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Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface. A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined. SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region. These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.

Invasive bacterial pathogens can capture host plasminogen (Plg) and allow it to form plasmin. This process is of medical importance as surface-bound plasmin promotes bacterial spread by cleaving tissue components and favors immune evasion by degrading opsonins. In Staphylococcus aureus, Plg binding is in part mediated by cell surface fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here, we use single-cell and single-molecule techniques to demonstrate that FnBPs capture Plg by a sophisticated activation mechanism involving fibrinogen (Fg), another ligand found in the blood. We show that while FnBPs bind to Plg through weak (∼200-pN) molecular bonds, direct interaction of the adhesins with Fg through the high-affinity dock, lock, and latch mechanism dramatically increases the strength of the FnBP-Plg bond (up to ∼2,000 pN). Our results point to a new model in which the binding of Fg triggers major conformational changes in the FnBP protein, resulting in the buried Plg-binding domains being projected and exposed away from the cell surface, thereby promoting strong interactions with Plg. This study demonstrated a previously unidentified role for a ligand-binding interaction by a staphylococcal cell surface protein, i.e., changing the protein orientation to activate a cryptic biological function.IMPORTANCEStaphylococcus aureus captures human plasminogen (Plg) via cell wall fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here we show that the forces involved in the interaction between Plg and FnBPs on the S. aureus surface are weak. However, we discovered that binding of fibrinogen to FnBPs dramatically strengthens the FnBP-Plg bond, therefore revealing an unanticipated role for Fg in the capture of Plg by S. aureus These experiments favor a model where Fg-induced conformational changes in FnBPs promote their interaction with Plg. This work uncovers a previously undescribed activation me

In plants, members of the ATP-Binding Cassette (ABC) superfamily of proteins play a wide range of essential functions, including hormone transport or defense against (a)biotic stresses. Despite their physiological importance, very little information is available about their biochemical properties. Our laboratory previously demonstrated that Nicotiana tabacum NtPDR1, a full-size ABCG transporter is involved in constitutive defense against pathogens. In vivo experiments suggested that the protein could secrete cembrene and sclareol (toxic cyclic diterpenes) out of tobacco trichomes. Yet, direct evidence associating NtPDR1 with its putative substrates was lacking. Our project aimed at providing such data as well as enzymatic and structural information about NtPDR1. To do so, we first expressed the protein provided with a purification tag in N. tabacum Bright Yellow 2 (BY-2) suspension cells under the control of a strong constitutive transcriptional promoter. We then optimized the solubilization and purification of NtPDR1 and obtained purified enzyme with sufficient yields to perform biochemical studies. Purified NtPDR1 was monodispersed and monomeric as observed by gel filtration and electron microscopy followed by single particle analysis. In addition, purified NtPDR1 retained vanadate sensitive ATP hydrolytic activity that was stimulated by both sclareol and cembrene. The transporter was further reconstituted in asolectin liposomes. Upon reconstitution, NtPDR1 ATPase activity was increased from 21 to 38 nmol Pi.mg-1.min-1 and the reconstituted transporter was again stimulated by sclareol and cembrene. Interestingly, capsidiol, a sesquiterpene involved in induced defense against pathogens, was also found to stimulate NtPDR1 ATPase activity. In contrast, other terpenoids (geranylgeraniol and eucalyptol) did not stimulate the transporter. In conclusion, our work provides direct evidence of the interaction between a plant ABC transporter and its substrates. We demonstrat, (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2017

An ultrasensitive balance has been developed to weigh single or multiple cells, at high time and mass resolution — revealing fast and subtle mass fluctuations during the cell cycle and viral infection.

Studying the structure, properties, and interactions of microbial cells is key to understanding the functions of the microbiome. Recent advances in nanotechnology have offered new tools to probe microbes at the single-molecule and single-cell levels. In this issue of ACS Nano, Kumar et al. present an atomic force microscopy method that is capable of imaging the nanoscale organization of bacterial proteins in native, curved membranes. This study represents an important step forward in the development of nanoscopy techniques for analyzing biological systems with large curvature and vertical dimensions, such as membrane vesicles and bacterial cells.

Staphylococcus epidermidis and Staphylococcus aureus are two important nosocomial pathogens that form biofilms on indwelling medical devices. Biofilm infections are difficult to fight as cells within the biofilm show increased resistance to antibiotics. Our understanding of the molecular interactions driving bacterial adhesion, the first stage of biofilm formation, has long been hampered by the paucity of appropriate force-measuring techniques. In this minireview, we discuss how atomic force microscopy techniques have enabled to shed light on the molecular forces at play during staphylococcal adhesion. Specific highlights include the study of the binding mechanisms of adhesion molecules by means of single-molecule force spectroscopy, the measurement of the forces involved in whole cell interactions using single-cell force spectroscopy, and the probing of the nanobiophysical properties of living bacteria via multiparametric imaging. Collectively, these findings emphasize the notion that force and function are tightly connected in staphylococcal adhesion.

