Effect of a specific supplement enriched with n-3 polyunsaturated fatty acids on markers of inflammation, oxidative stress and metabolic status of ear, nose and throat cancer patients.

Malnutrition affects 40-50% of patients with ear, nose and throat (ENT) cancer. The aim of this study was to assess changes induced by a specific nutritional supplement enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes, as compared with a standard nutritional supplement, on markers of inflammation, oxidative stress and metabolic status of ENT cancer patients undergoing radiotherapy (RT). Fourteen days after starting RT, 26 patients were randomly allocated to one of two groups, 13 supplemented with Prosure, an oncologic formula enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes (specific supplement), and 13 supplemented with Standard-Isosource (standard supplement). Patients were evaluated before RT, and 14, 28 and 90 days after starting RT. The results showed that there were no significant differences between the groups, but greater changes were observed in the standard supplement group, such as a decline in body mass index (BMI), reductions in hematocrit, erythrocyte, eosinophil and albumin levels, and a rise in creatinine and urea levels. We concluded that metabolic, inflammatory and oxidative stress parameters were altered during RT, and began to normalize at the end of the study. Patients supplemented with Prosure showed an earlier normalization of these parameters, with more favorable changes in oxidative stress markers and a more balanced evolution, although the difference was not significant.

Long-term effects of varying consumption of ��3 fatty acids in ear, nose and throat cancer patients: assessment 1 year after radiotherapy.

Abstract A prospective 1-year follow-up study in ear, nose, and throat (ENT) cancer patients was carried out one year after radiotherapy to assess the effect of varying consumption of ��3 fatty acid according to whether they consumed more or less than the 50th percentile of ��3 fatty acids. Clinical, analytical, inflammatory (CRP and IL-6), and oxidative variables (TAC, GPx, GST, and SOD) were evaluated. The study comprised 31 patients (87.1% men), with a mean age of 61.3��������9.1 years. Hematological variables showed significant differences in the patients with a lower consumption of ��3 fatty acids. A lower mortality and longer survival were found in the group with ��3 fatty acid consumption ���50th percentile but the differences were not significant. No significant difference was reached in toxicity, inflammation, and oxidative stress markers. The group with ��3 fatty acid consumption <50th percentile significantly experienced more hematological and immune changes.

Connexins in lymphatic vessel physiology and disease.

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (&lt;1kDa) between the extracellular space and the cytosol. Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes.

Dental Phenotype in Jalili Syndrome Due to a c.1312 dupC Homozygous Mutation in the CNNM4 Gene.

Jalili syndrome denotes a recessively inherited combination of an eye disease (cone-rod dystrophy) and a dental disorder (amelogenesis imperfecta), which is caused by mutations in the CNNM4 gene. Whereas the ophthalmic consequences of these mutations have been studied comprehensively, the dental phenotype has obtained less attention. A defective transport of magnesium ions by the photoreceptors of the retina is assumed to account for the progressive visual impairment. Since magnesium is also incorporated in the mineral of dental hard tissues, we hypothesized that magnesium concentrations in defective enamel resulting from mutations in CNNM4 would be abnormal, if a similar deficiency of magnesium transport also accounted for the amelogenesis imperfecta. Thus, a detailed analysis of the dental hard tissues was performed in two boys of Kosovan origin affected by Jalili syndrome. Retinal dystrophy of the patients was diagnosed by a comprehensive eye examination and full-field electroretinography. A mutational analysis revealed a c.1312 dupC homozygous mutation in CNNM4, a genetic defect which had already been identified in other Kosovan families and putatively results in loss-of-function of the protein. The evaluation of six primary teeth using light and scanning electron microscopy as well as energy-dispersive X-ray spectroscopy showed that dental enamel was thin and deficient in mineral, suggesting a hypoplastic/hypomineralized type of amelogenesis imperfecta. The reduced mineral density of enamel was accompanied by decreased amounts of calcium, but significantly elevated levels of magnesium. In dentin, however, a similar mineral deficiency was associated with reduced magnesium and normal calcium levels. It is concluded that the c.1312 dupC mutation of CNNM4 results in mineralization defects of both enamel and dentin, which are associated with significantly abnormal magnesium concentrations. Thus, we could not disprove the hypothesis that a disrupted magnesium transport is involved in the development of the dental abnormalities observed in Jalili syndrome.

