Identification of Novel Compounds Inhibiting the Kinase Activity of the CDK5/p25 Complex via Direct Binding to p25.

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.

Estrogenic hazards of short chain phthalates and bisphenols found in cosmetic products.

For several years, environmental exogenous agents, called endocrine disruptors, are suspected to interfere with the essential functions of reproduction and development in many living organisms. In this study, endocrine disruptors including five phthalates and two bisphenols contents in finished products were assayed and their estrogenic activity were measured by using the Yeast Estrogen Screen system with respect to human and trout estrogen receptors hERα and rtERS. Independently of the estrogen receptor, only short-chain phthalates (DBP and BBP) and the two bisphenols exhibited an estrogenic activity. Besides, the risk of three end-products (agro-food, cosmetics, and pharmaceutical) was evaluated before and after forced aging. Only two cosmetics the face cream and the perfume presented a hazard which increases with aging. These results are consistent with the compounds identified by Gas chromatography-mass spectrometry. These findings confirmed that the YES system can be routinely used to evaluate the estrogenic hazards within finished products.

Microplastics elutriation system: Part B: Insight of the next generation.

Elutriation is an efficient process for extracting microplastics. The development of a numerical model has shown the need for optimizing aspects of the design of the actual elutriation protocol as well as the dimensioning of the column to increase its efficiency. The study aims to propose new dimensioning data and protocol elements for designing an efficient column. Using a numerical model, the filling velocity was calculated as a function of the size and the density of the particles to prevent sand suspension. The sieving protocol was adapted to increase the density limit for the extraction of plastic particles from 1460 to >1800 kg·m-3. The durations of the elutriation and the column height were calculated to improve the control of the particle suspension. These results contribute to the development of the next generation of elutriation system and will accelerate the study of plasticome in the context of sandy sediments.

Threat of plastic ageing in marine environment. Adsorption/desorption of micropollutants.

Ageing of various plastics in marine environment was monitored after immersion of two synthetic (polyvinylchloride, PVC, and polyethylene terephthalate, PET) and one biodegradable (poly(butylene adipate co-terephtalate), PBAT) plastics for 502days in the bay of Lorient (Brittany, France). Data analysis indicates that aged PVC rapidly releases estrogenic compounds in seawater with a later adsorption of heavy metals; PET undergoes a low weakening of the surface whereas no estrogenic activity is detected; PBAT ages faster in marine environment than PVC. Aged PBAT exhibits heterogeneous surface with some cavities likely containing clay minerals from the chlorite group. Besides, this degraded material occasionally shows a high estrogenic activity. Overall, this study reports, for the first time, that some aged plastics, without being cytotoxic, can release estrogenic compounds in marine environment.

Microplastics elutriation system. Part A: Numerical modeling.

The elutriation process has shown its efficiency to extract microplastics from sand and began to spread in the scientific community. This extraction technic requires knowing with accuracy the extraction velocities of particles. This study aims to test whether numerical modeling could help to calculate these velocities. From hydrodynamic equations, a numerical model has been developed and the outputs are compared to experimental extraction data. The results show, for the calculated velocities, the experimental plastic extraction yields will be higher than 90% for <10% of sand contamination. The model also allows determining that, with the actual protocol, the maximum plastic density which can be extracted is about 1450kg·m-3 whereas the detrimental resuspension, which may occur during the column filling step, is highlighted. From model calculations, it arises that changes in the column dimensioning and the protocol operations need to be considered.

Efficient microplastics extraction from sand. A cost effective methodology based on sodium iodide recycling.

