New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1 × 10−5 and 5.5 × 10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

Selective recognition in potentiometric transduction of amoxicillin by molecularly imprinted materials

The indiscriminate use of antibiotics in food-producing animals has received increasing attention as a contributory factor in the international emergence of antibiotic-resistant bacteria (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004). Numerous analytical methods for quantifying antibacterial residues in edible animal products have been developed over years (Woodward in Pesticide, veterinary and other residues in food, CRC Press, Boca Raton, 2004; Botsoglou and Fletouris in Handbook of food analysis, residues and other food component analysis, Marcel Dekker, Ghent, 2004). Being Amoxicillin (AMOX) one of those critical veterinary drugs, efforts have been made to develop simple and expeditious methods for its control in food samples. In literature, only one AMOX-selective electrode has been reported so far. In that work, phosphotungstate:amoxycillinium ion exchanger was used as electroactive material (Shoukry et al. in Electroanalysis 6:914–917, 1994). Designing new materials based on molecularly imprinted polymers (MIPs) which are complementary to the size and charge of AMOX could lead to very selective interactions, thus enhancing the selectivity of the sensing unit. AMOX-selective electrodes used imprinted polymers as electroactive materials having AMOX as target molecule to design a biomimetic imprinted cavity. Poly(vinyl chloride), sensors of methacrylic acid displayed Nernstian slopes (60.7 mV/decade) and low detection limits (2.9 × 10−5 mol/L). The potentiometric responses were not affected by pH within 4–5 and showed good selectivity. The electrodes were applied successfully to the analysis of real samples.

Nitrite biosensing via selective enzymes—a long but promising route

Sensors 2010, 10, 11530-11555

How to sell successfully a perfume in the non-selective market?

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Frequency-domain receiver design for doubly-selective channels

This work is devoted to the broadband wireless transmission techniques, which are serious candidates to be implemented in future broadband wireless and cellular systems, aiming at providing high and reliable data transmission and concomitantly high mobility. In order to cope with doubly-selective channels, receiver structures based on OFDM and SC-FDE block transmission techniques, are proposed, which allow cost-effective implementations, using FFT-based signal processing. The first subject to be addressed is the impact of the number of multipath components, and the diversity order, on the asymptotic performance of OFDM and SC-FDE, in uncoded and for different channel coding schemes. The obtained results show that the number of relevant separable multipath components is a key element that influences the performance of OFDM and SC-FDE schemes. Then, the improved estimation and detection performance of OFDM-based broadcasting systems, is introduced employing SFN (Single Frequency Network) operation. An initial coarse channel is obtained with resort to low-power training sequences estimation, and an iterative receiver with joint detection and channel estimation is presented. The achieved results have shown very good performance, close to that with perfect channel estimation. The next topic is related to SFN systems, devoting special attention to time-distortion effects inherent to these networks. Typically, the SFN broadcast wireless systems employ OFDM schemes to cope with severely time-dispersive channels. However, frequency errors, due to CFO, compromises the orthogonality between subcarriers. As an alternative approach, the possibility of using SC-FDE schemes (characterized by reduced envelope fluctuations and higher robustness to carrier frequency errors) is evaluated, and a technique, employing joint CFO estimation and compensation over the severe time-distortion effects, is proposed. Finally, broadband mobile wireless systems, in which the relative motion between the transmitter and receiver induces Doppler shift which is different or each propagation path, is considered, depending on the angle of incidence of that path in relation to the direction of travel. This represents a severe impairment in wireless digital communications systems, since that multipath propagation combined with the Doppler effects, lead to drastic and unpredictable fluctuations of the envelope of the received signal, severely affecting the detection performance. The channel variations due this effect are very difficult to estimate and compensate. In this work we propose a set of SC-FDE iterative receivers implementing efficient estimation and tracking techniques. The performance results show that the proposed receivers have very good performance, even in the presence of significant Doppler spread between the different groups of multipath components.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1.

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis.

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.

