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Background Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis.Results The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X.Conclusion Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

Background Acquired von Willebrand syndrome (AVWS) is frequent in patients with myeloproliferative neoplasms (MPNs). For von Willebrand factor (VWF) functional evaluation, ristocetin cofactor activity by aggregometry (VWF:RCo) is considered the gold standard but has limitations, and automated activity measurement has been developed such as the HemosIL VWF:RCo Werfen with particle agglutination (VWF:GPIbR). Objectives To evaluate the performance of VWF:GPIbR with HemosIL VWF:RCo Werfen (VWF:GPIbR) versus VWF:RCo in patients with thrombocytosis in the context of MPNs (T-MPNs) and in patients with secondary thrombocytosis (ST). Methods MPN patients with thrombocytosis >450 G/L (T-MPNs) were compared with patients with ST due to inflammation or iron deficiency. VWF activity (VWF:Act) was analyzed using VWF:RCo or VWF:GPIbR. VWF analysis was completed by analysis of VWF multimers and VWF collagen binding (CB) assay (VWF:CB). Results A total of 33 T-MPNs and 18 ST patients were included. Compared with aggregometry, evaluation of VWF:Act by VWF:GPIbR led to lower values in T-MPN patients, but also in ST patients. Interestingly, although the VWF:RCo/VWF:Ag ratio did not reveal differences between T-MPNs and ST patients, the VWF:GPIbR/VWF:Ag ratio analysis allowed us to suspect AVWS only in T-MPN patients. Using the distribution of VWF multimer analysis and VWF:CB, we here demonstrated that VWF:GPIbR allows AVWS diagnosis in nine T-MPNs as opposed to aggregometry. Conclusion Evaluation of VWF:Act using VWF:GPIbR has a greater sensitivity compared with aggregometry to detect AVWS in T-MPN patients.

The identification of bacterial metabolites produced by the microbiota is a key point to understand its role in human health. Among them, lipo-amino acids (LpAA), which are able to cross the epithelial barrier and to act on the host, are poorly identified. Structural elucidation of few of them was performed by high-resolution tandem mass spectrometry based on electrospray combined with selective ion dissociations reach by collision-induced dissociation (CID). The negative ions were used for their advantages of yielding only few fragment ions sufficient to specify each part of LpAA with sensitivity. To find specific processes that help structural assignment, the negative ion dissociations have been scrutinized for an LpAA: the N-palmitoyl acyl group linked to glutamic acid (C16Glu). The singular behavior of [C16Glu-H]¯ towards CID showed tenth product ions, eight were described by expected fragment ions. In contrast, instead of the expected product ions due to CONH-CH bond cleavage, an abundant complementary dehydrated glutamic acid and fatty acid anion pair were observed. Specific to glutamic moiety, they were formed by a stepwise dissociation via molecular isomerization through ion–dipole formation prior to dissociation. This complex dissociated by partner splitting either directly or after inter-partner proton transfer. By this pathway, surprising regeneration of deprotonated fatty acid takes place. Such regeneration is comparable to that occurred from dissociation to peptides containing acid amino-acid. Modeling allow to confirm the proposed mechanisms explaining the unexpected behavior of this glutamate conjugate.

Oxylipins are formed in vivo from polyunsaturated fatty acids (PUFAs). A large structural variety of compounds is grouped under the term oxylipins, which differ from their formation mechanism (involving enzymes or not), as well as their chemical structures (cyclopentane, tetrahydrofuran, hydroxylated-PUFA, etc.). All structures of oxylipins are of great biological interest. Directly correlated to oxidative stress phenomenon, non-enzymatic oxylipins are used as systemic and/or specific biomarkers in various pathologies, and more especially, they were found to have their own biological properties. Produced in vivo as a non-separable mixture of isomers, their total synthesis is a keystone to answer biological questions. In this work, the total synthesis of three non-enzymatic oxylipins derived from docosahexaenoic acid (DHA) and docosapentanoic acid (DPAn-3) is described using a unique and convergent synthetic strategy.

