Critical role of Aquaporin-1 and telocytes in infantile hemangioma response to propranolol beta blockade.

Propranolol, a nonselective ß-adrenergic receptor (ADRB) antagonist, is the first-line therapy for severe infantile hemangiomas (IH). Since the incidental discovery of propranolol efficacy in IH, preclinical and clinical investigations have shown evidence of adjuvant propranolol response in some malignant tumors. However, the mechanism for propranolol antitumor effect is still largely unknown, owing to the absence of a tumor model responsive to propranolol at nontoxic concentrations. Immunodeficient mice engrafted with different human tumor cell lines were treated with anti-VEGF bevacizumab to create a model sensitive to propranolol. Proteomics analysis was used to reveal propranolol-mediated protein alteration correlating with tumor growth inhibition, and Aquaporin-1 (AQP1), a water channel modulated in tumor cell migration and invasion, was identified. IH tissues and cells were then functionally investigated. Our functional protein association networks analysis and knockdown of ADRB2 and AQP1 indicated that propranolol treatment and AQP1 down-regulation trigger the same pathway, suggesting that AQP1 is a major driver of beta-blocker antitumor response. Examining AQP1 in human hemangioma samples, we found it exclusively in a perivascular layer, so far unrecognized in IH, made of telocytes (TCs). Functional in vitro studies showed that AQP1-positive TCs play a critical role in IH response to propranolol and that modulation of AQP1 in IH-TC by propranolol or shAQP1 decreases capillary-like tube formation in a Matrigel-based angiogenesis assay. We conclude that IH sensitivity to propranolol may rely, at least in part, on a cross talk between lesional vascular cells and stromal TCs.

A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum.

Goldenhar syndrome or oculo-auriculo-vertebral spectrum (OAVS) is a complex developmental disorder characterized by asymmetric ear anomalies, hemifacial microsomia, ocular and vertebral defects. We aimed at identifying and characterizing a new gene associated with OAVS. Two affected brothers with OAVS were analyzed by exome sequencing that revealed a missense variant (p.(Asn358Ser)) in the EYA3 gene. EYA3 screening was then performed in 122 OAVS patients that identified the same variant in one individual from an unrelated family. Segregation assessment in both families showed incomplete penetrance and variable expressivity. We investigated this variant in cellular models to determine its pathogenicity and demonstrated an increased half-life of the mutated protein without impact on its ability to dephosphorylate H2AFX following DNA repair pathway induction. Proteomics performed on this cellular model revealed four significantly predicted upstream regulators which are PPARGC1B, YAP1, NFE2L2 and MYC. Moreover, eya3 knocked-down zebrafish embryos developed specific craniofacial abnormalities corroborating previous animal models and supporting its involvement in the OAVS. Additionally, EYA3 gene expression was deregulated in vitro by retinoic acid exposure. EYA3 is the second recurrent gene identified to be associated with OAVS. Moreover, based on protein interactions and related diseases, we suggest the DNA repair as a key molecular pathway involved in craniofacial development.

Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F.

The basic understanding of the biological effects of eukaryotic translation initiation factors (EIFs) remains incomplete, notably for their roles independent of protein translation. Different EIFs exhibit nuclear localization and DNA-related functions have been proposed, but the understanding of EIFs novel functions beyond protein translation lacks of integrative analyses between the genomic and the proteomic levels. Here, the noncanonical function of EIF3F was studied in human lung adenocarcinoma by combining methods that revealed both the protein-protein and the protein-DNA interactions of this factor. We discovered that EIF3F promotes cell metastasis in vivo. The underpinning molecular mechanisms involved the regulation of a cluster of 34 metastasis-promoting genes including Snail2, as revealed by proteomics combined with immuno-affinity purification of EIF3F and ChIP-seq/Q-PCR analyses. The interaction between EIF3F and signal transducer and activator of transcription 3 (STAT3) controlled the EIF3F-mediated increase in Snail2 expression and cellular invasion, which were specifically abrogated using the STAT3 inhibitor Nifuroxazide or knockdown approaches. Furthermore, EIF3F overexpression reprogrammed energy metabolism through the activation of AMP-activated protein kinase and the stimulation of oxidative phosphorylation. Our findings demonstrate the role of EIF3F in the molecular control of cell migration, invasion, bioenergetics, and metastasis. The discovery of a role for EIF3F-STAT3 interaction in the genetic control of cell migration and metastasis in human lung adenocarcinoma could lead to the development of diagnosis and therapeutic strategies.