Fast screening and confirmation of doping agents by UHPLC-QTOF-MS/MS

Introduction: The general strategy to perform anti-doping analysis starts with a screening followed by a confirmatory step when a sample is suspected to be positive. The screening step should be fast, generic and able to highlight any sample that may contain a prohibited substance by avoiding false negative and reducing false positive results. The confirmatory step is a dedicated procedure comprising a selective sample preparation and detection mode. Aim: The purpose of the study is to develop rapid screening and selective confirmatory strategies to detect and identify 103 doping agents in urine. Methods: For the screening, urine samples were simply diluted by a factor 2 with ultra-pure water and directly injected ("dilute and shoot") in the ultrahigh- pressure liquid chromatography (UHPLC). The UHPLC separation was performed in two gradients (ESI positive and negative) from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. The gradient analysis time is 9 min including 3 min reequilibration. Analytes detection was performed in full scan mode on a quadrupole time-of-flight (QTOF) mass spectrometer by acquiring the exact mass of the protonated (ESI positive) or deprotonated (ESI negative) molecular ion. For the confirmatory analysis, urine samples were extracted on SPE 96-well plate with mixed-mode cation (MCX) for basic and neutral compounds or anion exchange (MAX) sorbents for acidic molecules. The analytes were eluted in 3 min (including 1.5 min reequilibration) with a S1-25 Ann Toxicol Anal. 2009; 21(S1) Abstracts gradient from 5/95 to 95/5% of MeCN/Water containing 0.1% formic acid. Analytes confirmation was performed in MS and MS/MS mode on a QTOF mass spectrometer. Results: In the screening and confirmatory analysis, basic and neutral analytes were analysed in the positive ESI mode, whereas acidic compounds were analysed in the negative mode. The analyte identification was based on retention time (tR) and exact mass measurement. "Dilute and shoot" was used as a generic sample treatment in the screening procedure, but matrix effect (e.g., ion suppression) cannot be avoided. However, the sensitivity was sufficient for all analytes to reach the minimal required performance limit (MRPL) required by the World Anti Doping Agency (WADA). To avoid time-consuming confirmatory analysis of false positive samples, a pre-confirmatory step was added. It consists of the sample re-injection, the acquisition of MS/MS spectra and the comparison to reference material. For the confirmatory analysis, urine samples were extracted by SPE allowing a pre-concentration of the analyte. A fast chromatographic separation was developed as a single analyte has to be confirmed. A dedicated QTOF-MS and MS/MS acquisition was performed to acquire within the same run a parallel scanning of two functions. Low collision energy was applied in the first channel to obtain the protonated molecular ion (QTOF-MS), while dedicated collision energy was set in the second channel to obtain fragmented ions (QTOF-MS/MS). Enough identification points were obtained to compare the spectra with reference material and negative urine sample. Finally, the entire process was validated and matrix effects quantified. Conclusion: Thanks to the coupling of UHPLC with the QTOF mass spectrometer, high tR repeatability, sensitivity, mass accuracy and mass resolution over a broad mass range were obtained. The method was sensitive, robust and reliable enough to detect and identify doping agents in urine. Keywords: screening, confirmatory analysis, UHPLC, QTOF, doping agents

