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Summary In Parkinson’s disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery., Highlights • Derivation of distinct dopaminergic subpopulations from pluripotent stem cells • Wnt signaling inhibitors promote SN dopaminergic neuron specification • Modeling selective vulnerability of SN dopaminergic neurons in vitro • Proteomics reveals pathways that promote SN dopaminergic neuron survival, In this article, Panman and colleagues show that by modulating Wnt signaling levels, distinct subpopulations of dopaminergic neurons can be derived from mouse and human pluripotent stem cells. Cultures enriched for substantia nigra dopaminergic neurons recapitulate the selective sensitivity to mitochondrial toxins as observed in Parkinson’s disease patients. The developed strategy facilitates disease modeling and drug discovery.

Dysregulations of lipid metabolism in the liver may trigger steatosis progression, leading to potentially severe clinical consequences such as nonalcoholic fatty liver diseases (NAFLDs). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis. Mice with liver-specific Hmgb1 deficiency display exacerbated liver steatosis, while Hmgb1-overexpressing mice exhibited a protection from fatty liver progression when subjected to nutritional stress. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRα and PPARγ activity. HMGB1 repression is not mediated through nucleosome landscape reorganization but rather via a preferential DNA occupation in a region carrying genes regulated by LXRα and PPARγ. Together, these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target the LXRα-PPARγ axis during NAFLD. Published version This study was supported by grants from INSERM, Paul Sabatier University, the Agence Nationale de la Recherche (ANR-17-CE14-0016, to J.-P.P.), and the Région Occitanie and Association Française d’Etude et de Recherche sur l’Obésité (to J.-P.P.). J.P. was supported by a scholarship from Paul Sabatier University. E.P. was supported by a scholarship from Agence Nationale de la Recherche (ANR-17-CE14-0016). R.P. was supported by a scholarship from Région Midi-Pyrénées-INSERM (no. 15050341). O.B.-S. and L.C.-S. and the proteomic facility were supported, in part, by the Région Occitanie, European funds (Fonds Européens de Développement Régional, FEDER), Toulouse Métropole, and the French Ministry of Research with the Investissement d’Avenir Infrastructures Nationales en Biologie et Santé program (ProFI, Proteomics French Infrastructure project, ANR-10-INBS-08).

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention.

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity., Author summary The epidemic efficiency of tuberculosis bacilli is determined by their capacity to transmit via aerosol. Currently, the bacterial functions that favor Mycobacterium tuberculosis seeding in the lung of naïve host remain mostly unknown. Here we implemented a genome-wide approach to identify M. tuberculosis mutants deficient for seeding and early replication in the lung of mice. In addition to genes known to encode virulence factors, we identified three genes not previously described. We used complementary approaches to characterize the phenotype of a M. tuberculosis mutant with insertion within the rv0180c gene. We found that this mutant is impaired for seeding in the lung of mice and for invasion and replication in human macrophages. In macrophages, the defect relies on a lack of engagement of CR3 receptor. Although we did not detect any difference between the wild type strain and the rv0180c mutant with regard to potential CR3-ligand, we found that the bacterial cell envelope is altered in the rv0180c mutant. Our study provides new insight into bacterial genes required for early interaction of M. tuberculosis with the host and perspective to understand the bacterial functions enhancing infectivity.

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also demonstrate that EVs contain ACC in uterine fluid using spectroscopic analysis. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of bicarbonate ions (HCO23 ), for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first-time experimental evidence for the components of vesicular transport to supply ACC in a vertebrate model of biomineralization., Universite Francois Rabelais de Tours, Region Centre-Val de Loire, Natural Sciences and Engineering Research Council of Canada RGPIN-2016-04410, Le STUDIUM

Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where mineralization takes place. Herein, we report further experimental evidence for this model. We confirmed the presence of extracellular vesicles (EVs) that contain ACC in uterine fluid using transmission electron microscopy and elemental analysis. We also demonstrate high levels of expression of vesicular markers in the oviduct segments where eggshell is formed. Moreover, proteomics and immunofluorescence confirmed the presence of major vesicular, mineralization-specific and eggshell matrix proteins in the uterus and in purified EVs. We propose a comprehensive role for EVs in eggshell mineralization, in which annexins transfer calcium into vesicles and carbonic anhydrase 4 catalyzes the formation of HCO3−, for accumulation of ACC in vesicles. We hypothesize that ACC is stabilized by ovalbumin and/or lysozyme or additional vesicle proteins identified in this study. Finally, EDIL3 and MFGE8 are proposed to serve as guidance molecules to target EVs to the mineralization site. We therefore report for the first time experimental evidence for the components of vesicular transport to supply ACC in vertebrate biomineralization. These results could give insight to understand the mineralization of otoconia, which are calcium carbonate biomineralized structures present in all vertebrates and necessary for balance and sensing linear acceleration.

International audience; The Guinea fowl eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. Both eggshells are composed of 95% calcite and 3.5% organic matrix, which control its structural organization. Chicken eggshell is made of columnar calcite crystals arranged vertically. In the Guinea fowl, the same structure is observed in its inner half, followed by a dramatic change in crystal size and orientation in the outer region. Guinea fowl eggshell is thicker than chicken eggshell. Both structure and shell thickness confer a superior resistance to breakage compared to eggshells of other bird species. To understand the underlying mechanisms controlling the structural organization of this highly resistant material, we used quantitative proteomics to analyze the protein composition of the Guinea fowl eggshell organic matrix at key stages of the biomineralization process. We identified 149 proteins, which were compared to other bird eggshell proteomes and analyzed their potential functions. Among the 149 proteins, 9 are unique to Guinea fowl, some are involved in the control of the calcite precipitation (Lysozyme, Ovocleidin-17-like, Ovocleidin-116 and Ovalbumin), 61 are only found in the zone of microstructure shift and 17 are more abundant in this zone. Significance: The avian eggshell is a critical physical barrier to protect the contents of this autonomous reproductive enclosure from physical and microbial assault. The Guinea fowl (Numida meleagris) eggshell exhibits a unique microstructure (texture), which confers exceptional mechanical properties compared to eggshells of other species. In order to understand the mechanisms that regulate formation of this texture in the Guinea fowl eggshell, we performed comparative quantitative proteomics at key stages of shell mineralization and particularly during the dramatic shift in shell microstructure. We demonstrate that the Guinea fowl eggshell proteome comprises 149 proteins, of which 61 were specifically associated with the change in size and orientation of calcite crystals. Comparative proteomics analysis with eggshell of other bird species leads to new insights into the biomineralization process. Moreover, our data represents a list of organic compounds as potential additives to regulate material design for industrial fabrication of ceramics. This information also provides molecular markers for efficient genomic selection of chicken strains to lay eggs with improved shell mechanical properties for enhanced food safety.

