the BFCs combination compared to mice receiving radiotherapy only. Finally, the radiosensitization unbalanced the radio-induced DNA repair pathway to promote apoptosis in the PDAC tumor model. Conclusions: BFCs are potent radiosensitizers on digestive tumor cells. Resveratrol exhibits tumor-specific cytotoxicity with a functional relationship to UGT1A genes expression in digestive tumor cells and tissues. Moreover, BFCs and in particular resveratrol affect global tumor cell homeostasis, by impairing basic cell functions such as transcription, translation and DNA repair pathways. The combination of resveratrol and capsaicin radiosensitizes pancreatic and colorectal tumors in preclinical mouse models. This work positions BFCs as very potent alternative solutions for radiosensitization of tumors, without systemic side effect often observed with conventional chemotherapy., Contexte : La radiothérapie (RT) est couramment utilisée pour la prise en charge des tumeurs digestives, notamment le cancer du rectum (RC) et l'adénocarcinome du canal pancréatique (PDAC). La RT est souvent associée à la chimiothérapie pour en augmenter l'efficacité, au prix d'une toxicité importante. La radiochimiothérapie ne peut donc être proposée à tous les patients. Les composants bioactifs (BFC) d'origine naturelle, dont le resvératrol et la capsaïcine, peuvent potentialiser l'effet antitumoral des rayonnements ionisants. Cette radiosensibilisation peut être d'un grand intérêt si les tissus sains environnants sont épargnés. Objectifs : Ce travail a évalué la radiosensibilisation de modèles de PDAC et de RC par le resvératrol et la capsaïcine et a exploré les mécanismes impliqués dans leur action spécifique aux tumeurs, aux niveaux cellulaire et moléculaire. Méthodes : La survie cellulaire a été évaluée in vitro en présence de BFC sur différentes lignées cellulaires tumorales et non tumorales de PDAC et CRC. La métabolisation du resvératrol a été évaluée par spectrométrie de masse et corrélée avec l'expression des gènes des UDP-glucosyltransferases 1A (UGT1A ) par RT-qPCR. L'implication des enzymes UGT1A dans la sensibilité au resvératrol des cellules colorectales a été établie par modulation de l'expression des UGT1A. L’effet radiosensibilisant des BFCs a été évalué via l’exploration des processus d’apoptose et de leur impact sur le cycle cellulaire. Les voies moléculaires impliquées dans la radiosensibilisation ont été analysées par western blot. L'analyse de radiosensibilisation in vivo a été testée sur des xénogreffes de cellules tumorales humaines sous- cutanées. Résultats : Le resvératrol (R) et la capsaïcine (C) présentaient un effet cytotoxique sur les cellules tumorales et cet effet était renforcé par leur combinaison. Parallèlement, le cycle cellulaire était arrêté et l'apoptose induite. La résistance au resvératrol était liée à l'expression des gènes UGT1A dans les lignées digestives tumorales et non tumorales. L’inhibition de l'expression du gène UGT1A10 par shRNA permettait de réduire la métabolisation du resvératrol et augmentait sa toxicité. À l'inverse, lasurexpression du gène UGT1A10 augmentait la métabolisation et s’accompagnait d'une diminution de la cytotoxicité. Le resvératrol affectait l'homéostasie des cellules tumorales intestinales avec notamment une diminution des niveaux de nucléotides pyrimidiques, aboutissant à une réduction globale de la transcription et de la traduction. Cet effet n’était pas observé sur les cellules non-tumorales. Le resvératrol et la capsaïcine radiosensibilisaient les tumeurs xénogreffées en induisant un arrêt du cycle cellulaire et à une augmentation de l'apoptose. De plus, un meilleur contrôle de la croissance tumorale était obtenu pour les souris irradiées et traitées par les BFCs par rapport aux souris recevant uniquement la RT. Enfin, le traitement par BFCs engendrait un déséquilibre de la voie de réparation de l'ADN radio-induite amplifiant le phénomène apoptotique dans le modèle murin de tumeur PDAC. Conclusions : Les BFCs représentent des agents radiosensibilisants des tumeurs digestives prometteurs. Le resvératrol présente une cytotoxicité spécifique aux tumeurs lié fonctionnellement avec l'expression des gènes UGT1A dans les cellules et les tissus tumoraux digestifs. De plus, les BFCs et en particulier le resvératrol affectent l'homéostasie globale des cellules tumorales, en altérant les fonctions cellulaires de base. La combinaison du resvératrol et de la capsaïcine radiosensibilise les tumeurs pancréatiques et colorectales dans des modèles précliniques murins. Ces travaux positionnent les BFCs comme des solutions alternatives pour la radiosensibilisation des tumeurs, sans effet secondaire systémique souvent observé avec la chimiothérapie conventionnelle.