Evaluating the microplastics pollution on the shores requires overcoming the technological and economical challenge of efficient plastic extraction from sand. The recovery of dense microplastics requires the use of NaI solutions, a costly process. The aim of this study is to decrease this cost by recycling the NaI solutions and to determine the impact of NaI storage. For studying the NaI recyclability, the solution density and the salt mass have been monitored during ten life cycles. Density, pH and salt mass have been measured for 40days to assess the storage effect. The results show that NaI solutions are recyclable without any density alterations with a total loss of 35.9% after the 10cycles of use. During storage, chemical reactions may appear but are reversible. Consequently, the use of recycling methods allows for a significant cost reduction. How far the plastic extraction by dense solutions is representative is discussed.

Oyster's cells regulatory volume decrease: A new tool for evaluating the toxicity of low concentration hydrocarbons in marine waters.

Human activities require fossil fuels for transport and energy, a substantial part of which can accidentally or voluntarily (oil spillage) flow to the marine environment and cause adverse effects in human and ecosystems' health. This experiment was designed to estimate the suitability of an original cellular biomarker to early quantify the biological risk associated to hydrocarbons pollutants in seawater. Oocytes and hepatopancreas cells, isolated from oyster (Crassostrea gigas), were tested for their capacity to regulate their volume following a hypo-osmotic challenge. Cell volumes were estimated from cell images recorded at regular time intervals during a 90min-period. When exposed to diluted seawater (osmolalities from 895 to 712mosmkg(-1)), both cell types first swell and then undergo a shrinkage known as Regulatory Volume Decrease (RVD). This process is inversely proportional to the magnitude of the osmotic shock and is best fitted using a first-order exponential decay model. The Recovered Volume Factor (RVF) calculated from this model appears to be an accurate tool to compare cells responses. As shown by an about 50% decrease in RVF, the RVD process was significantly inhibited in cells sampled from oysters previously exposed to a low concentration of diesel oil (8.4mgL(-1) during 24h). This toxic effect was interpreted as a decreased permeability of the cell membranes resulting from an alteration of their lipidic structure by diesel oil compounds. In contrast, the previous contact of oysters with diesel did not induce any rise in the gills glutathione S-transferase specific activity. Therefore, this work demonstrates that the study of the RVD process of cells selected from sentinel animal species could be an alternative bioassay for the monitoring of hydrocarbons and probably, of various chemicals in the environment liable to alter the cellular regulations. Especially, given the high sensitivity of this biomarker compared with a proven one, it could become a relevant and accurate tool to estimate the biological hazards of micropollutants in the water.

Microplastics elutriation from sandy sediments: A granulometric approach.

Although relatively easy to extract in the marine environment, microplastics are very difficult to recover when they are trapped in sediments. The elutriation column is one of the best tools currently available for extracting plastics from sediment, but with a high sand recovery yield. This study aims to address the following questions: (i) is it possible to use a sedimentological approach to limit the sand recovery? (ii) does the extraction velocity of the sand and plastic particles vary according to density and granulometry? (iii) what is the relative recovery efficiency obtained for dense polymer particles mixed with marine sand? Based on a new granulometric classification, different plastic particle-size fractions are defined. Their extraction velocities are experimentally determined on particles of sediment and different plastics (PA, PVC). The particle recovery experiments indicate that it is possible to extract >90% of dense plastic particles in cases of negligible sand recovery.

Rapid and qualitative fluorescence-based method for the assessment of PHA production in marine bacteria during batch culture.

The expansion of polyhydroxyalkanoates (PHAs) into the biodegradable polymers market is mainly prevented by their production process which is still complicated with a low efficiency, resulting in relatively expensive products. In this study, we developed a method that used the lipophilic fluorescent probe Nile Red (1 mg l(-1) solution in DMSO) directly into the culture broth to stain the PHA inclusions inside bacterial cells followed by detection of the emitted fluorescence by both microscopic and spectrometric techniques. Epifluorescence microscopy provides a rapid tool to distinguish producing from non-producing bacterial species and the relative fluorescence intensity (FI) determined at the maximum of emission spectra in the wavelength region of 560-710 nm (λ(ex): 543 nm), allows a fast assessment of the cultural conditions that may enhance PHA production yield. During two-step cultivation in 500-ml flasks with glucose as the sole carbon source, the method aimed to select bacterial strains efficient for PHA synthesis among a marine collection. Subsequently, the NR assay was used to determine the C0/N0 ratio of the producing media that may improve the polymer yield as well as to follow the time course of fermentation. Characterization by GC-MS and DSC confirmed the production of the P(3-HB) homopolymer.

