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New approach based on immunochemical techniques for monitoring of selective estrogen receptor modulators (SERMs) in human urine

Salvador, J.-P., Vila-Roca, E., Monfort, N., Ventura, R., Marco, M.-P.

Journal of Pharmaceutical and Biomedical AnalysisVolume 156, 15 July 2018, Pages 147-152

 Antiestrogenic compounds such as tamoxifen, toremifen and chlomifen are used illegally by athletes to minimize physical impacts such as gynecomastia resulting from the secondary effects of anabolic androgenic steroids, used to increase athletic efficiency unlawfully. The use of these compounds is banned by the World Anti-Doping Agency (WADA) and controls are made through analytical methodologies such as HPLC–MS/MS, which do not fulfil the sample throughput requirements. Moreover, compounds such as tamoxifen are also used to treat hormone receptor-positive breast cancer (ER?+?).Therapeutic drug monitoring (TDM) of tamoxifen may also be clinically useful for guiding treatment decisions. An accurate determination of these drugs requires a solid phase extraction of patient serum followed by HPLC–MS/MS. In the context of an unmet need of high-throughput screening (HTS) and quantitative methods for antiestrogenic substances we have approached the development of antibodies and an immunochemical assay for the determination of these antiestrogenic compounds. The strategy applied has taken into consideration that these drugs are metabolized and excreted in urine as the corresponding 4-hydroxylated compounds. A microplate-based ELISA procedure has been developed for the analysis of these metabolites in urine with a LOD of 0.15, 0.16 and 0.63?µg/L for 4OH-tamoxifen, 4OH-toremifen and 4OH-clomifen, respectively, much lower than the MRPL established by WADA (20?µg/L).

 

Enzyme-linked immunosorbent assays for therapeutic drug monitoring coumarin oral anticoagulants in plasma

Salvador, J.-P., Tassies, D., Reverter, J.-C., Marco, M.-P.

Analytica Chimica Acta,2018

 The development of high-throughput immunochemical assays to assist on precision medicine for patients treated with coumarin oral anticoagulants (OA) is reported. The assays are able to quantitate Warfarin (W) and/or Acenocoumarol (ACL) directly in plasma samples without any previous sample pretreatment. The detectabilities (W, 3.52?±?2.25?nM and ACL, 1.56?±?0.64?nM) and the working ranges achieved (W, 1.19?±?0.73 to 12.05?±?2.99?nM and ACL 0.63?±?0.20 to 10.19?±?6.69?nM) are within the therapeutic levels usually found in patients treated with these drugs. The assays are specific with only cross-recognition of 4'-NH2-ACL on the ACL ELISA, which is one of the main metabolites of this drug. Moreover, accuracy studies performed with blind spiked samples show very good correlation between the spiked and the measured concentrations. Finally, a small clinical pilot study has been performed analyzing 96 plasma samples from treated and untreated patients, showing that the assay is able to quantitate ACL. The results obtained allow envisaging the possibility to use these assays for pharmacokinetic studies, dosage assessment or therapeutic drug monitoring.

 

Vesicular nanostructures composed of oleic acid and phosphatidylcholine:Effect of pH and molar ratio

K. Talló, V. Moner, M. De Cabo, M. Cócera, O. López

Chemistry and Physics of Lipids 213 (2018) 96–101

Phospholipids and fatty acids are the main building blocks of biological membranes. Oleic acid is a monounsaturated omega-9 fatty acid commonly found in many natural sources. Its characteristic kinked structure grants this molecule with a great number of biological properties. To better understand the role that this kind of fatty acids play into phospholipid membranes, nanostructured systems formed with hydrogenated soy phosphatidylcholine and oleic acid were studied in this work by means of cryo-electron microscopy, dynamic light scattering and differential scanning calorimetry. Differences concerning size, morphology and phase behavior were found when those systems were prepared at different conditions of pH and molar ratio between both compounds. Broadly, it was seen how alkaline mediums and high proportions of oleic acid reduced the size of the structures and increased the fluidity of the membranes. The ease of preparation of these lipid systems, and the response to pH suggests a future use of these systems as model membranes or delivery systems.