Live-cell nanoscopy has contributed significantly to assessing the inhibition of adhesion of uropathogenic Escherichia coli and Staphylococcus aureus by glycoconjugates and monoclonal antibodies, respectively, and of S. aureus surface attachment and cell-cell association by a synthetic peptide. This new technology shows promise for the development of antiadhesion therapies against bacterial pathogens.

Endocytosis is an essential cellular process required for uptake of nutrients from cell environment and turnover of plasma membrane components. Clathrin-mediated endocytosis is by far the best characterized endocytic process. Since the mid-90's, the existence of endocytic routes independent of clathrin emerged in mammalian cells. Therefore, the most challenging question in membrane biology rose: how can the plasma membrane be deformed in the absence of an organized clathrin coat? Until today, this question has not been fully solved. First attempts to shed light into this topic demonstrated the requirement of glycosphingolipids for membrane deformation in lectin-driven endocytosis (for instance, the endocytosis of the bacterial lectin Shiga toxin). Recently, BAR domain proteins (where BAR stands for Bin/Amphiphysin/Rvs) have been described as crucial for clathrin-independent endocytic routes. BAR domain proteins interact with membranes and act as curvature sensors/inducers. The function of many BAR domain proteins remains unclear in the landscape of clathrin-independent endocytosis, and especially regarding their role in the endocytic pit formation. The main part of my talk will be focused on the implication of a specific BAR domain protein, endophilin-A2, in the clathrin-independent endocytosis of Shiga toxin in mammalian cells (Renard et al. 2015. Nature, 517(7535):493-496). I will also say a few words about what I am doing now at the UCLouvain, and more particularly about the hypothesis according to which BAR domain proteins constitute a protein family that define cargo specificity and is able to deform plasma membrane in clathrin-independent endocytic processes.

Hox genes encode transcription factors which are well described for their implication during animal embryogenesis. In particular HOXA1 is involved in patterning the hindbrain and somitic derivatives as well as in inner ear and heart development. While the functions of HOXA1 are well known, the mode of action of this protein has been poorly characterised. In that context, a proteome-wide screening to identify proteins interacting with HOXA1 has been carried out in our laboratory and revealed OGT and PRDM14 as potential modulators of HOXA1 function. OGT is the only known mammalian enzyme able to add an O-GlcNAc group on target proteins. O-GlcNAcylation is involved in a variety of cellular processes from gene transcription to protein degradation. PRDM14 belongs to the PRDM family of proteins described to be histone methyltransferases. PRDM14 is involved in the maintenance of cell pluripotency and modulates the epigenetic landscape of target genes. Significantly to HOXA1 activity, both OGT and PRDM14 have also been described to be co-regulators of transcription. The goal of our thesis was therefore to characterize the modalities and the possible consequences of the interaction between HOXA1 and these two partners, OGT and PRDM14. At the molecular level, we showed that the N-terminal part of HOXA1 is required for its interaction with OGT while it is its homeodomain and a 11-His repeat motif which are necessary for its binding to PRDM14. On the other side, the TPR domain of OGT and the PR and zinc fingers domains of PRDM14 are required to interact with HOXA1. Next, we demonstrated that HOXA1 can be O-GlcNAcylated and phosphorylated and that the abundance of these modifications is dependent on the presence of OGT. However, the molecular consequences of HOXA1 O-GlcNAcylation still need to be identified. We also observed that PRDM14 can impact the transcriptional activity of HOXA1 and that this seems to be associated with a decrease of its stability. Finally, we overviewed w, (SC - Sciences) -- UCL, 2017

S'il est une problématique délicate et controversée, c'est bien celle des (r)empoissonnements ou repeuplements en poissons des lacs, rivières et ruisseaux. Pointés du doigt par les uns comme une pratique non durable voire néfaste visant à satisfaire le seul plaisir de quelques pêcheurs, et par les autres comme un moyen essentiel de soutien à la pêche ou à la restauration de nos populations piscicoles en difficulté démographique, les empoissonnements échappent souvent à une analyse rationnelle.

Watermelon (Citrullus lanatus) is one of the most economically important vegetable cucurbits. However, the genetic and taxonomic statuses of its closely related species remain scantly documented, limiting their full use for agronomic purposes. The genetic diversity and structure of 74 accessions covering 47 dessert type (C. lanatus subsp. vulgaris), 21 oilseed type (C. mucosospermus), and 6 citron melon (C. lanatus subsp. lanatus var citroides) collected from 36 countries throughout 4 continents, were analyzed using 18 polymorphic SSR markers. The mean values of proportion of polymorphic loci (P = 29.73), number of alleles per locus (A = 1.243), effective number of alleles per locus (Ae = 1.153) Shannon index (I = 0.191), observed and expected heterozygosities (Ho = 0.124; He = 0.149) confirmed the narrow genetic basis of C. lanatus. According to molecular variance analysis the most important component of the genetic variation was obtained among accessions (70%). On the contrary, lower genetic variation was noted among species (16%), countries (37%), and continents (14%). It is suggested that the cultivated forms of Citrullus spp. originated from or successive selection cycles aimed at few and/or common traits, in few ancestral populations. Clustering based on both Bayesian approach and an unweighted pair group method with arithmetic mean pointed out three groups of accessions corresponding to use types and collecting countries. Based on these results, future collecting missions could be focused mainly on representative ecological sites in Citrullus spp. distribution areas, and increasing the number of accessions and seeds per accession.