Investigating the in Vitro Thermal Stability and Conformational Flexibility of Estrogen Receptors as Potential Key Factors of Their in Vivo Activity.

Among hormone-inducible transcription factors, estrogen receptors (ERs) play important roles in tissue growth and differentiation, via either direct or indirect binding, in the nucleus, to specific DNA targets called estrogen responsive elements (EREs), or through nongenomic pathways. In humans, two estrogen receptor isoforms (hERs), designated hERα and hERß, have been identified. These two hERs, encoded by genes located on distinct chromosomes, exhibit divergent tissue-specific functions and different subcellular distributions depending on their binding status, free or complexed to their cognate ligands. Because it is hypothesized that such distinct behaviors may arise from various conformational stabilities and flexibilities, the effect of salt concentration and temperature was studied on the free and estrogen-activated hERα and hERß. Our results show that the conformational stability of hERß is weakly modulated by salt concentration as opposed to hERα. In addition, we show that the estrogen-bound hERs exhibit a more constrained structure than the unliganded ones and that their conformational flexibility is more affected by diethylstilbestrol binding than that of estradiol, 4-hydroxytamoxifen, or raloxifen. In line with these results, conformational analysis and computational docking were performed on hERα and hERß, which confer molecular support of a diethylstilbestrol-induced restrained flexibility as compared to other ligands. We found that Trp383 in hERα and Trp335 in hERß can closely interact with the NR-box motif of the H12 helix and act as a gatekeeper of the agonist-bound versus antagonist-bound conformations. Altogether, our study contributes to an improved knowledge of the diverse physicochemical properties of full-length hERs, which will help in our understanding of their distinct cellular roles in various cellular contexts.

Tamoxifen inhibits CDK5 kinase activity by interacting with p35/p25 and modulates the pattern of tau phosphorylation.

Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout.

In most of oviparous animals, vitellogenins (VTG) are the major egg yolk precursors. They are produced in the liver under the control of estrogens. In rainbow trout (Oncorhynchus mykiss), the vtg genes cluster contains an unusually large number of almost identical gene copies. In order to identify the regulatory elements in their promoters, we used a combination of reporter plasmids containing genomic sequences including putative estrogen response elements (EREs) and we performed transient transfection assays in MCF-7 and yeast cells. We found a functional ERE corresponding to the sequence GGGGCAnnnTAACCT (rtvtgERE), which differs from the consensus ERE (ERE(cs)) by three base pairs. This non-palindromic ERE is located in the env gene of a retrotransposon relic, 180 base pairs upstream of the transcriptional start site. Fluorescence anisotropy experiments confirmed that the purified human estrogen receptor alpha (hERalpha) can specifically bind to rtvtgERE. Furthermore, we observe that the stability of hERalpha-ERE(cs) and hERalpha-rtvtgERE complexes is similar with equilibrium dissociation constants of 3.0nM and 6.2nM respectively, under our experimental conditions. Additionally, this rtvtgERE sequence displays a high E2-responsiveness through ER activation in cellulo. In the rainbow trout, the functional ERE (rtvtgERE) lies within promoter sequences which are mostly composed of sequences derived from transposable elements (TEs), which therefore may have acted as an evolutionary buffer to secure the proper expression of these genes.

Antifouling activity of macroalgal extracts on Fragilaria pinnata (Bacillariophyceae): a comparison with Diuron.

