Identification of Novel Molecular Targets for Endometrial Cancer Using a Drill-Down LC-MS/MS Approach with iTRAQ

BACKGROUND: The number of patients with endometrial carcinoma (EmCa) with advanced stage or high histological grade is increasing and prognosis has not improved for over the last decade. There is an urgent need for the discovery of novel molecular targets for diagnosis, prognosis and treatment of EmCa, which will have the potential to improve the clinical strategy and outcome of this disease. METHODOLOGY AND RESULTS: We used a "drill-down" proteomics approach to facilitate the identification of novel molecular targets for diagnosis, prognosis and/or therapeutic intervention for EmCa. Based on peptide ions identified and their retention times in the first LC-MS/MS analysis, an exclusion list was generated for subsequent iterations. A total of 1529 proteins have been identified below the Proteinpilot® 5% error threshold from the seven sets of iTRAQ experiments performed. On average, the second iteration added 78% new peptides to those identified after the first run, while the third iteration added 36% additional peptides. Of the 1529 proteins identified, only 40 satisfied our criteria for significant differential expression in EmCa in comparison to normal proliferative tissues. These proteins included metabolic enzymes (pyruvate kinase M2 and lactate dehydrogenase A); calcium binding proteins (S100A6, calcyphosine and calumenin), and proteins involved in regulating inflammation, proliferation and invasion (annexin A1, interleukin enhancer-binding factor 3, alpha-1-antitrypsin, macrophage capping protein and cathepsin B). Network analyses revealed regulation of these molecular targets by c-myc, Her2/neu and TNF alpha, suggesting intervention with these pathways may be a promising strategy for the development of novel molecular targeted therapies for EmCa. CONCLUSIONS: Our analyses revealed the significance of drill-down proteomics approach in combination with iTRAQ to overcome some of the limitations of current proteomics strategies. This study led to the identification of a number of novel molecular targets having therapeutic potential for targeted molecular therapies for endometrial carcinoma

The PTEN Phosphatase Controls Intestinal Epithelial Cell Polarity and Barrier Function: Role in Colorectal Cancer Progression

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells

Parasite and Pesticide Impacts on the Bumblebee (Bombus terrestris) Haemolymph Proteome

International audiencePesticides pose a potential threat to bee health, especially in combination with other stressors, such as parasites. However, pesticide risk assessment tests pesticides in isolation from other stresses, i.e., on otherwise healthy bees. Through molecular analysis, the specific impacts of a pesticide or its interaction with another stressor can be elucidated. Molecular mass profiling by MALDI BeeTyping ® was used on bee haemolymph to explore the signature of pesticidal and parasitic stressor impacts. This approach was complemented by bottom-up proteomics to investigate the modulation of the haemoproteome. We tested acute oral doses of three pesticides-glyphosate, Amistar and sulfoxaflor-on the bumblebee Bombus terrestris, alongside the gut parasite Crithidia bombi. We found no impact of any pesticide on parasite intensity and no impact of sulfoxaflor or glyphosate on survival or weight change. Amistar caused weight loss and 19-41% mortality. Haemoproteome analysis showed various protein dysregulations. The major pathways dysregulated were those involved in insect defences and immune responses, with Amistar having the strongest impact on these dysregulated pathways. Our results show that even when no response can be seen at a whole organism level, MALDI BeeTyping ® can detect effects. Mass spectrometry analysis of bee haemolymph provides a pertinent tool to evaluate stressor impacts on bee health, even at the level of individuals

Chemical composition and antimicrobial activity of nine essential oils obtained by steam distillation of plants from the Souss-Massa Region (Morocco)

We would like to thank Professor Fouad Msanda from Faculty of Sciences of Agadir, Morocco, for the botanical identification of plant species.International audienceThe antimicrobial properties of nine essential oils (EOs) from Souss-Massa, Morocco, were assessed and compared. The studied plants were: Mentha piperita, Mentha pulegium, Mentha spicata, Pelargonium graveolens, Rosmarinus officinalis, Thymus leptobotrys, Thymus pallidus, Thymus satureioides and Citrus limon. EOs were extracted by steam distillation and their minimum inhibitory concentrations (MICs) were determined versus ten bacteria strains (Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus (MRSA), Enterococcus faecalis, Streptococcus equinus and Streptococcus pyogenes), two yeasts (Candida albicans and Candida glabrata) and two fungi (Aspergillus niger and Penicillium funiculosum). Thymus leptobotrys, P.graveolens and T.satureioides showed interesting antimicrobial properties (MIC = [0.7-5.9 mu g/mL]). Mentha piperita, T.pallidus and M.spicata gave intermediary results (MIC= [5.7-23.2 mu g/mL]) while the remaining EOs displayed poor results (MIC= [23.2-85.5 mu g/mL]). EOs that contained primarily, in this order, phenolic terpenes, terpenic alcohols and terpenic aldehydes displayed better antimicrobial properties

Strong reduction of exciton-phonon coupling in high crystalline quality single-wall carbon nanotubes: a new insight into broadening mechanisms and exciton localization