Game species like the red deer have been subjected to anthropogenic impacts for centuries. Translocations are often carried out-sometimes illegally-not only for sporting purposes, but also to increase trophy quality, reduce inbreeding, or mitigate bottlenecks after excessive persecution. Apart from the blurring of large-scale genetic structure, translocations without adequate quarantine measure risk introducing pathogens into potentially immunologically naïve populations. It is therefore important to understand the frequency of clandestine translocations. Identification of non-autochthonous animals and their potential origin is often difficult and, in red deer, has been hampered by the lack of large-scale genotypic datasets for comparison. In the present study, we make use of a recently published European-wide microsatellite dataset to detect and quantify the presence of non-autochthonous red deer in a large population sample (n = 1,780) from Central Europe (Belgium). Using factorial correspondence analysis, assignment tests and Bayesian clustering algorithms we arrive at an estimate of 3.7% non-autochthonous animals (or their descendants). Some of these animals were assigned to a nearby French population and may have immigrated into Belgium naturally, but the large majority must have been introduced by humans. Our analysis pointed to the British Isles and Germany/Poland as the potential origin of many introduced deer, regions known to have been source populations for translocations in Europe and beyond. We found evidence for recreational hunters using carcasses from farmed deer to fulfill mandatory hunting quotas. Our study is the first to quantify the extent of human-mediated introductions in a European game species at such a large scale with large and representative sample sizes.

For several decades now, an increasing phenomenon of enhanced honey bee colony mortality and morbidity is troubling beekeepers and naturalists. Such negative trends affect also wild pollinators and cast doubt about the sustainability of pollination both of wild plants and crops. These trends are also experienced in Belgium, country showing a level of annual colony losses above 18% in the best years. In addition, Walloon beekeepers describe for many years unspecific symptoms with their colonies like weaken colonies, queen losses, abnormal brood development or full death of the colony without any symptoms of disease. The aim of my work was to bring light in this situation, which revealed unexpected results. A prevalence case study carried out in the region of Wallonia by myself in collaboration with colleagues of the CRA-Wallonia, pointed at fungicides as the stressing factor linked to honey bee colony disorders. These pesticides considered harmless to bees, we started digging to understand the underlying reasons of our field observations. First we considered if they were just markers of intensive agriculture, not really having any toxicological effects on bees. Indeed, we found that colonies surrounded by conventionally-farmed crops were more likely to be contaminated by pesticides, in special fungicides. In addition, the colonies in these crop areas were more prone to show disorders. We also found that this contamination does not necessarily come from treated crops, but from wild plants or catch/cover crops in the surroundings or succeeding treated crops within a surface of at least 3 km radius. Then, I tested the direct toxicological effects fungicides on different colony individuals. I started by evaluating the effect of the most frequently found fungicide, boscalid, on be larvae, which did not show to induce increased mortality of immature bee stages. Then I studied the impact of chronic exposure of adult bees. Finally I suggest further research lines to better u, (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2017

ABC transporters mediate ATP-dependent translocation of a wide variety of structurally and functionally unrelated compounds across biological membranes. In plants, they were shown to be involved in many different physiological processes, such as pathogen resistance, heavy metal detoxification or hormone transport. This work aimed at the functional characterization of two plant ABC transporters, NtPDR3 and PhABCG1. NtPDR3 was identified in our laboratory as an ABC transporter expressed at the plasma membrane of Nicotiana tabacum root epidermal cells under conditions of iron deficiency. The characterization of transgenic plants silenced for NtPDR3 expression revealed that these plants were less tolerant to iron-deficient conditions than wild-type plants, displaying stronger chlorosis and growth retardation when not supplied with iron. However, identification of NtPDR3 substrate(s) is required to fully understand its physiological role. For this purpose, a metabolomic analysis of roots and root exudates was undertaken. This approach identified several O-methylated coumarins as putative substrates of the transporter. Interestingly, these metabolites are known to be abundant in iron-deficient exudates of other plant species, and some of them have the ability to mobilize iron in vitro. Flavins were also found to be secreted by N. tabacum roots upon iron depletion, but NtPDR3 does not seem to be involved in their export from the roots. To confirm O-methylated coumarins are indeed substrates of NtPDR3, over-expression of the transporter in N. tabacum BY-2 suspension cells was achieved and direct transport tests were performed using intact cells as well as microsomal vesicles. No transport of coumarins was observed. However, transport assays with cells indicated that glycosylated coumarins might be substrates of NtPDR3. On the other hand, PhABCG1 was suggested to be involved in the active emis-sion of volatile organic compounds (VOCs) from Petunia hybrida flowers. Heterolo-g, (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2017

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro–produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid–based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