In this work, we aimed to understand the biological activity and the mechanism of action of three polymer-'ruthenium-cyclopentadienyl' conjugates (RuPMC) and a low molecular weight parental compound (Ru1) in cancer cells. Several biological assays were performed in ovarian (A2780) and breast (MCF7, MDA-MB-231) human cancer derived cell lines as well as in A2780cis, a cisplatin resistant cancer cell line. Our results show that all compounds have high activity towards cancer cells with low IC50 values in the micromolar range. We observed that all Ru-PMC compounds are mainly found inside the cells, in contrast with the parental low molecular weight compound Ru1 that was mainly found at the membrane. All compounds induced mitochondrial alterations. PMC3 and Ru1 caused F-actin cytoskeleton morphology changes and reduced the clonogenic ability of the cells. The conjugate PMC3 induced apoptosis at low concentrations comparing to cisplatin and could overcame the platinum resistance of A2780cis cancer cells. A proteomic analysis showed that these compounds induce alterations in several cellular proteins which are related to the phenotypic disorders induced by them.Our results suggest that PMC3 is foreseen as a lead candidate to future studies and acting through a different mechanism of action than cisplatin. Here we established the potential of these Ru compounds as new metallodrugs for cancer chemotherapy., This work was financed by the Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e a Tecnologia, FCT) within the scope of projects UID/QUI/00100/2013 and PTDC/QUI-QIN/28662/2017. This work was supported by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). Andreia Valente acknowledges the COST action CM1302 (SIPs), the Investigator FCT2013 Initiative for the project IF/01302/2013 (acknowledging FCT, as well as POPH and FSE - European Social Fund) and the Royal Society of Chemistry's Research Fund. Pierre Falson and Elisabeta Comsa warmly acknowledge Thibault Andrieu from the cytometry plateau of SFR bioscience -UMS 3444- at Lyon-Gerland, France for assistance on CytoF. This work was also supported by the Marie Curie Initial Training Network: FP7-PEOPLE-2012-ITN proposal no 317297 - acronym GLYCOPHARM and PITN-GA-2012-317297. The high resolution mass spectrometer at CIRE-PAIB was financed (SMHART project no3069) by the European Regional Development Fund (ERDF), the Conseil Regional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm).

Rapeseed proteins are described to be poorly digestible in chickens. To further identify some molecular locks that may limit their use in poultry nutrition, we conducted a proteomic study on the various chicken digestive contents and proposed an integrative view of the proteins recruited in the crop, proventriculus/gizzard, duodenum, jejunum, and ileum for digestion of rapeseed by-products. Twenty-seven distinct rapeseed proteins were identified in the hydrosoluble fraction of the feed prior ingestion. The number of rapeseed proteins identified in digestive contents decreases throughout the digestion process while some are progressively solubilized in the most distal digestive segment, likely due to a combined effect of pH and activity of specific hydrolytic enzymes. Fifteen chicken proteins were identified in the hydrosoluble proventriculus/gizzard content, including chymotrypsin-like elastase and pepsin. Interestingly, on the 69 distinct proteins identified in duodenum, only 9 were proteolytic enzymes, whereas the others were associated with homeostasis, and carbohydrate, lipid, vitamin and hormone metabolisms. In contrast, chicken proteins identified in jejunal and ileal contents were mostly proteases and peptidases. The present work highlights the relevance of using integrative proteomics applied to the entire digestive tract to better appreciate the protein profile and functions of each digestive segment.

The interactions between oviductal fluid (OF) proteins and spermatozoa play major roles in sperm selection, storage and capacitation before fertilization. However, only a few sperm-interacting proteins in the OF has been identified and very little is known about the regulation of sperm-oviduct interactions across the estrous cycle. Samples of bovine frozen-thawed sperm from three bulls were incubated with OF at pre-, post-ovulatory stages (Pre-/Post-ov) or luteal phase (LP) of the estrous cycle (7 mg/mL proteins, treated groups) or with a protein-free media (control). The proteomes of sperm cells were assessed by nanoLC–MS/MS and quantified by label-free methods. A total of 27 sperm-interacting proteins originating in the OF were identified. Among those, 14 were detected at all stages, eight at Post-ov and LP and five only at LP. The sperm-interacting proteins detected at all stages or at LP and Post-ov were on average more abundant at LP than at other stages (P