This work focuses on the development and evaluation of a dual nanostructured lipid carrier (NLC)/Carbopol®-based hydrogel system as a potential transporter for the topical delivery of curcumin to the skin. Two populations of different sized negatively charged NLCs (P1, 70–90 nm and P2, 300–350 nm) were prepared and characterized by means of dynamic light scattering. NLCs presented an ovoid platelet shape confirmed by transmission electron microscopy techniques. Curcumin NLC entrapment efficiency and release profiles were assessed by HPLC (high pressure liquid chromatography) and spectrophotometric methods. Preservation and enhancement of curcumin (CUR) antioxidant activity in NLCs (up to 7-fold) was established and cell viability assays on fibroblasts and keratinocytes indicated that CUR-NLCs are non-cytotoxic for concentrations up to 10 μM and exhibited a moderate anti-migration/proliferation effect (20% gap reduction). CUR-NLCs were then embedded in a Carbopol®-based hydrogel without disturbing the mechanical properties of the gel. Penetration studies on Franz diffusion cells over 24 h in CUR-NLCs and CUR-NLCs/gels demonstrated an accumulation of CUR in Strat-M® membranes of 22% and 5%, respectively. All presented data support the use of this new dual CUR-NLC/hydrogel system as a promising candidate for adjuvant treatment in topical dermal applications.

Cancer stem cells (CSCs) alone can initiate and maintain tumors, but the function of non-cancer stem cells (non-CSCs) that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan fibromodulin (FMOD) in the conditioned medium of differentiated glioma cells (DGCs), the equivalent of glioma non-CSCs, compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

Spinal cord injury (SCI) is a debilitating condition, which leads to a permanent loss of functions below the injury site. The events which take place after SCI are characterized by cellular death, release of inhibitory factors, and inflammation. Many therapies have been studied to cure SCI, among them magnetic stimulation aims to reduce the secondary damages in particular by decreasing apoptosis, while, cellular transplantation promotes neuroregeneration by enhancing axonal regrowth. In the present study, we compared individually primary olfactory ensheathing cell (OEC) transplantation and repetitive trans‐spinal magnetic stimulation (rTSMS) and then, we combined these two therapeutic approaches on tissue repair and functional recovery after SCI. To do so, SCIs were performed at Th10 level on female C57BL/6 mice, which were randomized into four groups: SCI, SCI + primary bOECs, SCI + STM, SCI + primary bulbar olfactory ensheathing cells (bOECs) + stimulation (STM). On these animals bioluminescence, immunohistological, and behavioral experiments were performed after SCI. Our results show that rTSMS has beneficial effect on the modulation of spinal scar by reducing fibrosis, demyelination, and microglial cell activation and by increasing the astroglial component of the scar, while, primary bOEC transplantation decreases microglial reactivity. At the opposite, locotronic experiments show that both treatments induce functional recovery. We did not observed any additional effect by combining the two therapeutic approaches. Taken together, the present study indicates that primary bOEC transplantation and rTSMS treatment act through different mechanisms after SCI to induce functional recovery. In our experimental paradigm, the combination of the two therapies does not induce any additional benefit., Summary of the main results of the study.