The present study aims to discover the effect of exogenously applied of salicylic acid (SA) in the concentration 0, 1 and 15 mM, on the oxidative stress monitored through the concentration of PhytoPs and PhytoFs in immature and mature grains of three genotypes of rice ('R52', 'R45' and 'Yerua') and their influence on grains per panicle and chalkiness. Concentration of oxylipins showed differences in both harvest times (immature and mature) for each genotype. The cv 'Yerua' presented equal concentrations of PhytoPs in immature and mature grains, while the mature grains of the advanced lines 'R52' and 'R45' showed concentrations 24.0 and 79.0% lower than those of the immature grains, respectively. The PhytoFs were equivalent in the two harvest seasons, in "Yerua" and "R52", while in "R45", the concentration of these oxylipins was 46.0% lower in the mature grains. Regarding the effect of SA treatments, it was observed that a single application reduced the concentration of a single oxylipin of the ten quantified; while in mature grains, with two applications of SA, a significant decrease in the concentration of 6 of the ten biomarkers monitored was observed with the increase in the concentration of SA. The SA produced an increase in grains per panicle and a decrease in chalkiness, especially in "R52". The close relationship between the content of the oxylipins evaluated and the concentration of SA applied confirms the relationship of their participation in the redox balance and the ability to mitigate stress and improve performance parameters of grains.

The total synthesis of a docosahexaenoic-acid-derived prostaglandin, 4,11-diepi-4-F4t-neuroprostane, featuring a complex lateral chain was achieved for the first time. A novel prostaglandin cyclope...

International audience; Pistachio (Pistacia vera L.) is a drought-tolerant species grown under the semiarid conditions of the Mediterranean basin. For this reason, it is essential to make an exhaustive quantification of yield and quality benefits of the kernels because the regulated deficit irrigation will allow significant water savings with a minimum impact on yield while improving kernel quality. The goal of this scientific work was to study the influence of the rootstock, water deficit during pit hardening, and kernel roasting on pistachio (P. vera, cv. Kerman) fruit yield, fruit size, and kernel content of fatty acids phytoprostanes (PhytoPs) and phytofurans (PhytoFs) for the first time. Water stress during pit hardening did not affect the pistachio yield. The kernel cultivar showed a lower oleic acid and a higher linoleic acid contents than other cultivars. Kernels from plants grafted on the studied rootstocks showed very interesting characteristics. P. integerrima led to the highest percentage of monounsaturated fatty acids. Regarding the plant oxylipins, P. terebinthus led to the highest contents of PhytoPs and PhytoFs (1260 ng/100 g and 16.2 ng/100 g, respectively). In addition, nuts from trees cultivated under intermediate water deficit during pit hardening showed increased contents of the 9-series F1-phytoprostanes and ent-16(RS)-9-epi-ST-Δ14-10-phytofuran. However, roasting of pistachios led to PhytoP degradation. Therefore, plant cultivar, deficit irrigation, rootstock, and roasting must be considered to enhance biosynthesis of these secondary metabolites. New tools using agricultural strategies to produce hydroSOS pistachios have been opened thanks to the biological properties of these prostaglandin-like compounds linking agriculture, nutrition, and food science technology for further research initiatives.

Les maladies cardiovasculaires (MCV) sont la première cause de mortalité dans le monde. Les plaquettes jouent un rôle majeur dans le développement de ces maladies et la mise au point d’antiplaquettaires efficaces constitue une priorité dans le cadre de la lutte contre la mortalité liée aux MCV. L’aspirine et les médicaments de la famille des thiénopyridines sont les agents antiplaquettaires les plus utilisés actuellement. Ces médicaments ciblent des voies de signalisation impliquées dans l’initiation de l’agrégation, exerçant ainsi un effet antiplaquettaire modéré. D’autres médicaments aux effets plus importants, comme les molécules dirigées contre le récepteur GPIIb/IIIa, inhibent l’agrégation plaquettaire indépendamment de la voie de signalisation initiant l’activation plaquettaire, mais ils sont associés à des complications hémorragiques majorées. Étant données les caractéristiques spécifiques de chacun de ces agents antiplaquettaires, leur prescription nécessite de prendre en compte le type d’évènement cardio-vasculaire, l’âge et les comorbidités du patient traité et, bien sûr, les effets secondaires hémorragiques potentiels de la molécule qui est prescrite. Apparaît donc la nécessité de mettre au point de nouvelles molécules ayant un effet plus ciblé, gardant une efficacité optimale, mais permettant une réduction du risque hémorragique qui constitue la principale limite des médicaments antiplaquettaires.

