Bifunctional mannoside-glucosinolate glycoconjugates as enzymatically triggered isothiocyanates and FimH ligands.

Glucosinolates are sulfur-containing secondary metabolites found in plants of the Brassicale order. They are precursors of isothiocyanate species, resulting from C-S hydrolysis catalysed by the thioglucohydrolase myrosinase. We describe the synthesis of bifunctional glucosinolate-mannoside glycoconjugates combining both the structural features of a substrate of myrosinase and a ligand of the lectin FimH. We show that these glycoconjugates serve as enzyme substrates and that myrosinase can indeed hydrolyze the glucosinolate moiety with affinities (KM, Vmax) comparable to the natural substrates glucomoringin and sinigrin. This enzymatic hydrolysis of the thioglycosidic bond led to the efficient formation of an isothiocyanate which was assessed by the formation of the corresponding dithiocarbamate derivatives. Finally, we show that our synthetic bifunctional glycoconjugates also serve as FimH ligands where the glucosinolate moiety does not hamper the interaction with the lectin. Our findings set the stage for an original bioconjugation tool, allowing for myrosinase-triggered specific labelling of lectins using glucosinolate glycoconjugates as non-toxic, water soluble isothiocyanate precursors.

Baseline sensitivity of T cells to alpha-IFN correlates with sustained virological response to IFN-based triple therapy in HCV infection.

Chronic infection with HCV is a public health problem with approximately 170 million people infected worldwide. Interferon alpha (IFNα) sensitivity in liver and IL28B genotype has been identified as important determinants of HCV clearance in the setting of pegylated interferon/ribavirin treatment. Herein, we explored IFNα sensitivity in PBMC from 21 healthy donors and 21 HCV-infected patients treated with pegylated interferon/ribavirin and HCV nonstructural protein-3 inhibitors (i.e. telaprevir/boceprevir). We explored phospho-STAT1 level as read-out for IFN signalling pathway activation in PBMC, T cells and monocytes and correlated results with virological response. We found that PBMC from healthy donors are desensitized to IFNα after priming and challenged with IFNα, with a subsequent decrease of phospho-STAT1 and interferon-stimulated genes. Furthermore, we show that CD3+ T cells, but not monocytes, become desensitized after 4 weeks of treatment, with a significant decrease of phospho-STAT1 after ex vivo IFNα stimulation. Finally, we identified baseline phospho-STAT1 level in CD3+ T cells as a potential biomarker of sustained virological response, regardless of the IL28B genotype. In the upcoming costly era of IFN-sparing regimen, baseline IFNα sensitivity could act as biomarker to define cost-effectiveness strategies of treatment by identifying patients who will or will not respond to IFN-based treatments.

Unbiased proteomic analysis of proteins interacting with the HIV-1 5'LTR sequence: role of the transcription factor Meis.

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Facile and rapid access to linear and truncated microcystin analogues for the implementation of immunoassays.

A series of simplified microcystin-LR analogues based on Adda [(2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldecadienoic acid] or its corresponding aldol precursor linked to a polypeptide moiety have been synthesised and assessed for their binding affinity by the monoclonal antibody mAb MC159, an anti-microcystin-LR mAb recently selected by us for the detection of microcystins through various immunoassay formats. Some modifications have been brought to the enantiospecific synthesis of N-Boc-Adda developed by Pearson et al. (Org. Lett., 2000, 2, 2901) which enabled us to access in an economical and time-saving manner a small library of MC-LR linear analogues. Among which Adda was chosen to synthesise, as an illustrative example, a fluorescent probe derived from this beta-amino acid. This probe was subsequently solid-phase immobilised by means of oxime ligation in order to lead to biochips suitable for microcystin detection through the SPIT-FRI method.

Mapping Groundwater Potential Through an Ensemble of Big Data Methods.

Groundwater resources are crucial to safe drinking supplies in sub-Saharan Africa, and will be increasingly relied upon in a context of climate change. The need to better understand groundwater calls for innovative approaches to make the best out of the existing information. A methodology to map groundwater potential based on an ensemble of machine learning classifiers is presented. A large borehole database (n = 1848) was integrated into a Geographic Information Systems (GIS) environment and used to train, validate and test 12 machine learning algorithms. Each classifier predicts a binary target (positive or negative borehole) based on the minimum flow rate required for communal domestic supplies. Classification is based on a number of explanatory variables, including landforms, lineaments, soil, vegetation, geology and slope, among others. Correlations between the target and explanatory variables were then generalized to develop groundwater potential maps. Most algorithms attained success rates between 80% and 96% in terms of test score, which suggests that the outcomes provide an accurate picture of field conditions. Statistical learners were observed to perform better than most other algorithms, excepting random forests and support vector machines. Furthermore, it is concluded that the ensemble approach provides added value by incorporating a measure of uncertainty to the results. This technique may be used to rapidly map groundwater potential for rural supply or humanitarian emergencies in areas where there is sufficient historical data but where comprehensive field work is unfeasible.

Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Selection of egg peptide biomarkers in processed food products by high resolution mass spectrometry.

Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency.

Liquid chromatography coupled to tandem mass spectrometry for detecting ten allergens in complex and incurred foodstuffs.

Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories.

Development of a sensitive multi-well colorimetric assay for active NFkappaB.

The transcription factor nuclear factor kappaB (NFkappaB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFkappaB activation. Indeed, NFkappaB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

Is the effect of interleukin-1 on glutathione oxidation in cultured human fibroblasts involved in nuclear factor-kappaB activation?

Our understanding of the interleukin-1 (IL-1) signaling molecular mechanisms has recently made considerable progress, with the discovery of the IL-1 receptor-associated kinase and the downstream enzymatic cascade that leads to the activation of nuclear factor-kappaB (NF-kappaB). IL-1 signaling and especially NF-kappaB activation are thought to be redox-sensitive, even though the precise nature and the molecular targets of the oxidants/antioxidants involved remain largely unknown. Here, we investigated the possible role of cellular oxidized/reduced glutathione (GSSG/GSH) balance in IL-1 signaling. We describe a quantitative method based on capillary electrophoresis designed to assay both intracellular GSH and GSSG in adhering fibroblasts. This method allows the GSSG/GSH balance to be followed during IL-1 stimulation. Our data show that IL-1 induces rapid and transient oxidation of intracellular glutathione in human fibroblasts. Using various antioxidants, including pyrrolidine dithiocarbamate and curcumin, we were unable to show a direct relationship between this IL-1-induced glutathione oxidation and NF-kappaB activation. Of the five antioxidants tested, only curcumin was able to inhibit IkappaBalpha degradation upstream and, hence, NF-kappaB DNA-binding activity and NF-kappaB-dependent expression of IL-6 downstream.

A comparison of five different methods for the detection of TNP specific mouse IgE: ELISA, ELISA on cells, rosetting, granule enzyme release assay and passive cutaneous anaphylaxis.

Using the same anti-TNP hybridoma supernatant pool as IgE antibody source (2 micrograms/ml), several methods were compared for their sensitivity in IgE detection and convenience for screening purposes. Using rat basophilic leukemia (RBL) cells as specific receptor cells, one out of two rosetting methods with TNP-sheep red blood cells allowed the detection of 0.5 ng IgE/ml (50 pg/assay) while the other was much less sensitive (60 ng/ml). More convenient for screening was an ELISA method performed on cells, in which the IgE bound to the RBL cell surface could be detected either by enzyme-anti-Ig or by enzyme-antigen conjugates with a similar, albeit low, sensitivity of 10 ng IgE/ml (500 pg/assay). Using the same antibody and cell sources, a very convenient and more sensitive method for screening purposes was found to be the measurement of antigen-induced basophil granule beta-N-acetylglucosaminidase release by IgE sensitized RBL cells: 0.5 ng IgE/ml and 50 pg/assay. For the same anti-TNP IgE source, this compares with a detection limit by ELISA of 0.2 ng IgE/ml (10 pg/assay) and by passive cutaneous anaphylaxis in rats of 2 ng IgE/ml (100 pg/assay).

3,4-Dihydro-3-amino-2H-1-benzopyran derivatives as 5-HT1A receptor ligands and potential anxiolytic agents. 1. Synthesis and structure--activity relationship studies.

A series of 3,4-dihydro-3-amino-2H-1-benzopyran derivatives were prepared in order to determine the necessary structural requirements for good affinity for 5-HT1A receptors and high selectivity versus other receptors. Modifications of the extracyclic amino substituents, the length of the alkyl side chains, and their substituents were explored. The best compounds (9g, 9k, 15b, 15d) possess imido or sulfonamido functional groups with a preferential length of four methylenes for the side chain. After resolution, the dextrorotatory enantiomers showed better affinity and selectivity for 5-HT1A receptors. These compounds have been proven to be full agonists. 9g and its enantiomers showed anxiolytic activity in vivo in various comportemental models. The compound (+)-9g is currently under clinical investigation.