International audience; In many amniotes, the amniotic fluid is depicted as a dynamic milieu that participates in the protection of the embryo (cushioning, hydration, and immunity). However, in birds, the protein profile of the amniotic fluid remains unexplored, even though its proteomic signature is predicted to differ compared with that of humans. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development because of the massive inflow of egg white proteins, which are thereafter swallowed by the embryo to support its growth. Using GeLC-MS/MS and shotgun strategies, we identified 91 nonredundant proteins delineating the chicken amniotic fluid proteome at day 11 of development, before egg white transfer. These proteins were essentially associated with the metabolism of nutrients, immune response and developmental processes. Forty-eight proteins were common to both chicken and human amniotic fluids, including serum albumin, apolipoprotein A1 and alpha-fetoprotein. We further investigated the effective role of chicken amniotic fluid in innate defense and revealed that it exhibits significant antibacterial activity at day 11 of development. This antibacterial potential is drastically enhanced after egg white transfer, presumably due to lysozyme, avian beta-defensin 11, vitelline membrane outer layer protein 1, and beta-microseminoprotein-like as the most likely antibacterial candidates. Interestingly, several proteins recovered in the chicken amniotic fluid prior and after egg white transfer are uniquely found in birds (ovalbumin and related proteins X and Y, avian beta-defensin 11) or oviparous species (vitellogenins 1 and 2, riboflavin-binding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo.

Although essential for artificial insemination (AI) and MOET (multiple ovulation and embryo transfer), oestrus synchronisation and superovulation are associated with increased female reproductive tract mucus production and altered sperm transport. The effects of such breeding practices on the ovine cervicovaginal (CV) mucus proteome have not been detailed. The aim of this study was to qualitatively and quantitatively investigate the Merino CV mucus proteome in naturally cycling (NAT) ewes at oestrus and mid-luteal phase, and quantitatively compare CV oestrus mucus proteomes of NAT, progesterone synchronised (P4) and superovulated (SOV) ewes. Quantitative analysis revealed 60 proteins were more abundant during oestrus and 127 were more abundant during the luteal phase, with 27 oestrus specific and 40 luteal specific proteins identified. The oestrus proteins most disparate in abundance compared to mid-luteal phase were ceruloplasmin (CP), chitinase-3-like protein 1 (CHI3L1), clusterin (CLU), alkaline phosphatase (ALPL) and mucin-16 (MUC16). Exogenous hormones greatly altered the proteome with 51 and 32 proteins more abundant and 98 and 53 proteins less abundant, in P4 and SOV mucus, respectively when compared to NAT mucus. Investigation of the impact of these proteomic changes on sperm motility and longevity within mucus may help improve sperm transport and fertility following cervical AI. Significance This manuscript is the first to detail the proteome of ovine cervicovaginal mucus using qualitative and quantitative proteomic methods over the oestrous cycle in naturally cycling ewes, and also after application of common oestrus synchronisation and superovulation practices. The investigation of the mucus proteome throughout both the follicular and luteal periods of the oestrous cycle, and also after oestrous synchronisation and superovulation provides information about the endocrine control and the effects that exogenous hormones have on protein expression in the female reproductive tract. This information contributes to the field by providing important information on the changes that occur to the cervicovaginal mucus proteome after use of exogenous hormones in controlled breeding programs, which are commonly used on farm and also in a research setting.

Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography-tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.

Polymorphonuclear neutrophils (PMNs) can contribute to the regulation of the host immune response by crosstalk with innate and adaptive leukocytes, including NK cells. Mechanisms by which this immunoregulation process occurs remain incompletely understood. Here, we focused on the effect of human neutrophil-derived serine proteases on NKp46, a crucial activating receptor expressed on NK cells. We used flow cytometry, Western blotting, and mass spectrometry (MS) analysis to reveal that cathepsin G [CG; and not elastase or proteinase 3 (PR3)] induces a time- and concentration-dependent, down-regulatory effect on NKp46 expression through a restricted proteolytic mechanism. We also used a functional assay to demonstrate that NKp46 cleavage by CG severely impairs NKp46-mediated responses of NK cells, including IFN-γ production and cell degranulation. Importantly, sputa of cystic fibrosis (CF) patients, which have high concentrations of CG, also alter NKp46 on NK cells. Hence, we have identified a new immunoregulatory mechanism of neutrophils that proteolytically disarms NK cell responses.