Fasting animals must ration stored reserves appropriately for metabolic demands. Animals that experience fasting concomitant with other metabolically demanding activities are presented with conflicting demands of energy conservation and expenditure. Our objective was to understand how fasting northern elephant seals regulate the mobilization of lipid reserves and subsequently milk lipid content during lactation. We sampled 36 females early and 39 at the end of lactation. To determine the separate influences of lactation from fasting, we also sampled fasting but non-lactating females early and late (8 and 6 seals, respectively) in their molting fasting period. Mass and adiposity were measured, as well as circulating non-esterified fatty acid (NEFA), triacylglycerol (TAG), cortisol, insulin and growth hormone levels. Milk was collected from lactating females. Milk lipid content increased from 31% in early to 51% in late lactation. In lactating females plasma NEFA was positively related to cortisol and negatively related to insulin, but in molting seals, only variation in cortisol was related to NEFA. Milk lipid content varied with mass, adiposity, NEFA, TAG, cortisol and insulin. Surprisingly, growth hormone concentration was not related to lipid metabolites or milk lipid. Suppression of insulin release appears to be the differential regulator of lipolysis in lactating versus molting seals, facilitating mobilization of stored lipids and maintenance of high NEFA concentrations for milk synthesis. Milk lipid was strongly impacted by the supply of substrate to the mammary gland, indicating regulation at the level of mobilization of lipid reserves

Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).

Plum and cabbage are rich in carotenoids and polyphenols. However, their bioactivity depends on their release and intestinal uptake. Four varieties of Brassicaceae (Duchy, Scots Kale, Kale, Kalorama) and Prunus (Cherry Plum, Plum 620, Ersinger, Italian Plum) were studied; bioaccessibility following in vitro digestion, cellular uptake (Caco-2 vs. co-culture cell model: Caco-2:HT-29-MTX (90:10%) and colonic fermentation were determined for carotenoids/polyphenols; the influence of certain kitchen preparations was likewise studied. Carotenoids were non-significantly influenced by the latter, while for polyphenols, boiling and steaming significantly reduced total phenolics (p<0.05). Carotenoid bioaccessibility did not differ significantly between Prunus vs. Brassicaceae varieties, but xanthophyll was higher than carotene bioaccessibility (p<0.01). Polyphenol bioaccessibility was low (<10%), possibly compromised by the cream containing test meal. Total carotenoid cellular uptake varied between varieties (0.3-4.1%), being higher for carotenes (4.1%) than for xanthophylls (1.6%, p<0.01), and were higher for the co-culture cell model compared to Caco-2 cells (p<0.01). Total carotenoid recovery in the colonic fraction varied from 4% to 25%. Lower bioaccessibility of carotenes thus appeared to be somewhat counterbalanced by higher cellular uptake. The potential positive role of the mucus layer for cellular uptake and the fate of the colonic digesta deserve further attention in the future.

Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall-anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine-aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single-cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen-coated substrates through both weak and strong bonds. Single-molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand-binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well-characterized ‘dock, lock and latch’ mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins

Inflammation is a pathophysiological response to infection or tissue damage during which high levels of reactive oxygen and nitrogen species are produced by phagocytes to kill microorganisms. Reactive oxygen and nitrogen species serve also in the complex regulation of inflammatory processes. Recently, it has been proposed that peroxiredoxins may play key roles in innate immunity and inflammation. Indeed, peroxiredoxins are evolutionarily conserved peroxidases able to reduce, with high rate constants, hydrogen peroxide, alkyl hydroperoxides and peroxynitrite which are generated during inflammation. In this minireview, we point out different possible roles of peroxiredoxins during inflammatory processes such as cytoprotective enzymes against oxidative stress, modulators of redox signaling, and extracellular pathogen- or damage-associated molecular patterns. A better understanding of peroxiredoxin functions in inflammation could lead to the discovery of new therapeutic targets.

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Green tea, oolong tea and black tea were separately introduced to brew three kinds of tea beers. A model was designed to investigate the tea beer flavour character. Comparison of the volatiles between the sample of tea beer plus water mixture (TBW) and the sample of combination of tea infusion and normal beer (CTB) was accomplished by triangular sensory test and HS-SPME GC–MS analysis. The PCA of GC–MS data not only showed a significant difference between volatile features of each TBW and CTB group, but also suggested some key compounds to distinguish TBW from CTB. The results of GC–MS showed that the relative concentrations of many typical tea volatiles were significantly changed after the brewing process. More interestingly, the behaviour of yeast fermentation was influenced by tea components. A potential interaction between tea components and lager yeast could be suggested.