The Role of AtPHO1 in the Guard Cell Movements of Arabidopsis thaliana

In plants, stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume, a process that is in turn regulated by complex environ��mental and hormonal signals such as light and the phytohormone abscisic acid (ABA). With this study, we present genetic evidence that stomatal movements in response to ABA are influenced by PHOl expression in guard cells of Arabidopsis thaliana. PHOl is a phosphate exporter involved in phosphate loading into the root xylem ves��sels and, as a result, the phol mutant is characterized by low shoot phosphate lev��els. In leaves, PHOl was found expressed at higher level in guard cells, and was quickly up-regulated following treatment with ABA. The phol mutant was unaffected in ROS production following ABA treatment, and in stomatal movements in response to different light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Stomatal movements in re��sponse to hydrogen peroxide and reduced CO2 was altered as well. Micro-grafting a phol shoot scion onto wild-type root stock resulted in plants with normal shoot growth and Pi content, but failed to restore normal stomatal response to ABA treat-ment, showing that the impairment was not a simple pleiotropic consequence of phos��phate deficiency. PHOl knockdown using RNAi specifically in guard cells of wild-type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHOl in guard cells of phol plants complemented the mutant guard cell phenotype and re-established ABA sensitivity, although full functional complementation was co- dependent on shoot Pi sufficiency. Down-regulation of PHOl in guard cells did not alter the expression of ABA marker genes, indicating that PHOl does not affect the ABA signal transduction cascade at the transcriptional level. Together, these data reveal an important role for phosphate and PHOl action in the stomatal response to ABA. R��sum�� L'ouverture et la fermeture des stomates des plantes sont des mouvements contr��l��s par des flux d'ions causant des fluctuations de la turgescence des cellules de garde. Ce proc��d�� est en retour r��gul�� par des signaux environnementaux et hormonaux complexes, comme la lumi��re et l'hormone v��g��tale acide abscissique (ABA). Nous pr��sentons ici des preuves g��n��tiques montrant que les mouvements stomatiques en r��ponse �� l'ABA sont influenc��s par l'expression de PHOl dans les cellules de garde d'Arabidopsis thaliana. PHOl est un exporteur de phosphate, impliqu�� dans l'efflux de phosphate des cellules corticales racinaires vers les vaisseaux de xyl��me. En con��s��quence, le mutant phol est caract��ris�� par de faibles niveaux de phosphate dans les parties a��riennes. Dans les feuilles, PHOl est exprim�� pr��f��rentiellement dans les cellules de garde, compar�� au m��sophylle, et est rapidement induit par le traitement �� l'ABA. Le mutant phol n'est pas affect�� dans la perception de l'ABA, dans la pro��duction de ROS en r��ponse �� l'ABA, et dans la r��ponse des stomates aux traitements de lumi��re, �� l'auxine, �� la fusiccocine, et la forte concentration extracellulaire de cal��cium. En revanche, les mouvements de stomates en r��ponse aux traitements �� l'ABA sont fortement affect��s, dans l'induction de la fermeture des stomates comme dans l'inhibition de leur ouverture. De plus, les mouvements de stomates en r��ponse au p��roxyde d'hydrog��ne et �� la diminution du CO2 sont aussi compromis. La cr��ation de micro-greffes compos��es d'une partie a��rienne phol greff��s sur un syst��me racinaire sauvage g��n��re des plantes avec une croissance et une teneur en phosphate normale, mais ne permet pas de restaurer la r��ponse des stomates �� l'ABA, ce qui d��montre que le d��faut de r��ponse �� l'ABA n'est pas une simple cons��quence pl��iotropique de la carence en phosphate. La r��pression par RNAi de l'expression de PHOl dans les stomates de plantes sauvages provoque une r��duction de la r��ponse des stomates �� l'ABA, mais n'affecte pas la r��ponse de g��nes marqueurs �� l'ABA, ce qui sugg��re que PHOl n'agit pas au niveau transcriptionnel. Parall��lement, l'expression de PHOl dans les cellules de gardes de mutants phol compl��mente le ph��notype stomatique mutant et r��tablit la r��ponse �� l'ABA, bien que la totale compl��mentation n��cessite l'apport normal de phosphate aux parties a��riennes. Ensemble, ces r��sultats r��v����lent l'influence importante de PHOl et du phosphate dans la r��ponse des stomates �� l'ABA.