Melatonergic properties of the (+)- and (-)-enantiomers of N-(4-methoxy-2,3-dihydro-1H-phenalen-2-yl)amide derivatives.

N-(4-Methoxy-2,3-dihydro-1H-phenalen-2-yl)amide derivatives, conformationally restricted ligands for melatonin receptors, were synthesized by an alternative synthetic method from the corresponding 1,8-naphthalic anhydride which was transformed into the phenalenecarboxylic acid 7. A Curtius reaction on 7 gave the amino compound which was acylated to give compounds 4a-c. The (+)- and (-)-4a-c enantiomers were separated by semipreparative chiral HPLC. Compounds were evaluated for their affinity for chicken brain melatonin receptors in binding assays using 2-[125I]iodomelatonin and for their potency to lighten the skin of Xenopus laevis tadpoles. The butyramido derivative 4c was the most potent ligand (Ki = 1.7 nM). No enantioselectivity was observed with the enantiomers which were equipotent to the racemic mixture. In contrast to the reference compounds, melatonin, agomelatine (S 20098), and N-[2-(2, 7-dimethoxynaphth-1-yl)ethyl]acetamide, which were very potent at lightening the skin of X. laevis tadpoles, compounds 4a-c were inactive or weakly active (EC50 > 1 microM). In this bioassay, compound 4a was characterized as a putative antagonist of melatonin receptors.

A novel series of 2,6,7-substituted 2,3-dihydro-1,4-benzodioxin and 2,6,7-substituted 1,4-benzodioxin derivatives as lipid peroxidation inhibitors. Structure-activity relationships for high inhibition of human low-density lipoprotein peroxidation.

A series of 6- or 7-substituted 2-carboxamido- or 2-(aminomethyl)-1,4-benzodioxin and -2,3-dihydro-1,4-benzodioxin derivatives were synthesized and evaluated to determine the necessary structural requirements for a high inhibition of human low-density lipoprotein copper-induced peroxidation. The most active compounds (21, 25, 28, 36, and 37) were found between 5 and >45 times more active than probucol itself. Due to both their potency and their structural features, compounds 25 and 36 were selected with others for complementary in vitro and in vivo investigations. Both of them exhibit calcium antagonist properties in the same range of potency as flunarizine itself. Compound 36 was also found to have significant hypolipaemic activity in mice at 100 and 300 mg/kg po, while compound 25 proved to be clearly active in a normobar hypoxia test.

Synthesis and structure-activity relationships of novel naphthalenic and bioisosteric related amidic derivatives as melatonin receptor ligands.

A series of N-naphthylethyl amide derivatives were synthesized and evaluated as melatonin receptor ligands. The affinity of each compound for the melatonin receptor was determined by binding studies using [2-125I]iodomelatonin on ovine pars tuberalis membrane homogenates. Structure-activity relationships led to the conclusion that naphthalene is a bioisostere of the indole moiety of melatonin. Moreover it appears that the affinity is strongly affected by the size of the substituent of the nitrogen of the amidic function. Many of these ligands give biphasic dose-response curves which suggests that there may be two melatonin receptor subtypes within the ovine pars tuberalis cells. The replacement of naphthalene by benzofuran or benzothiophene did not strongly alter the affinity for the melatonin receptor. In contrast, the benzimidazole analogue was a poor ligand. Compound 7, the naphthalenic analogue of melatonin, a selective ligand of the melatonin receptor and an agonist derivative, has been selected for clinical development.

Novel and selective partial agonists of 5-HT3 receptors. 2. Synthesis and biological evaluation of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines.

In continuation of our previous work on piperazinopyrrolothienopyrazine derivatives, three series of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines were prepared and evaluated as 5-HT3 receptor ligands. The chemical modifications performed within these new series led to structure-activity relationships regarding both high affinity and selectivity for the 5-HT3 receptors that are in agreement with those established previously for the pyrrolothienopyrazine series. The best compound (8a) obtained in these new series is in the picomolar range of affinity for 5-HT3 receptors with a selectivity higher than 10(6). Four of the high-affinity 5-HT3 ligands (8a, 15a,b, and 16d) were selected in both the pyridopyrrolopyrazine and the pyrroloquinoxaline series and were characterized in vitro and in vivo as agonists or partial agonists. Compound 8a was also evaluated in the light/dark test where it showed potential anxiolytic-like activity at very low doses per os.