Among the specialized (secondary) plant metabolites, terpenoids represent the most diverse family and are often involved in the defense against pathogens and herbivores. Terpenoids can be produced both constitutively and in response to the environment. At the front line of this defense strategy are the glandular trichomes, which are organs dedicated primarily to the production of specialized metabolites. Mass spectrometry-based proteomics is a powerful tool, which is very useful to investigate enzymes involved in metabolic pathways, such as the synthesis and secretion of terpenoids in glandular trichomes. Here we review the strategies used to investigate the specific roles of these particular organs from non-model plant species, mainly belonging to the Lamiaceae, Solanaceae, and Cannabaceae families. We discuss how proteomics helps to accurately pinpoint candidate proteins to be functionally characterized, and how technological progresses create opportunities for studying low-abundance proteins, such as the ones related to the synthesis and transport of specialized metabolites. This article is part of a Special Issue entitled: Plant Proteomics - a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Yeast cells can grow on a wide variety of nitrogen sources, and, according to the quality of the source, modulate their set of nitrogen transporters present at the plasma membrane. The addition of a preferred nitrogen source to proline-grown cells induces the transcriptional repression of genes encoding transporters of non-preferred nitrogen sources. This gene regulation is called Nitrogen Catabolite Repression (NCR) and many transcriptomic studies were carried out to identify NCR-sensitive genes. Other regulations are possible at the post-translational level. For instance, the general amino acid permease Gap1 is rapidly ubiquitylated, endocytosed and degraded in the vacuole in an Rsp5-Bul1/2-dependent manner. However, little is known about the nitrogen-induced post-translational regulation of other plasma membrane transporters. In this study, we used a large-scale proteomic approach to identify plasma membrane proteins that are regulated by the nitrogen source. We found seventeen plasma membrane proteins more abundant when grown in proline-containing medium, and four proteins more abundant when grown in the presence of ammonium. Using the same global proteomic approach, we showed that five NCR-sensitive nitrogen transporters—Gap1, Put4, Opt2, Dal5 and Ptr2—are endocytosed and degraded after addition of a preferred nitrogen source to proline-grown cells. Although the degradation kinetics is different for each transporter, it is Rsp5-dependent for at least four of them—Gap1, Put4, Dal5 and Ptr2. Surprisingly, we found that the deletion of the Bul1 and Bul2 ubiquitin ligase adaptors increases vacuolar trafficking and degradation of Put4, Dal5 and Ptr2 in proline-grown cells. The exact role of Bul1 and Bul2 in this process remains to be determined. To conclude, we can argue that mass spectrometry is a technique of choice to monitor the nitrogen-induced remodeling of the plasma membrane proteome and to approach the study of endocytic mechanisms., (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2016

Calcium signaling depends on a tightly regulated set of pumps, exchangers, and channels that are responsible for controlling calcium fluxes between the different subcellular compartments of the eukaryotic cell. It have been recently reported in our lab that two members of the highly-conserved UPF0016 family, human TMEM165 and budding yeast Gdt1p, are functionally related and form a new group of Golgi-localized cation/Ca2+ exchangers. Both share topological characteristics reminiscent of the cation/calcium exchanger superfamily. They are composed of two hydrophobic regions containing a highly conserved motifs separated by a large cytosolic loop. Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. The Ca2+ transport activity of Gdt1p was demonstrated using an assay based on the heterologous expression of GDT1 in the bacterium Lactococcus lactis. The Ca2+ uptake activity in cell expressing GDT1 was dependent on the external pH, indicating that Gdt1p may act as a Ca2+/H+ antiporter. This work focuses on the functional and structural study of Gdt1p in the yeast Saccharomyces cerevisiae. We firstly found that Gdt1p controls cellular calcium content and plays a major role in the calcium response following an osmotic shock when the Golgi calcium pump, Pmr1p, is absent. Importantly, we also discovered that, in the presence of a high concentration of external Ca2+, Gdt1p is required for glycosylation of carboxypeptidase Y and the glucanosyltransferase Gas1p. Additionally, we highlighted that Gdt1p levels are controlled by external Ca2+ via a mechanism involving the calcineurin signaling pathway along with vacuolar proteolysis. Finally, by site-directed mutagenesis and characterization of the mutants, we found that the cations binding pocket of Gdt1p is most likely composed of glutamate, aspartate, serine and threonine residues located in the repeated motifs., (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2016

We analysed more than 600 red deer (Cervus elaphus) from large parts of its European distribution range at 13 microsatellite loci, presenting the first continent-wide study of this species using nuclear markers. Populations were clearly differentiated (overall FST = 0.166, Jost's Dest = 0.385), and the BAPS clustering algorithm yielded mainly geographically limited and adjacent genetic units. When forced into only three genetic clusters our data set produced a very similar geographic pattern as previously found in mtDNA phylogeographic studies: a western group from Iberia to central and parts of Eastern Europe, an eastern group from the Balkans to Eastern Europe and a third group including the threatened relict populations from Sardinia and Mesola in Italy. This result was also confirmed by a multivariate approach to analysing our data set, a discriminant analysis of principal components (DAPC). Calculations of genetic diversity and effective population sizes (linkage-disequilibrium approach) yielded the lowest results for Italian (Sardinia, Mesola; Ne between two and eight) and Scandinavian red deer, in line with known bottlenecks in these populations. Our study is the first to present comparative nuclear genetic data in red deer across Europe and may serve as a baseline for future analyses of genetic diversity and structuring in this widespread ungulate.