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

ASIC1a oligomerization structure-function of its extracellular vestibule and functional characterization of &#945;S243P&#946;&#611; ENaC

Quatre cristaux du canal ASIC1a ont ��t�� publi��s et soutiennent une stoechiom��trie trim��rique. Cependant, ces donn��es contredisant de pr��c��dentes analyses fonctionnelles effectu��es sur des canaux de la m��me famille, notre int��r��t fut port�� sur l'oligom��risation d'ASIC1a. Dans ce sens, un nouvel essai couplant la m��thode d'analyse par substitution de cyst��ines (SCAM) avec l'utilisation de r��actifs sulfhydryls bifonctionnels (crosslinkers) a ��t�� mis en place. Le but ��tant de stabiliser, puis s��lectionner les canaux fonctionnels, pour ensuite les s��parer selon leur taille par SDS-PAGE. Gr��ce �� cette technique, nous avons d��montr�� que le complexe stabilis�� a une taille co��ncidant avec une organisation t��tram��rique. En plus de son oligom��risation, le chemin emprunt�� par les ions pour traverser le canal n'est pas clairement d��fini dans ces structures. De ce fait, utilisant une approche ��lectrophysiologique, nous avons ��tudi�� le lien entre la structure et la fonction du vestibule extracellulaire d'ASIC1a. Dans ce but, nous nous sommes int��ress��s l'accessibilit�� de cyst��ines sp��cifiques localis��es dans ce vestibule pour des r��actifs m��thanethiosulfonates (MTS). Ainsi, nous avons pu corr��ler les cin��tiques de modification de ces cyst��ines par les MTS avec les effets sur le courant sodique, et donc avoir des informations suppl��mentaires sur la voie emprunt��e par les ions. De plus, la simulation informatique de liaison de ces r��actifs illustre le remplissage total de ce vestibule. Fonctionnellement, cette interaction ne perturbe pas le passage de ions, c'est pourquoi il nous appara��t probable que le vestibule pr��sente une taille plus large que celle illustr��e par les cristaux. Dans un deuxi��me temps, notre int��r��t fut port�� sur ENaC. Ce canal est compos�� des trois sous-unit��s (a, �� et y) et est exprim�� dans divers ��pith��liums, dont les tubules des reins. Il participe �� l'hom��ostasie sodique et est essentiellement r��gul�� par voie hormonale via l'aldost��rone et la Vasopressine, mais ��galement par des s��rines prot��ases ou le Na+. Nous avons ��tudi�� la r��percussion fonctionnelle de la mutation aS243P, d��couverte chez un nouveau-n�� pr��matur�� atteint de pseudohypoaldost��ronisme de type 1. Cette maladie autosomale r��cessive se caract��rise, g��n��ralement, par une hyponatr��mie li��e �� d'importantes pertes de sel dans les urines, une hyperkali��mie, ainsi qu'un niveau ��lev�� d'aldost��rone. Tout d'abord aucune des exp��riences biochimiques et ��lectrophysiologiques n'a pu d��montrer un d��faut d'expression ou une forte diminution de l'activit�� soutenant les donn��es cliniques. Cependant, en challengeant aS243P��yENaC avec une forte concentration de Na+ externe, une hypersensibilit�� de canal fut observ��e. En effet, ni les ph��nom��nes r��gulateurs de �� feedback inhibition �� ou de �� Na+ self-inhibition �� n'��taient semblables au canal sauvage. De ce fait, ils apparaissaient exacerb��s en pr��sence de la mutation, amenant ainsi �� une diminution de la r��absorption de Na+. Ceci corrobore enti��rement l'hyponatr��mie diagnostiqu��e. Le rein d'un pr��matur�� ��tant immature, la quantit�� de Na+ atteignant la partie distale du n��phron est plus ��lev��e, du fait que les autres m��canismes de r��absorption en amont ne sont probablement pas encore en place. Cette hypoth��se est renforc��e par l'existence d'un fr��re pr��sentant la m��me mutation, mais qui, n�� �� terme, ne pr��sentait aucun signe d'hyponatr��mie. - The main topic of my thesis is the structure-function relationship of the ENaC/Deg family of ion channels, namely the Acid-Sensing Ion Channel ASIC1a and the Epithelial Na Channel ENaC. The primary part of this research is dedicated to the structure of ASIC1a. Four channel crystals have been published, which support a trimeric stoichiometry, although these data contradict previous functional experiments on other ENaC/Deg members. We are therefore interested in ASIC1a oligomerization and have set up a new assay combining the Substituted- Cysteine Accessibility Method (SCAM) with Afunctional sulfhydryl reagents (crosslinkers) allowing its study. The aim was to first stabilize the channels, then select those that are functional and then resolve them according to their size on SDS-PAGE. We demonstrated that the stabilized complex has a molecular weight corresponding to a tetrameric stoichiometry. In addition to our interest in the oligomerization of the ENaC/Deg family of ion channels, we also wanted to investigate the thus far undefined way of permeation for these channels. Therefore, taking the advantage of a more electrophysiological approach, we studied the accessibility of specific