On 1 August 2018, the Democratic Republic of the Congo (DRC) declared its tenth Ebola virus disease (EVD) outbreak. To aid the epidemiologic response, the Institut National de Recherche Biomedicale (INRB) implemented an end-to-end genomic surveillance system, including sequencing, bioinformatic analysis and dissemination of genomic epidemiologic results to frontline public health workers. We report 744 new genomes sampled between 27 July 2018 and 27 April 2020 generated by this surveillance effort. Together with previously available sequence data (n = 48 genomes), these data represent almost 24% of all laboratory-confirmed Ebola virus (EBOV) infections in DRC in the period analyzed. We inferred spatiotemporal transmission dynamics from the genomic data as new sequences were generated, and disseminated the results to support epidemiologic response efforts. Here we provide an overview of how this genomic surveillance system functioned, present a full phylodynamic analysis of 792 Ebola genomes from the Nord Kivu outbreak and discuss how the genomic surveillance data informed response efforts and public health decision making. Phylogeographic analysis of 792 Ebola virus genomes from the 2018 oubreak in the Democratic Republic of the Congo integrated into an end-to-end surveillance program demonstrates the feasibility of using genomic sequencing data to inform the public health epidemic response in near-real time.

Background Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. Method To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. Results We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1–6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. Conclusion MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Summary Bipolar disorder (BD) is characterized by cyclical mood shifts. Studies indicate that BD patients have a peripheral pro-inflammatory state and alterations in glial populations in the brain. We utilized an in vitro model to study inflammation-related phenotypes of astrocytes derived from induced pluripotent stem cells (iPSCs) generated from BD patients and healthy controls. BD astrocytes showed changes in transcriptome and induced a reduction in neuronal activity when co-cultured with neurons. IL-1β-stimulated BD astrocytes displayed a unique inflammatory gene expression signature and increased secretion of IL-6. Conditioned medium from stimulated BD astrocytes reduced neuronal activity, and this effect was partially blocked by IL-6 inactivating antibody. Our results suggest that BD astrocytes are functionally less supportive of neuronal excitability and this effect is partially mediated by IL-6. We confirmed higher IL-6 in blood in a distinct cohort of BD patients, highlighting the potential role of astrocyte-mediated inflammatory signaling in BD neuropathology., Highlights • Bipolar disorder astrocytes are functionally less supportive of neuronal activity • Bipolar disorder astrocytes response to IL-1β is transcriptionally distinct • IL-6 secretion in bipolar disorder astrocytes reduces neuronal activity • Bipolar disorder patients show higher circulating levels of IL-6 in blood, In this article, Gage and collaborators show that astrocytes differentiated from induced pluripotent stem cells generated from bipolar disorder patients are functionally less supportive of neuronal activity. Bipolar disorder astrocytes' response to pro-inflammatory cytokines is characterized by a unique transcriptional response and increased IL-6 secretion that directly and negatively impacted on neuronal activity. Increased peripheral IL-6 was confirmed in a distinct clinical cohort highlighting the potential role of astrocyte-mediated inflammatory signaling in the neuropathology of bipolar disorder.

International audience; In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca 2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP QL , by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

International audience; Idiopathic CD4 T cell lymphocytopenia (ICL) is a rare entity characterized by CD4 T cell count of

Cell death can be executed through different subroutines. Since the description of ferroptosis as an iron-dependent form of non-apoptotic cell death in 2012, there has been mounting interest in the process and function of ferroptosis. Ferroptosis can occur through two major pathways, the extrinsic or transporter-dependent pathway and the intrinsic or enzyme-regulated pathway. Ferroptosis is caused by a redox imbalance between the production of oxidants and antioxidants, which is driven by the abnormal expression and activity of multiple redox-active enzymes that produce or detoxify free radicals and lipid oxidation products. Accordingly, ferroptosis is precisely regulated at multiple levels, including epigenetic, transcriptional, posttranscriptional and posttranslational layers. The transcription factor NFE2L2 plays a central role in upregulating anti-ferroptotic defense, whereas selective autophagy may promote ferroptotic death. Here, we review current knowledge on the integrated molecular machinery of ferroptosis and describe how dysregulated ferroptosis is involved in cancer, neurodegeneration, tissue injury, inflammation, and infection.

The continuing efforts to exploit the death receptor agonists, such as the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), for cancer therapy, have largely been impaired by the anti-apoptotic and pro-survival signalling pathways leading to drug resistance. Cell migration, invasion, differentiation, immune evasion and anoikis resistance are plastic processes sharing features of the epithelial-to-mesenchymal transition (EMT) that have been shown to give cancer cells the ability to escape cell death upon cytotoxic treatments. EMT has recently been suggested to drive a heterogeneous cellular environment that appears favourable for tumour progression. Recent studies have highlighted a link between EMT and cell sensitivity to TRAIL, whereas others have highlighted their effects on the induction of EMT. This review aims to explore the molecular mechanisms by which death signals can elicit an increase in response heterogeneity in the metastasis context, and to evaluate the impact of these processes on cell responses to cancer therapeutics.