Glutamatergic regulation of mitochondrial ion dynamics in astrocytes

Résumé grand public :Le cerveau se compose de cellules nerveuses appelées neurones et de cellules gliales dont font partie les astrocytes. Les neurones communiquent entre eux par signaux électriques et en libérant des molécules de signalisation comme le glutamate. Les astrocytes ont eux pour charge de capter le glucose depuis le sang circulant dans les vaisseaux sanguins, de le transformer et de le transmettre aux neurones pour qu'ils puissent l'utiliser comme source d'énergie. L'astrocyte peut ensuite utiliser ce glucose de deux façons différentes pour produire de l'énergie : la première s'opère dans des structures appelées mitochondries qui sont capables de produire plus de trente molécules riches en énergie (ATP) à partir d'une seule molécule de glucose ; la seconde possibilité appelée glycolyse peut produire deux molécules d'ATP et un dérivé du glucose appelé lactate. Une théorie couramment débattue propose que lorsque les astrocytes capturent le glutamate libéré par les neurones, ils libèrent en réponse du lactate qui servirait de base énergétique aux neurones. Cependant, ce mécanisme n'envisage pas une augmentation de l'activité des mitochondries des astrocytes, ce qui serait pourtant bien plus efficace pour produire de l'énergie.En utilisant la microscopie par fluorescence, nous avons pu mesurer les changements de concentrations ioniques dans les mitochondries d'astrocytes soumis à une stimulation glutamatergique. Nous avons démontré que les mitochondries des astrocytes manifestent des augmentations spontanées et transitoires de leur concentrations ioniques, dont la fréquence était diminuée au cours d'une stimulation avec du glutamate. Nous avons ensuite montré que la capture de glutamate augmentait la concentration en sodium et acidifiait les mitochondries des astrocytes. En approfondissant ces mécanismes, plusieurs éléments ont suggéré que l'acidification induite diminuerait le potentiel de synthèse d'énergie d'origine mitochondriale et la consommation d'oxygène dans les astrocytes. En résumé, l'ensemble de ces travaux suggère que la signalisation neuronale impliquant le glutamate dicte aux astrocytes de sacrifier temporairement l'efficacité de leur métabolisme énergétique, en diminuant l'activité de leurs mitochondries, afin d'augmenter la disponibilité des ressources énergétiques utiles aux neurones.Résumé :La remarquable efficacité du cerveau à compiler et propager des informations coûte au corps humain 20% de son budget énergétique total. Par conséquent, les mécanismes cellulaires responsables du métabolisme énergétique cérébral se sont adéquatement développés pour répondre aux besoins énergétiques du cerveau. Les dernières découvertes en neuroénergétique tendent à démontrer que le site principal de consommation d'énergie dans le cerveau est situé dans les processus astrocytaires qui entourent les synapses excitatrices. Un nombre croissant de preuves scientifiques a maintenant montré que le transport astrocytaire de glutamate est responsable d'un coût métabolique important qui est majoritairement pris en charge par une augmentation de l'activité glycolytique. Cependant, les astrocytes possèdent également un important métabolisme énergétique de type mitochondrial. Par conséquent, la localisation spatiale des mitochondries à proximité des transporteurs de glutamate suggère l'existence d'un mécanisme régulant le métabolisme énergétique astrocytaire, en particulier le métabolisme mitochondrial.Afin de fournir une explication à ce paradoxe énergétique, nous avons utilisé des techniques d'imagerie par fluorescence pour mesurer les modifications de concentrations ioniques spontanées et évoquées par une stimulation glutamatergique dans des astrocytes corticaux de souris. Nous avons montré que les mitochondries d'astrocytes au repos manifestaient des changements individuels, spontanés et sélectifs de leur potentiel électrique, de leur pH et de leur concentration en sodium. Nous avons trouvé que le glutamate diminuait la fréquence des augmentations spontanées de sodium en diminuant le niveau cellulaire d'ATP. Nous avons ensuite étudié la possibilité d'une régulation du métabolisme mitochondrial astrocytaire par le glutamate. Nous avons montré que le glutamate initie dans la population mitochondriale une augmentation rapide de la concentration en sodium due à l'augmentation cytosolique de sodium. Nous avons également montré que le relâchement neuronal de glutamate induit une acidification mitochondriale dans les astrocytes. Nos résultats ont indiqué que l'acidification induite par le glutamate induit une diminution de la production de radicaux libres et de la consommation d'oxygène par les astrocytes. Ces études ont montré que les mitochondries des astrocytes sont régulées individuellement et adaptent leur activité selon l'environnement intracellulaire. L'adaptation dynamique du métabolisme énergétique mitochondrial opéré par le glutamate permet d'augmenter la quantité d'oxygène disponible et amène au relâchement de lactate, tous deux bénéfiques pour les neurones.Abstract :The remarkable efficiency of the brain to compute and communicate information costs the body 20% of its total energy budget. Therefore, the cellular mechanisms responsible for brain energy metabolism developed adequately to face the energy needs. Recent advances in neuroenergetics tend to indicate that the main site of energy consumption in the brain is the astroglial process ensheating activated excitatory synapses. A large body of evidence has now shown that glutamate uptake by astrocytes surrounding synapses is responsible for a significant metabolic cost, whose metabolic response is apparently mainly glycolytic. However, astrocytes have also a significant mitochondrial oxidative metabolism. Therefore, the location of mitochondria close to glutamate transporters raises the question of the existence of mechanisms for tuning their energy metabolism, in particular their mitochondrial metabolism.To tackle these issues, we used real time imaging techniques to study mitochondrial ionic alterations occurring at resting state and during glutamatergic stimulation of mouse cortical astrocytes. We showed that mitochondria of intact resting astrocytes exhibited individual spontaneous and selective alterations of their electrical potential, pH and Na+ concentration. We found that glutamate decreased the frequency of mitochondrial Na+ transient activity by decreasing the cellular level of ATP. We then investigated a possible link between glutamatergic transmission and mitochondrial metabolism in astrocytes. We showed that glutamate triggered a rapid Na+ concentration increase in the mitochondrial population as a result of plasma-membrane Na+-dependent uptake. We then demonstrated that neuronally released glutamate also induced a mitochondrial acidification in astrocytes. Glutamate induced a pH-mediated and cytoprotective decrease of mitochondrial metabolism that diminished oxygen consumption. Taken together, these studies showed that astrocytes contain mitochondria that are individually regulated and sense the intracellular environment to modulate their own activity. The dynamic regulation of astrocyte mitochondrial energy output operated by glutamate allows increasing oxygen availability and lactate production both being beneficial for neurons.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.