Proteomic and metabolomic profiling underlines the stage- and time-dependent effects of high temperature on grape berry metabolism.

Climate change scenarios predict an increase in mean air temperatures and in the frequency, intensity, and length of extreme temperature events in many wine-growing regions worldwide. Because elevated temperature has detrimental effects on berry growth and composition, it threatens the economic and environmental sustainability of wine production. Using Cabernet Sauvignon fruit-bearing cuttings, we investigated the effects of high temperature (HT) on grapevine berries through a label-free shotgun proteomic analysis coupled to a complementary metabolomic study. Among the 2,279 proteins identified, 592 differentially abundant proteins were found in berries exposed to HT. The gene ontology categories "stress," "protein," "secondary metabolism," and "cell wall" were predominantly altered under HT. High temperatures strongly impaired carbohydrate and energy metabolism, and the effects depended on the stage of development and duration of treatment. Transcript amounts correlated poorly with protein expression levels in HT berries, highlighting the value of proteomic studies in the context of heat stress. Furthermore, this work reveals that HT alters key proteins driving berry development and ripening. Finally, we provide a list of differentially abundant proteins that can be considered as potential markers for developing or selecting grape varieties that are better adapted to warmer climates or extreme heat waves.

Normalization of hippocampal retinoic acid level corrects age-related memory deficits in rats.

Dietary micronutrients constitute a major environmental factor influencing aging processes. Vitamin A (vit. A) is the precursor of retinoic acid, a bioactive molecule that controls the expression of several genes involved in brain function. Evidence suggests a reduction of vit. A bioavailability with aging, but its impact on neuronal network is poorly understood. We investigated the mechanisms linking memory impairments with specific alterations of retinoic acid metabolism in the hippocampus. We compared young (10 weeks) and aged (16 months) rats, supplemented or not with dietary vit. A (20 IU retinol/g) for 4 weeks. Our study reveals that aging induced dysregulation of gene expression involved in vit. A and retinoic acid metabolism in the liver. Furthermore, vit. A supplementation restored the integrity of the hippocampal neuronal morphology altered by aging. Importantly, we found a high correlation between hippocampal levels of retinoic acid and memory performance. The present work establishes the link between collapse of retinoid metabolism and age-related cognitive decline, highlighting the role of vit. A in maintaining memory through aging.

Metabolomics and proteomics identify the toxic form and the associated cellular binding targets of the anti-proliferative drug AICAR.

5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-ß Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.

Prenatal retinoic acid exposure reveals candidate genes for craniofacial disorders.

Syndromes that display craniofacial anomalies comprise a major class of birth defects. Both genetic and environmental factors, including prenatal retinoic acid (RA) exposure, have been associated with these syndromes. While next generation sequencing has allowed the discovery of new genes implicated in these syndromes, some are still poorly characterized such as Oculo-Auriculo-Vertebral Spectrum (OAVS). Due to the lack of clear diagnosis for patients, developing new strategies to identify novel genes involved in these syndromes is warranted. Thus, our study aimed to explore the link between genetic and environmental factors. Owing to a similar phenotype of OAVS reported after gestational RA exposures in humans and animals, we explored RA targets in a craniofacial developmental context to reveal new candidate genes for these related disorders. Using a proteomics approach, we detected 553 dysregulated proteins in the head region of mouse embryos following their exposure to prenatal RA treatment. This novel proteomic approach implicates changes in proteins that are critical for cell survival/apoptosis and cellular metabolism which could ultimately lead to the observed phenotype. We also identified potential molecular links between three major environmental factors known to contribute to craniofacial defects including maternal diabetes, prenatal hypoxia and RA exposure. Understanding these links could help reveal common key pathogenic mechanisms leading to craniofacial disorders. Using both in vitro and in vivo approaches, this work identified two new RA targets, Gnai3 and Eftud2, proteins known to be involved in craniofacial disorders, highlighting the power of this proteomic approach to uncover new genes whose dysregulation leads to craniofacial defects.

Characterization of a Cell-Assembled extracellular Matrix and the effect of the devitalization process.