Hydrostatic pressure (HP) increases by about 1 atmosphere (0.1 MPa) for each ten-meter depth increasein the water column. This thermodynamical parameter could well influence the response to and effects ofxenobiotics in the deep-sea biota, but this possibility remains largely overlooked. To grasp the extent of HPadaptation in deep-sea fish, comparative studies with living cells of surface species exposed to chemicalsat high HP are required. We initially conducted experiments with precision-cut liver slices of a deep-sea fish (Coryphaenoides rupestris), co-exposed for 15 h to the aryl hydrocarbon receptor (AhR) agonist3-methylcholanthrene at HP levels representative of the surface (0.1 MPa) and deep-sea (5–15 MPa; i.e.,500–1500 m depth) environments. The transcript levels of a suite of stress-responsive genes, such asthe AhR battery CYP1A, were subsequently measured (Lemaire et al., 2012; Environ. Sci. Technol. 46,10310–10316). Strikingly, the AhR agonist-mediated increase of CYP1A mRNA content was pressure-dependently reduced in C. rupestris. Here, the same co-exposure scenario was applied for 6 or 15 h toliver slices of a surface fish, Dicentrarchus labrax, a coastal species presumably not adapted to high HP.Precision-cut liver slices of D. labrax were also used in 1 h co-exposure studies with the pro-oxidant tert-butylhydroperoxide (tBHP) as to investigate the pressure-dependence of the oxidative stress response(i.e., reactive oxygen production, glutathione and lipid peroxidation status). Liver cells remained viable inall experiments (adenosine triphosphate content). High HP precluded the AhR agonist-mediated increaseof CYP1A mRNA expression in D. labrax, as well as that of glutathione peroxidase, and significantly reducedthat of heat shock protein 70. High HP (1 h) also tended per se to increase the level of oxidative stress inliver cells of the surface fish. Trends to an increased resistance to tBHP were also noted. Whether thelatter observation truly reflects a

Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

A collaborative consortium, named "TRANSAUTOPHAGY," has been created among European research groups, comprising more than 150 scientists from 21 countries studying diverse branches of basic and translational autophagy. The consortium was approved in the framework of the Horizon 2020 Program in November 2015 as a COST Action of the European Union (COST means: CO-operation in Science and Technology), and will be sponsored for 4 years. TRANSAUTOPHAGY will form an interdisciplinary platform for basic and translational researchers, enterprises and stakeholders of diverse disciplines (including nanotechnology, bioinformatics, physics, chemistry, biology and various medical disciplines). TRANSAUTOPHAGY will establish 5 different thematic working groups, formulated to cooperate in research projects, share ideas, and results through workshops, meetings and short term exchanges of personnel (among other initiatives). TRANSAUTOPHAGY aims to generate breakthrough multidisciplinary knowledge about autophagy regulation, and to boost translation of this knowledge into biomedical and biotechnological applications.

Mapping of the microbial molecules underlying microbiota-host interactions is key to understand how microbiota preserve mucosal homeostasis. A pivotal family of such bacterial molecules are pili. Pili are proteinaceous cell wall appendages with a well-documented role in adhesion, whilst their role in immune interaction with the host is less established. Gram-positive pili are often posttranslationally modified by sortase-specific cleavage reactions and the formation of intramolecular peptide bonds. Here we report glycosylation as a new level of posttranslational modification of sortase-dependent pili of a beneficial microbiota species and its role in immune modulation. We focused on the SpaCBA pili of the model probiotic and beneficial human gut microbiota isolate Lactobacillus rhamnosus GG. A unique combination of molecular techniques, nanoscale mechanical and immunological approaches led to the identification of mannose and fucose residues on the SpaCBA pili. These glycans on the pili are recognized by human dendritic cells via the C-type lectin receptor DC-SIGN, a key carbohydrate-dependent immune tailoring pattern recognition receptor. This specific lectin-sugar interaction is moreover of functional importance and modulated the cytokine response of dendritic cells. This provides insight into the direct role bacterial glycoproteins can play in the immunomodulation of the host. Modification of the complex heterotrimeric pili of a model probiotic and microbiota isolate with mannose and fucose is of importance for the functional interaction with the host immune lectin receptor DC-SIGN on human dendritic cells. Our findings shed light on the yet underappreciated role of glycoconjugates in bacteria-host interactions

ATP-Binding Cassette (ABC) transporters are ubiquitous membrane proteins found in all living organisms, from bacteria to higher eukaryotes. They are involved in a wide variety of biological processes such as nutrients uptake, macromolecules export or ions transport. Some ABC transporters, particularly those involved in human diseases, have been well studied since decades. However, less is known about plant ABC transporters. Our laboratory is focused on one of the three subfamilies of full-size ABC transporters found in plants: ABCG or pleiotropic drug resistance (PDR) transporters. PDRs have been shown to be involved in many physiological processes at the whole plant level. Nevertheless, their biochemical properties have been barely characterized. In this work, we aimed to characterize two PDR transporters, MpPDR1 from Mentha x piperita and NpPDR5 from Nicotiana plumbaginifolia at the biochemical level. We sought to produce and purify both transporters, and then identify their enzymatic and structural properties. Nicotiana tabacum BY-2 suspension cells combined with a strong transcription promoter were used as an expression system. Both PDR genes provided with a double purification tag sequence (10-His/StrepII) were successfully expressed in N. tabacum BY-2 cells and solubilized from a microsomal fraction with dodecyl maltoside. Unlike MpPDR1, NpPDR5 was purified to homogeneity at a concentration (15 mg/ml) suitable for structural analysis. Exclusion chromatography and single particle analysis by electron microscopy showed that purified NpPDR5 is monodisperse and monomeric. At pH 7.5, NpPDR5 displayed an ATPase activity of 27.0 nmol.min-1.mg-1, which was increased up to 0.25 µmol.mg-1.min-1 in the presence of 0.1 % (w/v) asolectin and 0.1 % (w/v) dodecyl maltoside. This activity was inhibited by vanadate, miconazole, and verapamil. In conclusion, N. tabacum BY-2 suspension cells were shown to be a convenient expression system for PDR transporters. Moreover, the firs, (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2016