cysteines for methanethiosulfonate reagents (MTS) and were able to correlate the MTS association kinetics on cysteine residues with Na+ currents. These results have given us an insight into ion permeation and our functional evidence indicates that the extracellular is larger than that depicted by the crystal structures. As a side project, we focused on ENaC, which is made up of three subunits (a, �� and y) and is expressed in various epithelia, especially in the distal nephron of the kidneys. It plays a role in Na+ homeostasis and is essentially regulated by hormones via aldosterone and vasopressin, but also by serine proteases or Na+. We have studied the functional impact of the aS243P mutation, discovered in a premature baby suffering from pseudohypoaldosteronism of type 1. This autosomal recessive disease is characterized by hyponatremia, hyperkalemia and high aldosterone levels. Firstly, neither biochemical nor electrophysiological experiments indicated an expression defect or a strong decrease in activity. However, challenging aS243P��yENaC with increased external Na+ concentration showed channel hypersensitivity. Indeed, both the "feedback inhibition" and the "Na+ self-inhibition" regulatory mechanisms are impaired, leading to a decrease in Na+ reabsorption, entirely supports the diagnosis. The kidneys in preterm infants are immature and Na+ levels reaching the distal nephron are higher than normally observed. We hypothesize that the upstream reabsorption machinery is unlikely to be sufficiently matured and this assumption is supported by an asymptomatic sibling carrying the same mutation, but born at term. - La cellule, unit�� fonctionnelle du corps humain, est d��limit��e par une membrane plasmique servant de barri��re biologique entre les milieux intra et extracellulaires. Une communication entre cellules est indispensable pour un fonctionnement ad��quat. Sa survie d��pend, entre autres, du maintien de la teneur en ions dans chacun des milieux qui doivent pouvoir ��tre r��absorb��s, ou s��cr��t��s, selon les besoins. Les prot��ines ins��r��es dans la membrane forment un canal et sont un moyen de communication permettant sp��cifiquement �� des ions tel que le sodium (Na+) de traverser. Le Na+ se trouve dans la plupart des aliments et le sel, et est sp��cifiquement r��absorb�� au niveau des reins gr��ce au canal sodique ��pith��lial ENaC. Cette r��absorption se fait de l'urine primaire vers l'int��rieur de la cellule, puis est transport�� vers le sang. Pour maintenir un ��quilibre, une r��gulation de ce canal est n��cessaire. En effet, des dysfonctionnements impliquant la r��gulation ou l'activit�� d'ENaC lui-m��me sont �� l'origine de maladies telles que la mucoviscidose, l'hypertension ou encore, le pseudohypoaldost��ronisme (PHA). Cette maladie est caract��ris��e, notamment, par d'importantes pertes de sel dans les urines. Des p��diatres ont diagnostiqu�� un PHA chez un nouveau-n��, ce dernier pr��sentant une modification du canal ENaC, nous avons recr���� cette prot��ine afin d'��tudier l'impact de ce changement sur son activit��. Nous avons d��montr�� que la r��gulation d'ENaC ��tait effectivement perturb��e, conduisant ainsi �� une forte r��duction de la r��absorption sodique. Afin de d��velopper des mol��cules capables de moduler l'activit�� de prot��ines. Il est n��cessaire d'en conna��tre la structure. Celle du canal sodique sensible �� l'acidification ASIC1, un canal cousin d'ENaC, est connue. Ces donn��es structurales contredisant cependant les analyses fonctionnelles, nous nous sommes pench��s une nouvelle fois sur ASIC1. Une prot��ine est une macromol��cule biologique compos��e d'une cha��ne d'acides amin��s (aa). De l'encha��nement d'aa �� la prot��ine fonctionnelle, quatre niveaux de structuration existent. Chaque aa donne une indication quant au repliement et plus particuli��rement la cyst��ine. Arborant un groupe sulfhydryle (SH) capable de former une liaison sp��cifique et stable avec un autre SH, celle-ci est souvent impliqu��e dans la structure tridimensionnelle de la prot��ine. Ce type de liaison intervient ��galement dans la stabilisation de la structure quaternaire, qui est l'association de plusieurs prot��ines identiques (homom��re), ou pas (h��t��rom��re). Dans cette partie, nous avons remplac�� des aa par des cyst��ines �� des endroits sp��cifiques. Le but ��tait de stabiliser plusieurs homom��res d'ASICl ensemble avec des r��actifs cr��ant des ponts entre deux SH. Ainsi, nous avons pu d��terminer le nombre de prot��ines ASIC1 participant �� la formation d'un canal fonctionnel. Nos r��sultats corroborent les donn��es fonctionnelles soutenant un canal t��tram��rique. Nous avons ��galement ��tudi�� l'accessibilit�� de ces nouvelles cyst��ines afin d'obtenir des informations suppl��mentaires sur la structure du chemin emprunt�� par le Na+ �� travers ASIC1 et plus particuli��rement du vestibule extracellulaire.