Cométographie Ou Traité Historique Et Théorique Des Comètes, 1

ObjectivesClinical studies revealed that early-life adverse events contribute to the development of IBS in adulthood. The aim of our study was to investigate the relationship between prenatal stress (PS), gut microbiota and visceral hypersensitivity with a focus on bacterial lipopeptides containing γ-aminobutyric acid (GABA).DesignWe developed a model of PS in mice and evaluated, in adult offspring, visceral hypersensitivity to colorectal distension (CRD), colon inflammation, barrier function and gut microbiota taxonomy. We quantified the production of lipopeptides containing GABA by mass spectrometry in a specific strain of bacteria decreased in PS, in PS mouse colons, and in faeces of patients with IBS and healthy volunteers (HVs). Finally, we assessed their effect on PS-induced visceral hypersensitivity.ResultsPrenatally stressed mice of both sexes presented visceral hypersensitivity, no overt colon inflammation or barrier dysfunction but a gut microbiota dysbiosis. The dysbiosis was distinguished by a decreased abundance ofLigilactobacillus murinus, in both sexes, inversely correlated with visceral hypersensitivity to CRD in mice. An isolate from this bacterial species produced several lipopeptides containing GABA including C14AsnGABA. Interestingly, intracolonic treatment with C14AsnGABA decreased the visceral sensitivity of PS mice to CRD. The concentration of C16LeuGABA, a lipopeptide which inhibited sensory neurons activation, was decreased in faeces of patients with IBS compared with HVs.ConclusionPS impacts the gut microbiota composition and metabolic function in adulthood. The reduced capacity of the gut microbiota to produce GABA lipopeptides could be one of the mechanisms linking PS and visceral hypersensitivity in adulthood.

Cométographie Ou Traité Historique Et Théorique Des Comètes, 2

Date palm fruit has been considered for centuries as an ancient nutritional constituent in the human diet. Recently, global trade in dates increased at an average that, simultaneously, will be accompanied by an increase in date palm byproducts. Supported by date phytochemicals and their health benefits, the aim of this work is to evaluate for the first time the presence of special metabolites of plant called phytoprostanes (PhytoPs) in five different varieties of the Phoenix dactylifera L. pulps and pits using a microLC-ESI-QTrap-MS/MS methodology. Results obtained showed the interest of using these matrices as potential sources of several PhytoPs (ent-16-B1-PhytoP; ent-9-L1-PhytoP; and epimers of ent-16-F1t-PhytoP and of 9-F1t-PhytoP). The variation in concentration between different varieties and different DPF parts was also evaluated. Results obtained will help to unravel the biological activities associated with DPF consumption that could be related to these bioactive metabolites.

International audience; Les néoplasies myéloprolifératives (NMP) sans translocation de Philadelphie sont des maladies hématologiques acquises caractérisées par la prolifération d’une ou plusieurs lignées sanguines. Elles regroupent la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose primitive (MFP). La survenue de thromboses artérielles ou veineuses est un risque majeur au cours de ces maladies. Les facteurs de risque reconnus actuellement sont un âge supérieur à 60 ans et un antécédent de thrombose. Les mécanismes concourant à ce risque pro-thrombotique augmenté sont cependant multiples et complexes, impliquant l’ensemble des cellules sanguines, des facteurs plasmatiques et le compartiment endothélial. Ces dernières années, de nouveaux mécanismes physiopathologiques ont été révélés.

International audience; Cocoa has been widely discussed as a bioactive food rich in sensory stimulation and health benefits. However, no information has been provided concerning phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in cocoa. These compounds are of interest because they play a role in the regulation of immune function. The present study included 31 cocoa clones. The PhytoPs and PhytoFs were quantified by UHPLC-QqQ-MS/MS. The total PhytoPs and PhytoFs contents ranged from 221.46 to 1589.83 ng g −1 and from 1.18 to 13.13 ng g −1 , respectively. The profiles of the PhytoPs and PhytoFs identified in the cocoa beans showed significant differences among the clones analysed. The results indicate that dry fermented cocoa beans are rich in PhytoPs and PhytoFs, which may represent an additional benefit of the consumption of foods derived from cocoa.