Characterization of the molecular interactions between microbial cells and the human skin is essential to understand the functions of the skin microbiome, and to gain insight into the molecular basis of skin disorders. Although various molecular approaches have been used to studymicrobe–skin interactions, the underlying molecular forces were not accessible to study. Here we present a novel atomic force microscopy approach to localize and quantify the nanoscale interaction forces between the bacterial pathogen Staphylococcus aureus and human skin. A method combining nanoscale multiparametric imaging with single bacterial probes is developed to map simultaneously the topography and bacterial-binding properties of corneocytes at high spatiotemporal resolution. Further quantification of the forces between bacteria and corneocytes is achieved using single-cell force spectroscopy. The results show that the S. aureus–skin adhesion is strong (B500 pN) and originates frommultiple specific bonds between adhesins on the bacterial cell surface and target ligands on the corneocyte surface. Applicable to a wide variety of microbes and skin cells, our methodology offers exciting prospects for understanding the molecular details of skin colonization and infection.

NAN

The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of thediet, affects various cellular properties such as membrane structure and fluidity, energy metabolism andsusceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellularresponse to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicityinduced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has animpact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium.Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enrichingthe growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3),docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docos-apentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were thenchallenged for 24 h with increasing concentrations of methylmercury or cadmium. We observed that (i)the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA con-tent of the growth medium: major modifications were a high incorporation of the supplemented PUFA inthe cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellu-lar phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturatedfatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly protected theRTL-W1 cells against both methylmercury and cadmium; (iv) DHA enrichment significantly protectedthe cells against cadmium but not methylmercury; (v) AA and LA enrichment had no impact on the celltolerance to both methylmercury and cadmium; (vi) the abundance of 20:3n-6, a metabolite of the n-6 biotransformation pathway, in phospholipids was negati

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation ofCWrigidity.Ourresults indicate that conversion of L-aspartate (L-Asp) to N-carbamoyl-L-aspartate by PyrB may reduce the amount of L-Asp available forPGsynthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity.Wehypothesize that the concurrent utilization of L-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that L-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells themeansto ensure optimalCWplasticity without theneedto control the expression of PG synthesis genes.

Staphylococcus aureus and Staphylococcus epidermidis are two nosocomial pathogens. They are the leading cause of biofilm-associated infections in healthcare facilities. Understanding the molecular interactions lying behind the biofilm formation is essential for preventing nosocomial infections. The objective of this PhD thesis is to gain insights into the forces involved in staphylococcal adhesion, the first step of biofilm formation. The strategy involved the development and use of atomic force microscopy (AFM) techniques for characterizing adhesion forces at the single-molecule and single-cell levels. We investigated two serine-aspartate repeat proteins, SdrG and SdrF involved in fibrinogen (Fg) and collagen (Cn) binding respectively. The SdrG-Fg interaction involves strong forces and low dissociation rates leading to stable complexes. SdrG forms nanodomains at the cell surface highlighting how this bacterium might withstand shear forces on indwelling medical devices. Our next study shows that SdrF binds Cn through two types of molecular bonds involving two subdomains of the protein. Next, we studied the S. aureus fibronectin-binding protein A (FnBPA), which mediates cell-cell interactions during biofilm accumulation. FnBPA mediates low affinity, Zn-dependent homophilic bonds of moderate strength. This might be the basis for cell dissemination and participate to the biofilm dynamics. Lastly, we examined the forces at play in the interaction between S. epidermidis and Candida albicans. We highlighted the importance of the yeast-to-hyphae transition and the crucial role of Als adhesins and O-mannosylations in co-adhesion. This thesis sheds new light into the molecular forces at play during staphylococcal adhesion. In the future, AFM should contribute to the development of novel anti-adhesion therapies., (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2016