Metallic and nonmetallic shine in luster: An elastic ion backscattering study

Luster is a metal glass nanocomposite layer first produced in the Middle East in early Islamic times ( 9th AD) made of metal copper or silver nanoparticles embedded in a silica-based glassy matrix. These nanoparticles are produced by ion exchange between Cu+ and Ag+ and alkaline ions from the glassy matrix and further growth in a reducing atmosphere. The most striking property of luster is its capability of reflecting light like a continuous metal layer and it was unexpectedly found to be linked to one single production parameter: the presence of lead in the glassy matrix composition. The purpose of this article is to describe the characteristics and differences of the nanoparticle layers developed on lead rich and lead free glasses. Copper luster layers obtained using the ancient recipes and methods are analyzed by means of elastic ion backscattering spectroscopy associated with other analytical techniques. The depth profile of the different elements is determined, showing that the luster layer formed in lead rich glasses is 5���6 times thinner and 3���4 times Cu richer. Therefore, the metal nanoparticles are more densely packed in the layer and this fact is related to its higher reflectivity. It is shown that lead influences the structure of the metal nanoparticle layer through the change of the precipitation kinetics

A gating mutation in the internal pore of ASIC1a.

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.

Multifunctional ligands for application in Alzheimer���s Disease: molecular modelling and biological evaluation