Essentials Acquired Glanzmann thrombasthenia (GT) is generally caused by anti-αIIb β3 autoantibodies. We report the case of a man with an acquired GT phenotype associated with macrothrombocytopenia. Perturbed platelet function were associated with an activating anti-αIIb β3 IgM autoantibody. This novel clinical entity raises interesting questions about the αIIb β3 integrin signaling. SUMMARY: Background Acquired Glanzmann thrombasthenia (GT) is a bleeding disorder generally caused by anti-αII b β3 autoantibodies. Objectives We aimed to characterize the molecular mechanism leading to a progressive GT-like phenotype in a patient with chronic immune thrombocytopenia. Patient, Methods, and Results The patient suffered from repeated episodes of gastrointestinal bleeding; further studies indicated a moderate platelet aggregation defect. A few months later, platelet function showed abolished aggregation using all agonists, but normal agglutination with ristocetin. No platelet-bound antibodies were detected, but the presence of large amounts of an IgM type antibody detected together with αII b β3 in the patient permeabilized platelets suggested that this IgM was an autoantibody causing the internalization of the complex. This was confirmed by the fact that the patient IgM bound to normal platelets but not to platelets from GT type I patients. Moreover, patient's plasma activated αII b β3 on controls' platelets as evidenced by increased PAC-1 binding. We also demonstrated that the patient plasma triggered αII b β3 outside-in signaling, as β3 Tyr773 and FAK were phosphorylated, and increased the rate of actin polymerization in resting platelets reflecting an impairment of cytoskeletal reorganization. Because different signs of dysmegakaryopoiesis were also observed in our patient, we evaluated the ability of its serum to impair proplatelets formation and showed that it significantly decreased the number of proplatelet-bearing megakaryocytes in controls' bone marrow stem cells culture compared with normal serum. Conclusions We present the case of a patient with a progressive and severely perturbed platelet function associated with the presence of an IgM activating autoantibody directed against αII b β3 .

Background In Colombia, agro-industrial residues represent an enormous economic and environmental problem, which could be reduced if different techniques for the addition of value to such residues were implemented by this industrial sector. One of the fruits with the highest export rates is Physalis peruviana (goldenberry); however, this fruit is generally marketed without its calyx, generating a large amount of residues. To develop a strategy to add value to these residues, it is essential to know their chemical composition. Results In the present work, phytoprostanes (PhytoPs) - new active oxylipins - have been detected for the first time in Physalis peruviana calyces by ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS), F1t -phytoprostanes and D1t -phytoprostanes being the predominant and minor classes, respectively. In addition, we were able to characterize the phenolic compounds profile of this matrix using LC-IT-DAD-MS/MS, describing six phenolic derivatives for the first time therein. Conclusions This study increases our knowledge of the chemical composition of the calyces of this fruit and thereby supports the recycling of this class of residue. Consequently, goldenberry calyces could be used as phytotherapeutic, nutraceutic, or cosmetic ingredients for the development of diverse natural products. © 2018 Society of Chemical Industry.

BCR–ABL-fusion-negative myeloproliferative neoplasms (MPNs) with myelofibrosis (MF) include primary MF, post-polycythemia vera MF and post-essential thrombocythemia MF. Clonal extramedullary hematopoiesis (EMH) can occur during MPN pathogenesis. Although histopathological bone-marrow (BM) features during clonal EMH have been investigated, those of the spleen have been poorly described. We analyzed splenectomy samples from 28 patients with MF and BM samples from 20 of them. Slides were stained with hematoxylin and eosin, reticulin, and trichrome, with immunohistochemical labeling of glycophorin A, myeloperoxidase, CD61, CD34, and CD117. We also subjected splenectomy and BM samples from six patients and spleen samples from seven patients to next-generation sequencing (NGS). Megakaryocyte-rich spleen nodules (MRSNs), seen in seven of the 28 patients, were significantly associated with megakaryocyte proliferation in the spleen (p = 0.04). We devised a grading system for spleen fibrosis (SF) and found that SF was increased in 20 of 28 patients. Notably, patients with SF were more likely to have MRSNs, suggesting that megakaryocytes might participate in SF, as previously described in BM. Comparisons of spleen and BM NGS findings of six patients’ specimens revealed identical mutational status in the two organs for half of the patients. We observed additional mutations in the spleen of two patients. However, the meaning of this finding remains unknown since there was a long interval between BM and spleen samplings (68 and 82 months, respectively).

