Optimizing potentiometric ionophore and electrode design for environmental on-site control of antibiotic drugs: Application to sulfamethoxazole

Potentiometric sensors are typically unable to carry out on-site monitoring of environmental drug contaminants because of their high limits of detection (LODs). Designing a novel ligand material for the target analyte and managing the composition of the internal reference solution have been the strategies employed here to produce for the first time a potentiometric-based direct reading method for an environmental drug contaminant. This concept has been applied to sulfamethoxazole (SMX), one of the many antibiotics used in aquaculture practices that may occur in environmental waters. The novel ligand has been produced by imprinting SMX on the surface of graphitic carbon nanostructures (CN) < 500 nm. The imprinted carbon nanostructures (ICN) were dispersed in plasticizer and entrapped in a PVC matrix that included (or not) a small amount of a lipophilic additive. The membrane composition was optimized on solid-contact electrodes, allowing near-Nernstian responses down to 5.2 μg/mL and detecting 1.6 μg/mL. The membranes offered good selectivity against most of the ionic compounds in environmental water. The best membrane cocktail was applied on the smaller end of a 1000 μL micropipette tip made of polypropylene. The tip was then filled with inner reference solution containing SMX and chlorate (as interfering compound). The corresponding concentrations were studied for 1 × 10−5 to 1 × 10−10 and 1 × 10−3 to 1 × 10−8 mol/L. The best condition allowed the detection of 5.92 ng/L (or 2.3 × 10−8 mol/L) SMX for a sub-Nernstian slope of −40.3 mV/decade from 5.0 × 10−8 to 2.4 × 10−5 mol/L.

Rapid automated method for on-site determination of sulfadiazine in fish farming: a stainless steel veterinary syringe coated with a selective membrane of PVC serving as a potentiometric detector in a flow-injection-analysis system

Sulfadiazine is an antibiotic of the sulfonamide group and is used as a veterinary drug in fish farming. Monitoring it in the tanks is fundamental to control the applied doses and avoid environmental dissemination. Pursuing this goal, we included a novel potentiometric design in a flow-injection assembly. The electrode body was a stainless steel needle veterinary syringe of 0.8-mm inner diameter. A selective membrane of PVC acted as a sensory surface. Its composition, the length of the electrode, and other flow variables were optimized. The best performance was obtained for sensors of 1.5-cm length and a membrane composition of 33% PVC, 66% onitrophenyloctyl ether, 1% ion exchanger, and a small amount of a cationic additive. It exhibited Nernstian slopes of 61.0 mV decade-1 down to 1.0×10-5 mol L-1, with a limit of detection of 3.1×10-6 mol L-1 in flowing media. All necessary pH/ionic strength adjustments were performed online by merging the sample plug with a buffer carrier of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 4.9. The sensor exhibited the advantages of a fast response time (less than 15 s), long operational lifetime (60 days), and good selectivity for chloride, nitrite, acetate, tartrate, citrate, and ascorbate. The flow setup was successfully applied to the analysis of aquaculture waters. The analytical results were validated against those obtained with liquid chromatography–tandem mass spectrometry procedures. The sampling rate was about 84 samples per hour and recoveries ranged from 95.9 to 106.9%.

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Biochemistry, 2004, 43 (46), pp 14566–14576 DOI: 10.1021/bi0485833

Terapia celular com células mesenquimais por via intracoronária: contributos do estudo invasivo da microcirculação coronária