 

siRNA Modified with 2'-Deoxy-2'-C-methylpyrimidine Nucleosides

Dellafiore, M., Aviñó, A., Alagia, A., Montserrat, J.M., Iribarren, A.M., Eritja, R.

 ChemBioChem, 2018

 (2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

 

DNA-based nanoscaffolds as vehicles for 5-fluoro-2'-deoxyuridine oligomers in colorectal cancer therapy

Jorge, A.F., Aviñó, A., Pais, A.A.C.C., Eritja, R., Fàbrega, C.

 Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy, 196, pp. 185-195. 2018

 Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs, are considered one of the most successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain the major limiting bottlenecks. Here, we exploited for the first time the ability of two DNA nanoscaffolds, a DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2'-deoxyuridine (FdUn) oligomers. In addition, cholesterol moieties were synthetically attached to Td and DNA origami staples to enhance cellular uptake. DNA nanostructures functionalized with FdUn exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU, especially having cholesterol as an internalization helper. The cholesterol content mostly correlates with the increase of the FdUn nanostructure cytotoxicity. DNA nanoscaffolds bearing FdUn were able to circumvent the low sensitivity of colorectal cancer cells towards 5-FU. Both DNA nanostructures attained a comparable cytotoxic effect yet Td displays higher antiproliferative action. The ability to reduce the proliferation of cancer cells is mainly related to the concentration of DNA nanostructures. The present work suggests that self-assembled DNA nanoparticles are privileged vehicles for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment.

 

Chromonic self-assemblies in a series of dialkyl-thiacarbocyanine dyes and generalization of a facile route for the synthesis of fluorescent nanostructured silica fibers

Magana, J.R., Solans, C., Salonen, L.M., Carbó-Argibay, E., Gallo, J., Tiddy, G.J.T., Rodríguez-Abreu, C.

 Journal of the Taiwan Institute of Chemical Engineers, 2018

 In the search for new chromonic liquid crystals, we have explored the self-assembly behavior in water of a series of high-purity dialkylthiacarbocyanine dyes with different alkyl chain lengths and acetate as counterion. The dimer model was used to fit the UV–vis spectra and estimate the dissociation energy between molecules, which increases with the alkyl chain length suggesting that not only aromatic p–p interactions but also entropic/steric effects contribute to the aggregation process. NMR spectra suggest the existence of aromatic stacking interactions within the aggregates. At high concentrations, the dyes form nematic and hexagonal chromonic liquid crystals (CLC) in water within a limited range of alkyl chain lengths (ethyl to butyl); for longer lengths (pentyl) no liquid crystals were observed because of insufficient dye solubility. From small angle X-ray scattering patterns, it can be inferred that dye aggregates have cylindrical morphology with a multimolecular cross-section. The dye aggregates template the formation of silica nanofibers synthetized via sol–gel method in alkaline media. After removing excess dye, the silica/dye nanofibers showed high fluorescence emission with superb photochemical stability. The present report demonstrates a generalized route for the wet synthesis of nanostructured silica nanofibers with tunable optical properties.

 

Analysis of the neurotoxic effects of neuropathic organophosphorus compounds in adult zebrafish

Melissa Faria, Inmaculada Fuertes, Eva Prats, Jose Luis Abad, Francesc Padrós, Cristian Gomez-Canela, Josefina Casas, Jorge Estevez, Eugenio Vilanova, Benjamin Piña & Demetrio Raldúa

Scientific Reports volume 8, Article number: 4844 (2018)

 Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN.

 

Fluorescent microarray for multiplexed quantification of environmental contaminants in seawater samples

Sanchis, A., Salvador, J.-P., Campbell, K., Elliott, C.T., Shelver, W.L., Li, Q.X., Marco, M.-P.