Selective acquired long QT syndrome (saLQTS) upon risperidone treatment.

ABSTRACT: BACKGROUND: Numerous structurally unrelated drugs, including antipsychotics, can prolong QT interval and trigger the acquired long QT syndrome (aLQTS). All of them are thought to act at the level of KCNH2, a subunit of the potassium channel. Although the QT-prolonging drugs are proscribed in the subjects with aLQTS, the individual response to diverse QT-prolonging drugs may vary substantially. CASE PRESENTATION: We report here a case of aLQTS in response to small doses of risperidone that was confirmed at three independent drug challenges in the absence of other QT-prolonging drugs. On the other hand, the patient did not respond with QT prolongation to some other antipsychotics. In particular, the administration of clozapine, known to be associated with higher QT-prolongation risk than risperidone, had no effect on QT-length. A detailed genetic analysis revealed no mutations or polymorphisms in KCNH2, KCNE1, KCNE2, SCN5A and KCNQ1 genes. CONCLUSIONS: Our observation suggests that some patients may display a selective aLQTS to a single antipsychotic, without a potassium channel-related genetic substrate. Contrasting with the idea of a common target of the aLQTS-triggerring drugs, our data suggests existence of an alternative target protein, which unlike the KCNH2 would be drug-selective.

Genetic male infertility and mutation of CATSPER ion channels.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Role of the epithelial sodium channel (ENaC) and its positive regulator, the channel-activating protease 1 (CAP1) in skin