RESUMO: Introdução - A utilização de células e das suas propriedades para o tratamento das doenças cardiovasculares, é uma promessa para o futuro e talvez a única forma de ultrapassar algumas das insuficiências das terapêuticas atuais. A via de entrega das células mais utilizada na investigação tem sido a intracoronária, ganhando a microcirculação especial relevância, por ser onde ocorre a primeira interação com o tecido nativo. As células estaminais mesenquimais (CEM) têm propriedades que as tornam particularmente aptas para a Terapia Celular, mas as suas dimensões, superiores ao diâmetro dos capilares, tem motivado controvérsia quanto à sua entrega intracoronária. A cardiologia de intervenção tem atualmente técnicas que permitem a avaliação em tempo real e in vivo do estado da microcirculação coronária. A determinação do índice da resistência da microcirculação (IRM) fornece informação sobre a circulação dos pequenos vasos, de forma independente da circulação coronária e do estado hemodinâmico, mas a aplicabilidade clínica deste conhecimento encontra-se ainda por definir. Objectivos Esclarecer o potencial do IRM no estudo dos efeitos do transplante de CEM por via intracoronária. População e Métodos . Estudo pré-clínico com modelo animal (suíno) desenvolvido em 3 fases. Na Primeira Fase foram utilizados 8 animais saudáveis para estudar e validar a técnica de determinação de estudo da microcirculação. Efetuou-se a determinação do IRM com duas doses diferentes de papaverina para a indução da resposta hiperémica máxima (5 e 10 mg) e após a disfunção da microcirculação com injeção intracoronária de microesferas de embozene com 40 μm de diâmetro. Na Segunda Fase foram utilizados 18 animais saudáveis, randomizados em grupo controlo e grupo recetor de 30 x 106 CEM por via intracoronária. Foram avaliados de forma cega o IRM, a pressão aórtica, o fluxo coronário epicárdico e a ocorrência de alterações electrocardiográficas. Na Terceira Fase foram utilizados 18 animais, com enfarte agudo do miocárdio provocado (EAM), randomizados em grupo controlo, grupo recetor de CEM expandidas de forma convencional e grupo recetor de CEM expandidas com metodologia inovadora e de menores dimensões. Foi realizada uma exploração da dose/efeito com infusão faseada de 10 x 106, 15 x 106 e 20 x 106 CEM, com determinação do IRM, da pressão aórtica, do fluxo coronário epicárdico e da ocorrência de alterações eletrocardiográficas. Quatro semanas após a entrega das células foi novamente avaliado o IRM e foi efetuado o estudo anatomopatológico dos animais na procura de evidência de neoangiogénese e de regeneração miocárdica, ou de um efeito positivo da resposta reparadora após o enfarte. Resultados Nas 3 fases todos os animais mantiveram estabilidade hemodinâmica e eletrocardiográfica, com exceção da elevação de ST de V1-V3 verificada após a injeção das microesferas. Na Primeira Fase as duas doses de papaverina induziram uma resposta hiperémica eficaz, sem tradução com significado na determinação do IRM (variação da pressão distal de - 11,4 ± 5 e de - 10,6± 5 mmHg com as doses de 5 e 10 mg respetivamente (p=0,5). Com a injeção das microesferas o IRM teve uma elevação média de 310 ± 190 %, para um valor médio de 41,3 ± 16 U (p = 0,001). Na Segunda Fase não houve diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os dois grupos. O IRM de base foi semelhante e após a infusão intracoronária observou-se uma elevação expressiva do IRM nos animais que receberam células em comparação com o grupo controlo (8,8 U ± 1 vs. 14,2 U ± 1,8, P=0,02) e quanto ao seu valor de base (aumento de 112%, p=0,008). Na terceira Fase não houve novamente diferenças significativas dos parâmetros hemodinâmicos, do fluxo epicárdico e da avaliação eletrocardiográfica entre os três grupos. Houve uma elevação do IRM nos animais que receberam células a partir da 2ª dose (72% nas células convencionai e 108% nas células inovadoras) e que se manteve com a 3ª dose (100% nas células convencionais e 88% nas inovadoras) com significado estatístico em comparação com o grupo controlo (p=0,034 com a 2ªdose e p=0,024 com a 3ª dose). Quatro semanas após a entrega das CEM observou-se a descida do IRM nos dois grupos que receberam células, para valores sobreponíveis aos do grupo controlo e aos valores pós-EAM. Na avaliação anatomopatológica e histológica dos corações explantados não houve diferenças entre os três grupos. Conclusões O IRM permite distinguir alterações da microcirculação coronária motivadas pela entrega intracoronária de CEM, na ausência de alterações de outros parâmetros clínicos da circulação coronária utilizados em tempo real. As alterações do IRM são progressivas e passíveis de avaliar o efeito/dose, embora não tenha sido possível determinar diferenças com os dois tipos de CEM. No nosso modelo a injeção intracoronária não se associou a evidência de efeito benéfico na reparação ou regeneração miocárdica após o EAM.---------------------------- ABSTRACT: ABSTRACT Introduction The use of cells for the treatment of cardiovascular disease is a promise for the future and perhaps the only option to overcome some of the shortcomings of current therapies. The strategy for the delivery of cells most often used in current research has been the intracoronary route and due to this microcirculation gains special relevance, mainly because it is the first interaction site of transplanted cells with the native tissue. Mesenchymal stem cells (MSC) have properties that make them suitable for Cell Therapy, but its dimensions, larger than the diameter of capillaries, have prompted controversy about the safety of intracoronary delivery. The interventional cardiology currently has techniques that allow for real-time and in vivo assessment of coronary microcirculation state. The determination of the index of microcirculatory resistance index (IMR) provides information about small vessels, independently of the coronary circulation and hemodynamic status, but the clinical applicability of this knowledge is yet to be defined. Objectives To clarify the potential use of IMR in the study of the effects of MSC through intracoronary transplantation. Population and Methods Preclinical study with swine model developed in three phases. In Phase One 8 healthy animals were used to study and validate the IMR assessment in our animal model. IMR was assessed with two different doses of papaverine for inducing the maximal hyperaemic response (5 and 10 mg) and microcirculation dysfunction was achieved after intracoronary injection with embozene microspheres with 40 μm in diameter. In Phase Two we randomized 18 healthy animals divided between the control group and the one receiving 30 x 106 MSC through an intracoronary infusion. There we blindly evaluated IMR, the aortic pressure, the epicardial coronary flow and the occurrence of ECG changes. In Phase Three we used 18 animals with a provoked acute myocardial infarction (AMI), randomized into a control group, a MSC expanded conventionally receiver group and a MSC expanded with an innovative methodology receiver group. There was a stepwise infusion with doses of 10 x 106, 15 x 106 and 20 x 106 MSC with determination of IMR, the aortic pressure, the epicardial coronary flow and occurrence of electrocardiographic abnormalities. Four weeks after cell delivery we again measured the IMR and proceeded with the pathological study of animals in the search for evidence of neoangiogenesis and myocardial regeneration, or a positive effect in the reparative response following the infarction. Results All animals remained hemodynamically stable and with no electrocardiographic abnormalities, except for the ST elevation in V1-V3 observed after injection of the microspheres. In Phase One the two doses of papaverine achieved an hyperemic and effective response without significant differences in IMR (variation of the distal pressure -11.4 ± 5 and -10.6 ± 5 mmHg with the doses of 5 and 10 mg respectively (p = 0.5). With the injection of the microspheres the IMR had an average increase of 310 ± 190% for an average value of 41.3 ± 16 U (p = 0.001). In the second phase there were no significant differences in hemodynamic parameters, epicardial flow and electrocardiographic assessment between the two groups. The baseline IMR was similar and after intracoronary infusion there was a significant increase in animals receiving cells compared with the control group (8.8 ± U 1 vs. 14.2 ± 1.8, p = 0.02) and with their baseline (112% increase, p = 0.008). In the third phase again there were no significant differences in hemodynamic parameters, the epicardial flow and electrocardiographic evaluation between the three groups. There was a significant increase in IMR in animals that received cells from the 2nd dose (72% in conventional cells and 108% in the innovative cells) that remained with the 3rd dose (100% in conventional cells and 88% in the innovative) with statistical significance compared with the control group (p = 0.034 with 2nd dose, p = 0.024 with 3rd dose). Four weeks after delivery of the MSC we observed the fall of the IMR in the two groups that received cells with values overlapping those of the control group. In pathological and histological evaluation of removed hearts there were no differences among the three groups. Conclusions The IMR allows for the differentiation of changes in coronary microcirculation motivated by intracoronary delivery of MSC in the absence of modification in other clinical parameters. IMR changes are progressive and enable the evaluation of the effect / dose, though it has not been possible to determine differences in the two types of MSC. In our model, intracoronary injection of MSC was not associated with evidence of repair or myocardial regeneration after AMI.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