Differences between male and female preimplantation bovine embryos have been reported and concern numerous aspects as kinetics of development, cell number or metabolism. These differences are less or more obvious depending on the culture medium embryos are grown in and seem related to the expression of genes on both X chromosomes in female embryos at the blastocyst stage. This transcriptional sexual dimorphism can result in functional consequences and contribute to the differential ability of each sex to cope with its environment. This may in turn alter the sex ratio if embryo survival varies between sexes. We here evaluated developmental and apoptotic rates of in vitro produced male and female bovine embryos in two culture media, one containing fetal calf serum (FCS) and one containing bovine serum albumin (BSA). The levels of apoptosis were assessed in blastocysts collected 7 days after insemination (Day 7 pi) using simultaneously two staining techniques on the same embryos. Analysis by confocal microscopy and subsequent 3D reconstruction allowed a precise cell count and localization of apoptotic cells. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition, but with a more pronounced difference in FCS medium. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm and was more important in embryos of small size grown in presence of FCS. However, the higher level of apoptosis among female blastocysts at Day 7 pi did not result in a preferential degeneration of female embryos one day later since no shift in sex ratio was observed at Day 8 pi. In conclusion, female embryos are more prone to apoptosis than male ones. Moreover, culture conditions and embryonic size seem to affect the difference between sexes., (SC - Sciences) -- UCL, 2016

This work focuses on the physico-chemical characterization of kernels and oil of Terminalia catappa L. from Benin. The detailed physico-chemical properties are given including the nutritional composition and fatty acid profile, but also the phenolic content, phytochemical screening and antioxidant capacity which were determined for the first time. The kernel (100 g) contained 5.5 g of moisture, a high level of lipids (64.7–140.4 of Recommended Daily Intake (RDI)), proteins (36.0% RDI), sugars (6.0% RDI), and tannins (0.6%). The defatted kernels (100 g) contained high levels of manganese (184.8–236.1% RDI), magnesium (173.6–235.2% RDI), iron (89.7–201.9% RDI), zinc (87.9–120.9% RDI) and calcium (41.5% RDI), and contributed for 98.6% of RDI proteins. The kernel oil showed a high level of unsaturated fatty acids including oleic (27.1%) and linoleic acids (26.6%) and saturated fatty acids such as palmitic acid (40.0%) as well as several phytosterols and triterpenes. These kernels and their unsaturated oil are of interesting nutritional value but could also be used as a biofuel or lubricant. The presence of phenolic and terpenic derivatives may also explain at least in part their use in traditional medicine., [Composition chimique et propriétés nutritionnelles des amandes et huile de fruits de Terminalia catappa L. du Bénin] La caractérisation physico-chimique de l’huile et des amandes de fruits de Terminalia catappa du Bénin a été réalisée. Les propriétés physico-chimiques ont été analysées, notamment la composition nutritionnelle et le profil en acides gras, mais aussi la teneur en polyphénols, le criblage phytochimique et l’activité antioxydante, qui ont été déterminées pour la première fois. Les analyses réalisées montrent que 100 g d'amandes contiennent 5,5 g d'humidité, un taux élevé de lipides (contribuant pour 63,7 à 140,4% de la dose journalière recommandée (DJR)), de protéines (36,0% de DJR), de sucres (6,0% DJR) et de tanins (0,6%). L’amande délipidée contient des taux élevés de manganese (184,8–236,1% DJR), de magnésium (173,6-235,2% DJR), de fer (89,7-201,8% DJR), zinc (87.9–120.9% DJR) et calcium (41.5% DJR), et contribue pour 98,8% de la DJR en protéines. L’huile est riche en acides gras insaturés, tels que l'acide oléique (27,1%) et l'acide linoléique (26,6%), et en un acide gras saturé, l'acide palmitique (40,0%), et contient aussi des phytostérols et triterpènes. Ces amandes et cette huile de type insaturé ont une valeur nutritionnelle intéressante et peuvent aussi être utilisées comme biocarburants ou lubrifiants pour moteurs. La présence de composés phénoliques et terpéniques peut aussi expliquer, au moins en partie, leur usage en médecine traditionnelle.

Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.

In previous studies, we demonstrated that ammonium nutrition enhances the drought tolerance of rice seedlings compared to nitrate nutrition and contributes to a higher root water uptake ability. It remains unclear why rice seedlings maintain a higher water uptake ability when supplied with ammonium under drought stress. Here, we focused on the effects of nitrogen form and drought stress on root abscisic acid (ABA) concentration and aquaporin expression using hydroponics experiments and stimulating drought stress with 10% PEG6000. Drought stress decreased the leaf photosynthetic rate and stomatal conductivity and increased the leaf temperature of plants supplied with either ammonium or nitrate, but especially under nitrate supply. After 4 h of PEG treatment, the root protoplast water permeability and the expression of root PIP and TIP genes decreased in plants supplied with ammonium or nitrate. After 24 h of PEG treatment, the root hydraulic conductivity, the protoplast water permeability, and the expression of some aquaporin genes increased in plants supplied with ammonium compared to those under non-PEG treatment. Root ABA accumulation was induced by 24 h of PEG treatment, especially in plants supplied with ammonium. The addition of exogenous ABA decreased the expression of PIP and TIP genes under non-PEG treatment but increased the expression of some of them under PEG treatment. We concluded that drought stress induced a down-regulation of aquaporin expression, which appeared earlier than did root ABA accumulation. With continued drought stress, aquaporin expression and activity increased due to root ABA accumulation in plants supplied with ammonium.