International audience; Improving knowledge about metabolites produced by the microbiota is a key point to understand its role in human health and disease. Among them, lipoamino acid (LpAA) containing asparagine and their derivatives are bacterial metabolites which could have an impact on the host. In this study, our aim was to extend the characterization of this family. We developed a semi-targeted workflow to identify and quantify new candidates. First, the sample preparation and analytical conditions using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) were optimized. Using a theoretical homemade database, HRMS raw data were manually queried. This strategy allowed us to find 25 new LpAA conjugated to Asn, Gln, Asp, Glu, His, Leu, Ileu, Pro, Lys, Phe, Trp and Val amino acids. These metabolites were then fully characterized by MS2, and compared to the pure synthesized standards to validate annotation. Finally, a quantitative method was developed by LC coupled to a triple quadrupole instrument, and linearity and limit of quantification were determined. 14 new LpAA were quantified in gram positive bacteria, Lactobacilus animalis, and 12 LpAA in Escherichia coli strain Nissle 1917.

International audience; Lipid peroxidation is a primary event associated with oxidative stress in plants. This phenomenon secondarily generates bioactive and/or toxic compounds such as reactive carbonyl species (RCS), phytoprostanes, and phytofurans, as confirmed here in Arabidopsis plants exposed to photo‐oxidative stress conditions. We analyzed the effects of exogenous applications of secondary lipid oxidation products on Arabidopsis plants by luminescence techniques. Oxidative damage to attached leaves was measured by autoluminescence imaging, using a highly sensitive CCD camera, and the activity of the detoxification pathway, dependent on the transcription regulator SCARECROW‐LIKE 14 (SCL14), was monitored with a bioluminescent line expressing the firefly LUCIFERASE (LUC) gene under the control of the ALKENAL REDUCTASE (AER) gene promoter. We identified 4‐hydroxynonenal (HNE), and to a lesser extent 4‐hydroxyhexenal (HHE), as highly reactive compounds that are harmful to leaves and can trigger AER gene expression, contrary to other RCS (pentenal, hexenal) and to isoprostanoids. Although the levels of HNE and other RCS were enhanced in the SCL14‐deficient mutant (scl14), exogenously applied HNE was similarly damaging to this mutant, its wild‐type parent and a SCL14‐overexpressing transgenic line (OE:SCL14). However, strongly boosting the SCL14 detoxification pathway and AER expression by a pre‐treatment of OE:SCL14 with the signaling apocarotenoid β‐cyclocitral canceled the damaging effects of HNE. Conversely, in the scl14 mutant, the effects of β‐cyclocitral and HNE were additive, leading to enhanced leaf damage. These results indicate that the cellular detoxification pathway induced by the low‐toxicity β‐cyclocitral targets highly toxic compounds produced during lipid peroxidation, reminiscent of a safener‐type mode of action.

International audience; Thrombosis, both in arterial and venous territories, is the major complication of myeloproliferative neoplasms and is responsible for a high rate of morbidity and mortality. The currently accepted risk factors are an age over 60 years and a history of thrombosis. However, many complex mechanisms contribute to this increased prothrombotic risk, with involvement of all blood cell types, plasmatic factors, and endothelial cells. Besides, some cardiovascular events may originate from arterial vasospasm that could contribute to thrombotic complications. In this review, we discuss recent results obtained in mouse models in the light of data obtained from clinical studies. We emphasize on actors of thrombosis that are currently not targeted with current therapeutics but could be promising targets, i.e, neutrophil extracellular traps and vascular reactivity.

International audience; Mycobacterium abscessus (M. abscessus), a rapidly growing mycobacterium, is an emergent opportunistic pathogen responsible for chronic bronchopulmonary infections in individuals with respiratory diseases such as cystic fibrosis. Most treatments of M. abscessus pulmonary infections are poorly effective due to the intrinsic resistance of this bacteria against a broad range of antibiotics including anti-tuberculosis agents. Consequently, the number of drugs that are efficient against M. abscessus remains limited. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs impair extracellu-lar M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or act intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., compound iBpPPOX, via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis. Among them, the Ag85C protein has been confirmed as a vulnerable target of iBpPPOX. This study clearly emphasizes the potential of the OX derivatives to inhibit the extracellular and/or intracellular growth of M. abscessus by targeting various enzymes potentially involved in many physiological processes of this most drug-resistant mycobacterial species.