Talanta, 184, pp. 499-506. 2018

 The development of a fluorescent multiplexed microarray platform able to detect and quantify a wide variety of pollutants in seawater is reported. The microarray platform has been manufactured by spotting 6 different bioconjugate competitors and it uses a cocktail of 6 monoclonal or polyclonal antibodies raised against important families of chemical pollutants such as triazine biocide (i.e. Irgarol 1051®), sulfonamide and chloramphenicol antibiotics, polybrominated diphenyl ether flame-retardant (PBDE, i.e. BDE-47), hormone (17ß-estradiol), and algae toxin (domoic acid). These contaminants were selected as model analytes, however, the platform developed has the potential to detect a broader group of compounds based on the cross-reactivity of the immunoreagents used. The microarray chip is able to simultaneously determine these families of contaminants directly in seawater samples reaching limits of detection close to the levels found in contaminated areas (Irgarol 1051®, 0.19?±?0,06?µg?L-1; sulfapyridine, 0.17?±?0.07?µg?L-1; chloramphenicol, 0.11?±?0.03?µg?L-1; BDE-47, 2.71?±?1.13?µg?L-1; 17ß-estradiol, 0.94?±?0.30?µg?L-1 and domoic acid, 1.71?±?0.30?µg?L-1). Performance of the multiplexed microarray chip was assessed by measuring 38 blind spiked seawater samples containing either one of these contaminants or mixtures of them. The accuracy found was very good and the coefficient of variation was

 

Can white-rot fungi be a real wastewater treatment alternative for organic micropollutants removal? A review

Mir-Tutusaus, J.A., Baccar, R., Caminal, G., Sarrà, M.

Water Research, 138, pp. 137-151., 2018

Micropollutants are a diverse group of compounds that are detected at trace concentrations and may have a negative effect on the environment and/or human health. Most of them are unregulated contaminants, although they have raised a concern in the scientific and global community and future regulation might be written in the near future. Several approaches have been tested to remove micropollutants from wastewater streams. In this manuscript, a focus is placed in reactor biological treatments that use white-rot fungi. A critical review of white-rot fungal-based technologies for micropollutant removal from wastewater has been conducted, several capabilities and limitations of such approaches have been identified and a range of solutions to overcome most of the limitations have been reviewed and/or proposed. Overall, this review argues that white-rot fungal reactors could be an efficient technology to remove micropollutants from specific wastewater streams.

 

Direct coupling of detergent purified human mGlu5 receptor to the heterotrimeric G proteins Gq and Gs

Chady Nasrallah, Karine Rottier, Romain Marcellin, Vincent Compan, Joan Font, Amadeu Llebaria, Jean-Philippe Pin, Jean-Louis Banères & Guillaume Lebon

 Scientific Reports volume 8, Article number: 4407 (2018)

 The metabotropic glutamate (mGlu) receptors are class C G protein-coupled receptors (GPCRs) that modulate synaptic activity and plasticity throughout the mammalian brain. Signal transduction is initiated by glutamate binding to the venus flytrap domains (VFT), which initiates a conformational change that is transmitted to the conserved heptahelical domains (7TM) and results ultimately in the activation of intracellular G proteins. While both mGlu1 and mGlu5 activate Gαq G-proteins, they also increase intracellular cAMP concentration through an unknown mechanism. To study directly the G protein coupling properties of the human mGlu5 receptor homodimer, we purified the full-length receptor, which required careful optimisation of the expression, N-glycosylation and purification. We successfully purified functional mGlu5 that activated the heterotrimeric G protein Gq. The high-affinity agonist-PAM VU0424465 also activated the purified receptor in the absence of an orthosteric agonist. In addition, it was found that purified mGlu5 was capable of activating the G protein Gs either upon stimulation with VU0424465 or glutamate, although the later induced a much weaker response. Our findings provide important mechanistic insights into mGlu5 G protein-dependent activity and selectivity.

 


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