Projecte de recerca elaborat a partir d���una estada a la University of British Columbia, Canad��, entre 2010 i 2012 La malaltia d'Alzheimer (MA) representa avui la forma m��s comuna de dem��ncia en la poblaci�� envellida. Malgrat fa 100 anys que va ser descoberta, encara avui no existeix cap tractament preventiu i/o curatiu ni cap agent de diagn��stic que permeti valorar quantitativament l'evoluci�� d'aquesta malaltia. L'objectiu en el que s'emmarca aquest treball ��s contribuir a aportar solucions al problema de la manca d'agents terap��utics i de diagnosi, un��vocs i rigorosos, per a la MA. Des del camp de la qu��mica bioinorg��nica ��s f��cil fixar-se en l'excessiva concentraci�� d'ions Zn(II) i Cu(II) en els cervells de malalts de MA, plantejar-se la seva utilitzaci�� com a dianes terap��utica i, en conseq����ncia, cercar agents quelants que evitin la formaci�� de plaques senils o contribueixin a la seva dissoluci��. Si b�� aquest va ser el punt de partida d���aquest projecte, els m��ltiples factors implicats en la patog��nesi de la MA fan que el cl��ssic paradigma d��� ��una mol��cula, una diana�� limiti la capacitat de la mol��cula de combatre aquesta malaltia tan complexa. Per tant, un esfor�� considerable s���ha dedicat al disseny d���agentsmultifuncionals que combatin els m��ltiples factors que caracteritzen el desenvolupament de la MA. En el present treball s���han dissenyat agents multifuncionals inspirats en dos esquelets moleculars ben establers i coneguts en el camp de la qu��mica medicinal: la tioflavina-T (ThT) i la deferiprona (DFP). La utilitzaci�� de t��cniques in silico que inclouen c��lculs farmacocin��tics i modelatge molecular ha estat un proc��s cabdal per a l���avaluaci�� dels millors candidats en base als seg��ents requeriments: (a) compliment de determinades propietats farmacocin��tiques que estableixin el seu possible ��s com a f��rmac (b) hidrofobicitat adequada per travessar la BBB i (c) interacci�� amb el p��ptid A������en soluci��.

Quantification of 4 antidepressants and a metabolite by LC-MS for therapeutic drug monitoring.

A liquid chromatography method coupled to mass spectrometry was developed for the quantification of bupropion, its metabolite hydroxy-bupropion, moclobemide, reboxetine and trazodone in human plasma. The validation of the analytical procedure was assessed according to Soci��t�� Fran��aise des Sciences et Techniques Pharmaceutiques and the latest Food and Drug Administration guidelines. The sample preparation was performed with 0.5mL of plasma extracted on a cation-exchange solid phase 96-well plate. The separation was achieved in 14min on a C18 XBridge column (2.1mm��100mm, 3.5&#956;m) using a 50mM ammonium acetate pH 9/acetonitrile mobile phase in gradient mode. The compounds of interest were analysed in the single ion monitoring mode on a single quadrupole mass spectrometer working in positive electrospray ionisation mode. Two ions were selected per molecule to increase the number of identification points and to avoid as much as possible any false positives. Since selectivity is always a critical point for routine therapeutic drug monitoring, more than sixty common comedications for the psychiatric population were tested. For each analyte, the analytical procedure was validated to cover the common range of concentrations measured in plasma samples: 1-400ng/mL for reboxetine and bupropion, 2-2000ng/mL for hydroxy-bupropion, moclobemide, and trazodone. For all investigated compounds, reliable performance in terms of accuracy, precision, trueness, recovery, selectivity and stability was obtained. One year after its implementation in a routine process, this method demonstrated a high robustness with accurate values over the wide concentration range commonly observed among a psychiatric population.

Review article: Intestinal epithelia and barrier functions.

The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.

Selective regulation of acid-sensing ion channel 1 by serine proteases.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.

Practical considerations for conducting ecotoxicity test methods with manufactured nanomaterials: what have we learnt so far?

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench. [Authors]

The future key role of LC-high-resolution-MS analyses in clinical laboratories: a focus on quantification.

For the last decade, high-resolution (HR)-MS has been associated with qualitative analyses while triple quadrupole MS has been associated with routine quantitative analyses. However, a shift of this paradigm is taking place: quantitative and qualitative analyses will be increasingly performed by HR-MS, and it will become the common 'language' for most mass spectrometrists. Most analyses will be performed by full-scan acquisitions recording 'all' ions entering the HR-MS with subsequent construction of narrow-width extracted-ion chromatograms. Ions will be available for absolute quantification, profiling and data mining. In parallel to quantification, metabotyping will be the next step in clinical LC-MS analyses because it should help in personalized medicine. This article is aimed to help analytical chemists who perform targeted quantitative acquisitions with triple quadrupole MS make the transition to quantitative and qualitative analyses using HR-MS. Guidelines for the acceptance criteria of mass accuracy and for the determination of mass extraction windows in quantitative analyses are proposed.