Summary: The mammalian epidermis is a pluristratified epithelium composed of 90% keratinocytes, and its main function is to serve as barrier for the body. The epithelial sodium channel (ENaC), formed by three homologous subunits α, β and γ is found in a variety of epithelia including epidermis. Previous studies showed that ENaC modulates different aspects of epidermal differentiation, such as synthesis of differentiation-specific proteins and lipid secretion. ENaC plays also a critical role in sodium homeostasis of renal and pulmonary epithelia, and its activity is thereby well controlled by hormones and non-hormonal factors, such as the serine protease CAP1 (channel-activating protease 1), also termed prostasin encoded by Prss8 gene. Serine proteases are proteolytic enzymes involved in numerous physiological and pathological processes in the epidermis. In order to evaluate the role of β and γENaC in epidermis, we analyzed the skin phenotype of β and γENaC null mutant (βENaC-/- and γENaC-/-) mice in comparison with the phenotype of αENaC-deficient mice. Furthermore, keratin14-specific CAP1-deficient mice (Prss8lox/Δ /K14-Cre) were generated in order to unveil the role of the serine protease CAP1 in epidermal development and function. This study reveals that the skin phenotype of βENaC and γENaC null mutant mice is less severe than the one of αENaC-deficient mice. However, all these mice present a common premature lipid secretion in the mid-granular layer of the epidermis. Further, the composition of the lipids of the stratum corneum in αENaC-deficient mice is strongly altered, suggesting that epidermal barrier function is compromised. K14-specific CAP1-deficient newborn mice are born at the expected Mendelian ratio, but die soon after birth, showing that CAP1 is required for postnatal survival. The epidermis of these mice exhibits striking malformations of the stratum corneum showing hyperkeratosis. These defects seriously affect both inward and outward epidermal barrier function, leading to rapid and fatal dehydration. As in αENaC-deficient mice, the lipid composition of the stratum corneum of K14-specific CAP1-deficient mice is disturbed. Furthermore, lack of CAP1 leads to the selective loss of filaggrin monomers, important for keratins aggregation and skin moisturization, and to an increased of aberrant profilaggrin precursors. In conclusion, both ENaC and CAP1 expression in the epidermis are crucial for keratinocyte differentiation processes and/or barrier function. Since the abnormalities in K14-specific CAP1-deficient mice resemble key features of human skin ichthyosis, in particular Harlequin ichthyosis, the study of ENaC and CAP1 mutant mice might allow new insights into mechanisms underlying skin diseases. Résumé: L'épiderme des mammifères est un épithélium pluristratifié, protégeant le corps contre les perturbations extérieures et la déshydratation. Le canal épithélial à sodium (ENaC), formé de trois sous-unités α, β et γ, est exprimé dans de nombreux épithélia, comme l'épiderme. Des études ont montré que l'absence de la sous-unité αENaC modulait différents aspects de la différenciation des kératinocytes de l'épiderme, comme la synthèse de protéines spécifiques ou la sécrétion de lipides dans la couche granulaire de l'épiderme. ENaC joue également un rôle crucial dans l'homéostasie du sodium dans les épithélia électriquement étanches, comme l'épithélium rénal ou pulmonaire. L'activité de ENaC est par conséquent finement régulée, en partie par des hormones, mais aussi par des facteurs non-hormonaux, telle que la sérine protéase CAP1 (« channel-activating protease 1 >>) (nommée également prostasine et codée par le gène Prss8). Le but de ce travail a donc été d'étudier le rôle des sous-unités β et γENaC dans l'épiderme en comparaison avec celui de la sous-unité α en utilisant des souris mutantes βENaC-/- et γENaC-/-. Dans un deuxième temps, le phénotype d'une souris chez qui CAP1 a été spécifiquement invalidé dans l'épiderme (Prsslox/Δ/K14-Cre) a été analysé, dans le but de mettre en évidence le rôle de cette protéase dans l'épiderme. Comme déjà montré pour les souris αENaC-/-, la sécrétion des lipides dans la couche granulaire de l'épiderme des souris βENaC-/- et γENaC-/- est prématurée. Cependant, l'hyperplasie et l'expression anormale des