We have previously shown that the Cell-Assembled extracellular Matrix (CAM) synthesized by normal, human, skin fibroblasts in vitro can be assembled in a completely biological vascular graft that was successfully tested in the clinic. The goal of this study was to perform a detailed analysis of the composition and the organization of this truly bio-material. In addition, we investigated whether the devitalization process (dehydration) used to store the CAM, and thus, make the material available "off-the-shelf," could negatively affect its organization and mechanical properties. We demonstrated that neither the thickness nor the mechanical strength of CAM sheets were significantly changed by the dehydration/freezing/rehydration cycle. The identification of over 50 extracellular matrix proteins highlighted the complex composition of the CAM. Histology showed intense collagen and glycosaminoglycan staining throughout the CAM sheet. The distribution of collagen I, collagen VI, thrombospondin-1, fibronectin-1, fibrillin-1, biglycan, decorin, lumican and versican showed various patterns that were not affected by the devitalization process. Transmission electron microscopy analysis revealed that the remarkably dense collagen network was oriented in the plane of the sheet and that neither fibril density nor diameter was changed by devitalization. Second harmonic generation microscopy revealed an intricate, multi-scale, native-like collagen fiber orientation. In conclusion, this bio-material displayed many tissue-like properties that could support normal cell-ECM interactions and allow implantation without triggering degradative responses from the host's innate immune system. This is consistent with its success in vivo. In addition, the CAM can be devitalized without affecting its mechanical or unique biological architecture. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) defines biological function and mechanical properties of tissues and organs. A number of promising tissue engineering approaches have used processed ECM from cadaver/animal tissues or cell-assembled ECM in vitro combined with scaffolds. We have shown the clinical potential of a scaffold-free approach based on an entirely biological material produced by human cells in culture without chemical processing. Here, we perform a comprehensive analysis of the properties of what can truly be called a bio-material. We also demonstrate that this material can be stored dried without losing its remarkable biological architecture.

Energy Metabolism Rewiring Precedes UVB-Induced Primary Skin Tumor Formation.

Although growing evidence indicates that bioenergetic metabolism plays an important role in the progression of tumorigenesis, little information is available on the contribution of reprogramming of energy metabolism in cancer initiation. By applying a quantitative proteomic approach and targeted metabolomics, we find that specific metabolic modifications precede primary skin tumor formation. Using a multistage model of ultraviolet B (UVB) radiation-induced skin cancer, we show that glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid ß-oxidation are decreased at a very early stage of photocarcinogenesis, while the distal part of the electron transport chain (ETC) is upregulated. Reductive glutamine metabolism and the activity of dihydroorotate dehydrogenase (DHODH) are both necessary for maintaining high ETC. Mice with decreased DHODH activity or impaired ETC failed to develop pre-malignant and malignant lesions. DHODH activity represents a major link between DNA repair efficiency and bioenergetic patterning during skin carcinogenesis.

A tyrosine kinase-STAT5-miR21-PDCD4 regulatory axis in chronic and acute myeloid leukemia cells.

MicroRNAs (miRNAs) are regulators of several key patho-physiological processes, including cell cycle and apoptosis. Using microarray-based miRNA profiling in K562 cells, a model of chronic myeloid leukemia (CML), we found that the oncoprotein BCR-ABL1 regulates the expression of miR-21, an "onco-microRNA", found to be overexpressed in several cancers. This effect relies on the presence of two STAT binding sites on the promoter of miR-21, and on the phosphorylation status of STAT5, a transcription factor activated by the kinase activity of BCR-ABL1. Mir-21 regulates the expression of PDCD4 (programmed cell death protein 4), a tumor suppressor identified through a proteomics approach. The phosphoSTAT5 - miR-21 - PDCD4 pathway was active in CML primary CD34+ cells, but also in acute myeloid leukemia (AML) models like MV4.11 and MOLM13, where the constitutively active tyrosine kinase FLT3-ITD plays a similar role to BCR-ABL1 in the K562 cell line.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.

A second Wpl1 anti-cohesion pathway requires dephosphorylation of fission yeast kleisin Rad21 by PP4.