Algae result from a complex evolutionary history that shapes their metabolic network. For example, these organisms can synthesize different polyunsaturated fatty acids, such as those found in land plants and oily fish. Due to the presence of numerous double-bonds, such molecules can be oxidized nonenzymatically, and this results in the biosynthesis of high-value bioactive metabolites named isoprostanoids. So far, there have been only a few studies reporting isoprostanoid productions in algae. To fill this gap, the current investigation aimed at profiling isoprostanoids by liquid chromatography -mass spectrometry/mass spectrometry (LC-MS/MS) in four marine microalgae. A good correlation was observed between the most abundant polyunsaturated fatty acids (PUFAs) produced by the investigated microalgal species and their isoprostanoid profiles. No significant variations in the content of oxidized derivatives were observed for Rhodomonas salina and Chaetoceros gracilis under copper stress, whereas increases in the production of C18-, C20- and C22-derived isoprostanoids were monitored in Tisochrysis lutea and Phaeodactylum tricornutum. In the presence of hydrogen peroxide, no significant changes were observed for C. gracilis and for T. lutea, while variations were monitored for the other two algae. This study paves the way to further studying the physiological roles of isoprostanoids in marine microalgae and exploring these organisms as bioresources for isoprostanoid production.

Cardiovascular diseases are the leading cause of deaths in the world. Platelets play a major role in the occurrence of these diseases and the development of antiplatelet drugs is a priority in the fight against cardiovascular diseases-associated mortality. Aspirin and thienopyridine-based P2Y12 inhibitors are the main drugs currently used. These molecules target the initiation of platelets activation and are responsible for a moderate inhibitory action. Other antiplatelet agents, as glycoprotein (GP) IIb/IIIa antagonists, inhibit platelet aggregation independently of initial activation-associated pathways, but are responsible for increased hemorrhagic events. Regarding each antiplatelet agent's specific characteristics, the prescription of these drugs must take into account the type of cardiovascular event, the age of the patient, the past medical history, and the potential hemorrhagic adverse events. Thus, there is a need for the development of new molecules with a more targeted effect, maintaining optimal efficiency but with a reduction of the hemorrhagic risk, which is the principal limitation of these treatments.Antiplaquettaires actuels, en cours de développement et cibles thérapeutiques.Les maladies cardiovasculaires (MCV) sont la première cause de mortalité dans le monde. Les plaquettes jouent un rôle majeur dans le développement de ces maladies et la mise au point d’antiplaquettaires efficaces constitue une priorité dans le cadre de la lutte contre la mortalité liée aux MCV. L’aspirine et les médicaments de la famille des thiénopyridines sont les agents antiplaquettaires les plus utilisés actuellement. Ces médicaments ciblent des voies de signalisation impliquées dans l’initiation de l’agrégation, exerçant ainsi un effet antiplaquettaire modéré. D’autres médicaments aux effets plus importants, comme les molécules dirigées contre le récepteur GPIIb/IIIa, inhibent l’agrégation plaquettaire indépendamment de la voie de signalisation initiant l’activation plaquettaire, mais ils sont associés à des complications hémorragiques majorées. Étant données les caractéristiques spécifiques de chacun de ces agents antiplaquettaires, leur prescription nécessite de prendre en compte le type d’évènement cardio-vasculaire, l’âge et les comorbidités du patient traité et, bien sûr, les effets secondaires hémorragiques potentiels de la molécule qui est prescrite. Apparaît donc la nécessité de mettre au point de nouvelles molécules ayant un effet plus ciblé, gardant une efficacité optimale, mais permettant une réduction du risque hémorragique qui constitue la principale limite des médicaments antiplaquettaires.

International audience; Introduction: Philadelphia-negative myeloproliferative neoplasms (MPNs) greatly increase the risk of maternal and fetal complications during pregnancy. Currently, international agreements regarding the management of these women are lacking.Patients and methods: Our study aimed to assess the current management and outcomes of MPN pregnancies in a French cohort. We retrospectively analyzed 27 pregnancies in women with MPNs that were associated with a specific mutation. Nineteen pregnancies in nine women with essential thrombocythemia and eight pregnancies in five women with polycythemia vera were identified.Results: Our study showed 70% live births, but only 30% uneventful pregnancies. Fetal complications were mainly early spontaneous abortions (22%), fetal growth restriction (15%), and premature delivery (15%). Maternal issues were divided between thrombosis (15%) and hemorrhages (11%). High rates of preeclampsia and hemolysis, elevated liver enzymes, and low platelet count syndrome (15%) were reported. Uterine artery Doppler was performed in 70% pregnancies. Abnormal Doppler results were found in 43% pregnancies. Pregnancies with high platelet counts and packed cell volume remaining static or increasing ended with fetal death and utero-placental dysfunction. According to expert consensus, most of the pregnancies (67%) could be stratified in the high risk group and had a bad obstetrical outcome, with 50% standard-risk pregnancies versus 22% high-risk pregnancies that were uneventful. Higher risk pregnancies were prescribed heparin and/or interferon α in 72%.Conclusions: The prognosis of these pregnancies remains very bad and may be improved by a more effective collaboration between specialists as well as a therapeutic intensification including heparin and interferon α.