protéines marqueurs de la différenciation présents chez les souris αENaC-/- n'ont pas été observés dans l'épiderme des souris βENaC-/- et γENaC-/-. La composition lipidique de la couche cornée des souris αENaC-/- est fortement altérée suggérant que la fonction de barrière de l'épiderme de ces souris est compromise. Les souris mutantes CAP1 ont quant à elles révélé des malformations sévères de leur couche cornée, affectant la fonction de barrière de leur épiderme et conduisant à la mort de ces souris par déshydratation quelques jours après leur naissance. De plus, la composition en lipides de la couche cornée ainsi que la taille des cellules cornées, les cornéocytes, de ces souris sont modifiées par rapport aux souris contrôles. L'invalidation de la protéine CAP1 dans l'épiderme conduit aussi à la perte de la filaggrine, une protéine cruciale pour l'agrégation des kératines dans la couche cornée et le maintien du niveau d'hydratation de la peau, et à l'accumulation de ses précurseurs. En conclusion, l'expression de ENaC et de CAP1 est cruciale pour la différenciation de l'épiderme et/ou sa fonction de barrière. De plus, le phénotype des souris mutantes CAP1 présente des caractéristiques qui ressemblent à celles observées dans certaines pathologies humaines cutanées, comme l'ichthyose d'Harlequin. L'étude des souris mutantes ENaC et CAP1 pourrait donc apporter de nouvelles connaissances dans les mécanismes impliqués dans l'ichthyose d'Harlequin ou d'autres maladies de la peau chez l'homme. Résumé tout public: La peau est le plus grand organe vital du corps humain. Sa fonction principale est de protéger le corps comme une barrière, contre les agressions extérieures et la déshydratation. De nombreuses maladies de la peau résultent d'une perte de fonction de cette barrière. Bien que les pathologies cutanées soient très bien décrites, leur cause génétique n'est en général pas encore connue. La souris est alors un modèle de choix pour la recherche fondamentale. En effet, grâce aux progrès récents de la science, le génome de la souris peut aujourd'hui être modifié dans le but d'étudier le rôle de nombreuses protéines. Dans différents organes, comme le rein et le poumon, le canal épithélial à sodium (ENaC), composé de trois sous-unités protéiques homologues (α, β, et γ), joue un rôle essentiel dans la réabsorption du sodium. L'activité de ENaC est régulée par de nombreux facteurs hormonaux et non-hormonaux, telle que la protéase CAP1 (« channel-activating protease 1 »). L'invalidation de la sous-unité αENaC chez la souris a permis de montrer que dans la peau, le canal ENaC est impliqué dans la différenciation des cellules de l'épiderme et la croissance des poils. Durant ce travail, le phénotype des souris chez qui la protéine βENaC, γENaC ou CAP1 a été invalidée (souris mutantes), a été étudié dans le but de mieux comprendre le rôle des sous-unités du canal ENaC et de son régulateur CAP1 dans la peau. Les résultats de ce projet ont montré que les souris mutantes βENaC et γENaC présentent un épiderme anormal avec une synthèse prématurée de lipides dans la couche granulaire, suggérant l'implication de ENaC dans la fonction de barrière de la peau. De plus, quand CAP1 est invalidé de manière totale chez les souris, le développement embryonnaire est perturbé et ces souris meurent avant la naissance. CAP1 a donc été invalidé spécifiquement dans l'épiderme des souris. Ces souris mutantes « épiderme-spécifique » naissent normalement, mais meurent peu après la naissance de déshydratation. La couche superficielle de l'épiderme, appelée couche cornée, de ces souris est malformée et ne confère plus à la peau sa fonction de barrière. De plus, les composants de la couche cornée, les cellules cornées entourées de lipides, sont sévèrement altérés. Le phénotype de ces souris ressemble aux caractéristiques présentes chez les patients atteints d'ichthyoses, en particulier l'ichthyose d'Harlequin. En conclusion, le canal ENaC ainsi que son régulateur CAP1 jouent un rôle clé dans les processus de différenciation de l'épiderme et/ou de sa fonction de barrière. De plus, les souris mutantes pour CAP1 et ENaC se révéleront peut-être comme des modèles appropriés dans l'étude de l'ichthyose d'Harlequin ou d'autres maladies cutanées.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.