Cohesin mediates sister chromatid cohesion which is essential for chromosome segregation and repair. Sister chromatid cohesion requires an acetyl-transferase (Eso1 in fission yeast) counteracting Wpl1, promoting cohesin release from DNA We report here that Wpl1 anti-cohesion function includes an additional mechanism. A genetic screen uncovered that Protein Phosphatase 4 (PP4) mutants allowed cell survival in the complete absence of Eso1. PP4 co-immunoprecipitated Wpl1 and cohesin and Wpl1 triggered Rad21 de-phosphorylation in a PP4-dependent manner. Relevant residues were identified and mapped within the central domain of Rad21. Phospho-mimicking alleles dampened Wpl1 anti-cohesion activity, while alanine mutants were neutral indicating that Rad21 phosphorylation would shelter cohesin from Wpl1 unless erased by PP4. Experiments in post-replicative cells lacking Eso1 revealed two cohesin populations. Type 1 was released from DNA by Wpl1 in a PP4-independent manner. Type 2 cohesin, however, remained DNA-bound and lost its cohesiveness in a manner depending on Wpl1- and PP4-mediated Rad21 de-phosphorylation. These results reveal that Wpl1 antagonizes sister chromatid cohesion by a novel pathway regulated by the phosphorylation status of the cohesin kleisin subunit.

High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy.

Podocytes play a key role in diabetic nephropathy pathogenesis, but alteration of their metabolism remains unknown in human kidney. By using a conditionally differentiating human podocyte cell line, we addressed the functional and molecular changes in podocyte energetics during in vitro development or under high glucose conditions. In 5 mM glucose medium, we observed a stepwise activation of oxidative metabolism during cell differentiation that was characterized by peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-dependent stimulation of mitochondrial biogenesis and function, with concomitant reduction of the glycolytic enzyme content. Conversely, when podocytes were cultured in high glucose (20 mM), stepwise oxidative phosphorylation biogenesis was aborted, and a glycolytic switch occurred, with consecutive lactic acidosis. Expression of the master regulators of oxidative metabolism transcription factor A mitochondrial, PGC-1α, AMPK, and serine-threonine liver kinase B1 was altered by high glucose, as well as their downstream signaling networks. Focused transcriptomics revealed that myocyte-specific enhancer factor 2C (MEF2C) and myogenic factor 5 (MYF5) expression was inhibited by high glucose levels, and endoribonuclease-prepared small interfering RNA-mediated combined inhibition of those transcription factors phenocopied the glycolytic shift that was observed in high glucose conditions. Accordingly, a reduced expression of MEF2C, MYF5, and PGC-1α was found in kidney tissue sections that were obtained from patients with diabetic nephropathy. These findings obtained in human samples demonstrate that MEF2C-MYF5-dependent bioenergetic dedifferentiation occurs in podocytes that are confronted with a high-glucose milieu.-Imasawa, T., Obre, E., Bellance, N., Lavie, J., Imasawa, T., Rigothier, C., Delmas, Y., Combe, C., Lacombe, D., Benard, G., Claverol, S., Bonneu, M., Rossignol, R. High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy.

Copper stress-induced changes in leaf soluble proteome of Cu-sensitive and tolerant Agrostis capillaris L. populations.

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 µM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930).

Beyond plant defense: insights on the potential of salicylic and methylsalicylic acid to contain growth of the phytopathogen Botrytis cinerea.

Using Botrytis cinerea we confirmed in the present work several previous studies showing that salicylic acid, a main plant hormone, inhibits fungal growth in vitro. Such an inhibitory effect was also observed for the two salicylic acid derivatives, methylsalicylic and acetylsalicylic acid. In marked contrast, 5-sulfosalicylic acid was totally inactive. Comparative proteomics from treated vs. control mycelia showed that both the intracellular and extracellular proteomes were affected in the presence of salicylic acid or methylsalicylic acid. These data suggest several mechanisms that could potentially account for the observed fungal growth inhibition, notably pH regulation, metal homeostasis, mitochondrial respiration, ROS accumulation and cell wall remodeling. The present observations support a role played by the phytohormone SA and derivatives in directly containing the pathogen. Data are available via ProteomeXchange with identifier PXD002873.

Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR(ER)) to restore ER homeostasis. The AAA(+) ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR(ER) genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA(+) ATPase, as a novel repressor of a subset of UPR(ER) genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR(ER) genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, ß-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology.

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4-5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4-5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. ß-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.

Exploring early steps in biofilm formation: set-up of an experimental system for molecular studies.

BACKGROUND: Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth. RESULTS: Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated. CONCLUSIONS: Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.