International audience; Given the growing interest in phytoprostanes (PhytoPs) and phytofurans (PhytoFs) in the fields of plant physiology, biotechnology, and biological function, the present study aims to optimize a method of enzymatic hydrolysis that utilizes bacterial and yeast esterases that allow the appropriate quantification of PhytoPs and PhytoFs. To obtain the highest concentration of PhytoPs and PhytoFs, a response surface methodology/Box−Behnken design was used to optimize the hydrolysis conditions. Based on the information available in the literature on the most critical parameters that influence the activity of esterases, the three variables selected for the study were temperature (°C), time (min), and enzyme concentration (%). The optimal hydrolysis conditions retrieved differed between PhytoPs (21.5°C, 5.7 min, and 0.61 μg of enzyme per reaction) and PhytoFs (20.0°C, 5.0 min, and 2.17 μg of enzyme per reaction) and provided up to 25.1-and 1.7-fold higher contents relative to nonhydrolyzed extracts. The models were validated by comparing theoretical and experimental values for PhytoP and PhytoF yields (1.01 and 1.06 theoretical/experimental rates, respectively). The optimal conditions were evaluated for their relative influence on the yield of individual nonesterified PhytoPs and PhytoFs to define the limitations of the models for obtaining the highest concentration of most considered compounds. In conclusion, the models developed provided valuable alternatives to the currently applied methods using unspecific alkaline hydrolysis to obtain free nonesterified PhytoPs and PhytoFs, which give rise to more specific hydrolysis of PhytoP and PhytoF esters, reducing the degradation of free compounds by classical chemical procedures.

Enzymatically formed oxygenated metabolites of polyunsaturated fatty acids (PUFA) are of great interest for the scientific community being mediators and biomarkers in the physiological and pathological regulation of many key biological processes. More recently; metabolites of PUFA formed through a non-enzymatic free radical pathway have gained interest in diseases linked with oxidative stress. Thus, synthetic strategies leading to the total synthesis of such metabolites are an essential field of research, and this review will cover a structural presentation, will discuss their biological interests, and present total syntheses reported between 2008 and 2017.1 Introduction2 Non-Enzymatic Metabolites of PUFAs2.1 Where Do They Come From?2.2 Why Are They Interesting Metabolites?2.3 A Huge Family of Natural Compounds2.3.1 Presentation of All Families2.3.1.1 Isoprostanoids2.3.1.2 Isofuranoids2.3.1.3 Isoketals2.3.2 Nomenclature of These Metabolites2.3.2.1 Isoprostanoids2.3.2.2 Isofuranoids2.3.2.3 Isoketals3 Total Synthesis Strategies3.1 Isoprostanoids3.2 Isofuranoids3.3 Isoketals4 The Discoveries Behind the Synthesis4.1 Recent Identification and Biomarkers Roles of Isoprostanes and Isofuranes4.2 Biological Properties4.2.1 Phytoprostanes, Isoprostanes, and Neuroprostanes4.2.2 Epoxy-isoprostanes5 Conclusion