The tributyltin-based products and organic biocides which are incorporated into antifouling paints have had a negative impact on the marine environment, and the ban on tributyltin-based antifouling products has urged the industry to find substitutes to prevent the development of fouling on ship hulls. Natural antifouling agents could be isolated from marine resources, providing an alternative option for the industry. The effects of different marine seaweed extracts from Sargassum muticum and Ceramium botryocarpum on the growth, pigment content and photosynthetic apparatus of the marine diatom Fragilaria pinnata were compared with those of Diuron, a biocide widely used in antifouling paints. The addition of the macroalgal extracts in the culture medium resulted in an inhibition of the growth of F. pinnata, but this inhibition was lower than that obtained with Diuron. After transfer to a biocide-free medium, F. pinnata cells previously exposed to the macroalgal extracts exhibited normal growth, in contrast to Diuron-treated cells, which died, demonstrating that the effects of the natural antifouling agents were reversible. Macroalgal extracts and Diuron-induced modifications in F. pinnata cellular pigment content. Chlorophyll a, fucoxanthin, and the xanthophyll pool, diadinoxanthin and diatoxanthin, were the most affected. Changes in the structure and function of the photosynthetic apparatus were studied by microspectrofluorimetry, and provided a comprehensive evaluation of the inhibition of the diatom Photosystem II (PSII) by the biocides. This study confirms that natural extracts from the macroalgae studied have the potential to be used as a substitute to commercial biocides in antifouling paints.

Role of the heat-induced whey protein/kappa-casein complexes in the formation of acid milk gels: a kinetic study using rheology and confocal microscopy.

The effect of heat treatment of milk on the formation of acid gel was examined using confocal scanning laser microscopy and low-amplitude dynamic oscillation throughout acidification. Milk samples were reconstituted by mixing colloidal phase from unheated or preheated skim milk, labeled with rhodamine B isothiocyanate, with the aqueous phase from unheated or preheated milk, labeled with fluorescein isothiocyanate. Gels were made by acidification with glucono-delta-lactone. The presence of material from preheated milk, that is, either the colloidal or the aqueous phase or both, led to an increase in the gelation pH and in the final elastic modulus and to a more branched network with larger pores. During acidification, the heat-induced serum complexes and the casein micelles did not appear to form separated gels with time or in space. Moreover, the colocalization in the final network of serum heat-induced complexes and casein micelles is particularly well observed in the presence of an aqueous phase obtained from preheated milk. Finally, because the rheological and microstructural properties of acid gels containing either micelle-bound or serum heat-induced complexes were similar, it was suggested that the serum heat-induced complexes interacted with the casein micelles early in the course of acidification and that formation of the network did not differ significantly whether the heat-induced complexes were initially found in the aqueous phase of milk or bound to casein micelles.

Molecular interaction between apo or holo alpha-lactalbumin and lysozyme: formation of heterodimers as assessed by fluorescence measurements.

In a previous work, we reported that contrary to native calcium-loaded alpha-lactalbumin (holo alpha-LA), calcium-depleted form (apo alpha-LA) has the ability to self-assemble with lysozyme (LYS) to form different supramolecular structures in temperature-dependent manner. In this study, we examine what happens at molecular scale using fluorescence techniques. Fluorescence anisotropy coupled with fluorescence lifetime measurements provides a means to measure intermolecular interactions. We showed that LYS interacts with both apo alpha-LA and holo alpha-LA to form oligomers, assumed to be heterodimers, at 10 degrees C and 45 degrees C. The dissociation constants for dimerization were found to be in the muM range and increased significantly with increasing ionic strength from 39 to 124 mM. Although the binding constants of holo alpha-LA-LYS and apo alpha-LA-LYS complexes were of the same order of magnitude, the shape or conformation of formed heterodimers differed as assessed by fluorescence parameters in particular correlation time calculations. Such conformation differences could explain why holo alpha-LA-LYS complexes are trapped as heterodimers while the apo alpha-LA-LYS complexes have the ability to further self-assemble into various supramolecular structures.