International audience; The natural C-13 abundance (delta C-13) in plant leaves has been used for decades with great success in agronomy to monitor water-use efficiency and select modern cultivars adapted to dry conditions. However, in wheat, it is also important to find genotypes with high carbon allocation to spikes and grains, and thus with a high harvest index (HI) and/or low carbon losses via respiration. Finding isotope-based markers of carbon partitioning to grains would be extremely useful since isotope analyses are inexpensive and can be performed routinely at high throughput. Here, we took the advantage of a set of field trials made of more than 600 plots with several wheat cultivars and measured agronomic parameters as well as delta C-13 values in leaves and grains. We find a linear relationship between the apparent isotope discrimination between leaves and grain (denoted as Delta delta(corr)), and the respiration use efficiency-to-HI ratio. It means that overall, efficient carbon allocation to grains is associated with a small isotopic difference between leaves and grains. This effect is explained by postphotosynthetic isotope fractionations, and we show that this can be modelled by equations describing the carbon isotope composition in grains along the wheat growth cycle. Our results show that C-13 natural abundance in grains could be useful to find genotypes with better carbon allocation properties and assist current wheat breeding technologies.

International audience; We propose to use numerical simulations and Monte Carlo approaches to test 40 MR fingerprinting sequences for their ability to quantify the BOLD effect and provide maps of baseline blood oxygen saturation without the need for contrast agent injection. Our results suggest that several sequences can produce SO2 maps with less that 3% error on average even in the presence of B1 and B0 inhomogeneities. T2 and blood vessel radius could also be estimated with the same acquisitions.

Pulmonary arterial hypertension (PAH) is a complex multifactorial disease with poor prognosis characterized by functional and structural alterations of the pulmonary circulation causing marked increase in pulmonary vascular resistance (PVR), ultimately leading to right heart failure and death. Mutations in the gene encoding Bone Morphogenetic Protein Receptor type 2 (BMPR2), a receptor for the transforming growth factor-beta (TGF-β) superfamily, account for over 70% of families with PAH, and approximately 20% of sporadic cases. In recent years, however, less common or rare mutations in other genes have been identified. This review will consider how these newly discovered PAH genes could help to provide a better understanding of the molecular and cellular bases of the maintenance of the pulmonary vascular integrity, as well as their role in the PAH pathogenesis underlying occlusion of arterioles in the lung. We will also discuss how insights into the genetic contributions of these new PAH-related genes may open up new therapeutic targets for this, currently incurable, cardiopulmonary disorder.

Escherichia coli strains are responsible for a majority of human extra-intestinal infections, resulting in huge direct medical and social costs. We had previously shown that HlyF encoded by a large virulence plasmid harbored by pathogenic E. coli is not a hemolysin but a cytoplasmic enzyme leading to the overproduction of outer membrane vesicles (OMVs). Here, we showed that these specific OMVs inhibit the macroautophagic/autophagic flux by impairing the autophagosome-lysosome fusion, thus preventing the formation of acidic autolysosomes and autophagosome clearance. Furthermore, HlyF-associated OMVs were more prone to activate the non-canonical inflammasome pathway. Because autophagy and inflammation are crucial in the host’s response to infection especially during sepsis, our findings revealed an unsuspected role of OMVs in the crosstalk between bacteria and their host, highlighting the fact that these extracellular vesicles have exacerbated pathogenic properties. Abbreviations: AIEC: adherent-invasive E. coliBDI: bright detail intensityBMDM: bone marrow‐derived macrophagesCASP: caspaseE. coli: Escherichia coliEHEC: enterohemorrhagic E. coliExPEC: extra-intestinal pathogenic E. coliGSDMD: gasdermin DGFP: green fluorescent proteinHBSS: Hanks’ balanced salt solutionHlyF: hemolysin FIL1B/IL‐1B: interleukin 1 betaISX: ImageStreamX systemLPS: lipopolysaccharideMut: mutatedOMV: outer membrane vesicleRFP: red fluorescent proteinTEM: transmission electron microscopyWT: wild-type