International audience; Aims:Hedgehog (Hh) signalling has been shown to be re-activated in ischaemic tissues and participate in ischaemia-induced angiogenesis. Sonic Hedgehog (Shh) is upregulated by more than 80-fold in the ischaemic skeletal muscle, however its specific role in ischaemia-induced angiogenesis has not yet been fully investigated. The purpose of the present study was to investigate the role of endogenous Shh in ischaemia-induced angiogenesis.Methods and results:To this aim, we used inducible Shh knock-out (KO) mice and unexpectedly found that capillary density was significantly increased in re-generating muscle of Shh deficient mice 5 days after hind limb ischaemia was induced, demonstrating that endogenous Shh does not promote angiogenesis but more likely limits it. Myosin and MyoD expression were equivalent in Shh deficient mice and control mice, indicating that endogenous Shh is not required for ischaemia-induced myogenesis. Additionally, we observed a significant increase in macrophage infiltration in the ischaemic muscle of Shh deficient mice. Our data indicate that this was due to an increase in chemokine expression by myoblasts in the setting of impaired Hh signalling, using tissue specific Smoothened conditional KO mice. The increased macrophage infiltration in mice deficient for Hh signalling in myocytes was associated with increased VEGFA expression and a transiently increased angiogenesis, demonstrating that Shh limits inflammation and angiogenesis indirectly by signalling to myocytes.Conclusion:Although ectopic administration of Shh has previously been shown to promote ischaemia-induced angiogenesis, the present study reveals that endogenous Shh does not promote ischaemia-induced angiogenesis. On the contrary, the absence of Shh leads to aberrant ischaemic tissue inflammation and a transiently increased angiogenesis.

Metabolic reprogramming is one of the hallmarks of cancer, as these rapidly dividing cells need to adapt their metabolism to cope with an increased energy demand. We have previously showed that in mouse models of myeloproliferative neoplasms (MPN), JAK2-mutant cells display metabolic alterations, including increased oxidative phosphorylation and glycolysis. The glutamine-glutamate-alphaKetoglutarate (aKG) axis, besides fueling the Krebs cycle and anabolic processes, contributes to the synthesis of the heme precursor 5-aminolevulinic acid (5-ALA), thus making glutaminolysis a potential target for MPN therapy. Here, we examined the effects of pharmacological inhibition of the conversion of glutamine to glutamate by glutaminase 1 by Telaglenastat (CB-839) in a mouse model of JAK2-V617F driven MPN. We performed competitive transplantations of bone marrow co-expressing JAK2-V617F with a GFP-reporter and wildtype mice in a 1:10 mixture into lethally irradiated mice. At 8 weeks post-transplantation, the mice developed MPN phenotype and were randomized into 4 treatment arms. They were then treated for 6 weeks with vehicle, CB-839, 3PO (6-phosphofructo-2-kinase inhibitor) and the combination of CB-839 + 3PO. We found that hemoglobin levels were significantly reduced in CB-839 treated mice and were fully normalised in mice that received a combination of CB-839 + 3PO (Fig. 1A). Other peripheral blood cell counts did not change (not shown). Hypoglycemia was noted in the vehicle group, as previously described, but an increase in blood glucose levels was seen already after one week of treatment with CB-839. 3PO alone had less impact on hypoglycemia and mice receiving CB-839 + 3PO had similar values to CB-839 alone (Fig. 1A). In line with these findings, splenomegaly was also reduced (Fig. 1A). To characterise the changes in metabolic activity, we determined the metabolic profiles of unfractionated bone marrow (BM) cells using seahorse assays. As expected, basal and maximal glycolysis and oxidative phosphorylation were higher in vehicle treated MPN cells than cells from wildtype controls. However, when BM cells from CB-839 or CB-839 + 3PO groups were examined, they displayed a strong downmodulation of both glycolysis and oxidative phosphorylation, indicating that chronic exposure to CB-839 reprograms the metabolic machinery by reducing not only the basal glycolytic and respiratory levels, but also the maximal capacity (Fig. 1B). Glutamine is the primary source of aKG needed to produce the heme precursor 5-ALA, and hemoglobin synthesis consumes the majority of ATP in erythroid progenitors. We therefore hypothesize that 5-ALA shortage caused by CB-839 limits hemoglobin production, leading to reduced catabolic activity and drop in ATP demand. To evaluate this model, we examined the stages of erythroid differentiation from vehicle or treated mice. Early and intermediate erythroid cells were enriched at the expense of later stages in CB-839 and CB-839+3PO treatment arms (Fig. 1C). The accumulation of erythroid precursors at the stage before hemoglobin is assembled supports the hypothesis that targeting glutamine usage impairs excessive erythropoiesis by limiting hemoglobinization rate. Our data show that inhibiting glutaminolysis ameliorated MPN phenotype and that the combination with the glycolytic inhibitor 3PO was able to induce complete hematological response. Since CB-839 is already in clinical trials for other cancers, our data suggest that it could also be tested for treatment of patients with polycythemia vera. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.