Safe dissection of the distally based anterolateral thigh flap.

Background The distally based anterolateral thigh (ALT) flap is an interesting reconstructive solution for complex soft tissue defects of the knee. In spite of a low donor site morbidity and wide covering surface as well as arch of rotation, it has never gained popularity among reconstructive surgeons. Venous congestion and difficult flap dissection in the presence of a variable anatomy of the vascular pedicle are the possible reasons.Methods An anatomical study of 15 cadaver legs was performed to further clarify the blood supply of the distally based ALT. Our early experience with the use of preoperative angiography and a safe flap design modification that avoids distal intramuscular skeletonization of the vascular pedicle and includes a subcutaneous strip ranging from the distal end of the flap to the pivot point is presented.Results The distally based ALT presents a constant and reliable retrograde vascular contribution from the superior genicular artery. Preoperative angiography reliably identified and avoided critical Shieh Type II pedicled flaps. The preservation of a subcutaneous strip ranging from the distal flap end to the upper knee was associated with the absence of venous congestion in a short case series.Conclusions Preoperative angiography and a flap design modification are proposed to allow the safe transfer of the distally based ALT to reconstruct soft tissue defects of the knee.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Evaluation of the anti-arenaviral activity of the subtilisin kexin isozyme-1/site-1 protease inhibitor PF-429242.

The cellular protease subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in the proteolytic processing of the viral envelope glycoprotein precursor (GPC) of arenaviruses, a step strictly required for production of infectious progeny. The small molecule SKI-1/S1P inhibitor PF-429242 was shown to have anti-viral activity against Old World arenaviruses. Here we extended these studies and show that PF-429242 also inhibits GPC processing and productive infection of New World arenaviruses, making PF-429242 a broadly active anti-arenaviral drug. In combination therapy, PF-429242 potentiated the anti-viral activity of ribavirin, indicating a synergism between the two drugs. A hallmark of arenaviruses is their ability to establish persistent infection in vitro and in vivo. Notably, PF-429242 was able to efficiently and rapidly clear persistent infection by arenaviruses. Interruption of drug treatment did not result in re-emergence of infection, indicating that PF-429242 treatment leads to virus extinction.

Genetic dissection of sodium and potassium transport along the aldosterone-sensitive distal nephron: Importance in the control of blood pressure and hypertension.

In this review, we discuss genetic evidence supporting Guyton's hypothesis stating that blood pressure control is critically depending on fluid handling by the kidney. The review is focused on the genetic dissection of sodium and potassium transport in the distal nephron and the collecting duct that are the most important sites for the control of sodium and potassium balance by aldosterone and angiotensin II. Thanks to the study of Mendelian forms of hypertension and their corresponding transgenic mouse models, three main classes of diuretic receptors (furosemide, thiazide, amiloride) and the main components of the aldosterone- and angiotensin-dependent signaling pathways were molecularly identified over the past 20years. This will allow to design rational strategies for the treatment of hypertension and for the development of the next generation of diuretics.

Methods for integration site distribution analyses in animal cell genomes.

The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host-virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.