International audienceCarbon nanotubes are quantum sources whose emission can be tuned at telecommunication wavelengths by choosing the diameter appropriately. Most applications require the smallest possible linewidth. Therefore, the study of the underlying dephasing mechanisms is of utmost interest. Here, we report on the low-temperature photoluminescence of high crystalline quality individual single-wall carbon nanotubes synthesized by laser ablation (L-SWNTs) and emitting at telecom-munication wavelengths. A thorough statistical analysis of their emission spectra reveals a typical linewidth one order of magnitude narrower than that of most samples reported in the literature. The narrowing of the PL line of L-SWNTs is due to a weaker effective exciton-phonon coupling subsequent to a weaker localization of the exciton. These results suggest that exciton localization in SWNTs not only arises from interfacial effects, but that the intrinsic crystalline quality of the SWNT plays an important role. Photoluminescence (PL) emission in semiconducting carbon nanotubes arises from exciton recombination [1–3] and has been extensively studied in view of possible applications in opto-electronics, bio-imaging or photovoltaics [4–7]. Observation of photon antibunching in the near infrared [8, 9] suggests that SWNTs are also promising single-photon sources for the implementation of quantum information protocols. Interestingly, the PL emission energy (i.e. the excitonic recombination energy) strongly depends on the tube diameter and can be easily tuned in the telecommunication bands at 0.83 eV (1.5µm) by choosing SWNTs with a diameter of about 1-1.2 nm [10]. SWNTs could therefore make up a very versatile light source for quantum optics. Several studies suggested that the optical properties of SWNTs at low temperature are best described in terms of localized excitons (zero-dimensional confinement), leading to a quantum dot like behavior [11, 12]. Nevertheless, the nature of the traps responsible for this exciton localization is not elucidated yet. In order to address the issue of exciton localization, we studied carbon nanotubes produced by high-temperature synthesis methods such as electric arc or laser ablation methods, which are known for their higher crystalline quality, with a lower density of defects [13–17]

The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward β-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus. We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the β-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate β-defensins. IMPORTANCE β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus. Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations

Towards the development of safer by design TiO 2 -based photocatalytic paint: impacts and performances

International audienceAddition of titanium dioxide (TiO 2) (nano)particles into photocatalytic paints represents a promising alternative aiming to mineralize gaseous pollutants, such as volatile organic compounds (VOCs) into innocuous species (H 2 O and CO 2). Despite important industrial and economic benefits, some concerns were raised regarding the risks associated with nano-objects and their human and environmental impacts. To mitigate potential risks associated with the use of these nano-objects, we report a safer by design strategy to develop a photocatalytic paint containing TiO 2 nanoparticles (NPs) taking into consideration the safety aspects over its life cycle. Specific innovative types of TiO 2 NPs were synthesized. These nanoparticles were then incorporated into an organic matrix-based paint. These paints were applied on standard substrates and underwent artificial weathering in an accelerated weathering chamber with controlled parameters. Photocatalytic efficiency towards airborne VOCs was measured for all the paints. Mechanical solicitation through abrasion and incineration tests were performed to assess the potential emission of airborne particles that could lead to human or environmental exposure. In parallel, toxicology studies were conducted to assess the hazards associated with the pristine particles and paint residues. Using this safer by design strategy, we succeeded in decreasing the negative impact of TiO 2 on the paint matrix while keeping a good photocatalytic efficiency and reducing the NP release. Taken together, these results show that we succeeded in generating safer by design paints, thanks to the use of these specifically developed TiO 2 NPs, which exhibit similar photocatalytic properties and enhanced physical properties as compared to paints containing the reference TiO 2 NPs, while reducing their potential hazards

Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor

In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a “signal” (such as fragments of infecting bacteria), mRNA and/or protein/peptide effectors to protect their eggs against two natural bacterial pathogens, namely the Gram-positive Bacillus thuringiensis and the Gram-negative Serratia entomophila. By taking the mealworm beetle Tenebrio molitor as a biological model, our results suggest that eggs are mainly protected by an active direct transfer of a restricted number of immune proteins and of antimicrobial peptides. In contrast, the present data do not support the involvement of mRNA transfer while the transmission of a “signal”, if it happens, is marginal and only occurs within 24h after maternal exposure to bacteria. This work exemplifies how combining global approaches helps to disentangle the different scenarios of a complex trait, providing a comprehensive characterization of TGIP mechanisms in T. molitor. It also paves the way for future alike studies focusing on TGIP in a wide range of invertebrates and vertebrates to identify additional candidates that could be specific to TGIP and to investigate whether the TGIP mechanisms found herein are specific or common to all insect species. Author summary   All living organisms are regularly exposed to a wide and diverse range of pathogens. To protect themselves, many species have developed an immune system able to detect and eradicate these pathogens. Most interestingly, this immunological experience can be transferred by parents to their offspring to protect them from pathogens that may persist in the environment and to which they could be exposed during their life. While extensively studied in vertebrates, this phenomenon–called trans-generational immune priming (TGIP)–has only been identified a decade ago in invertebrates and the supporting molecular mechanisms are still largely unknown. Recently, we proposed four different scenarios as a practical framework to investigate the mechanisms supporting this complex phenomenon. In the present study, we combined different molecular approaches to disentangle these different scenarios and provide a comprehensive characterization of maternal TGIP mechanisms in a model insect, the mealworm beetle Tenebrio molitor