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Cet article fait le point sur l'état actuel des connaissances concernant les résines époxy réticulées par des polyamines, et plus particulièrement sur : - la réticulation ; - la structure et la morphologie des systèmes réticulés et les paramètres qui les influencent; - les relations entre la structure et les propriétés sur une très large gamme de températures. Malgré les nombreuses études réalisées, beaucoup de points apparaissent encore controversés, notamment en ce qui concerne les relations structure/propriétés dans le domaine des températures d'utilisation, c'est-à-dire entre la transition secondaire ß et la transition alpha (transition vitreuse). S'il est maintenant admis que le module à l'état vitreux est gouverné par l'importance des mouvements des séquences hydroxypropyléther formées lors de la réticulation, les résultats concernant les propriétés mécaniques aux grandes déformations (allongement et contrainte à rupture, énergie de propagation de fissures) sont beaucoup plus discutés. Les tendances obtenues semblent dépendre de la manière utilisée pour faire varier la densité de réticulation (cuisson, proportions du mélange, extenseurs de mailles, prépolymères de différentes longueurs, etc. ), du système ou encore de l'auteur. Les désaccords entre chercheurs pourraient provenir des conditions de préparation des échantillons, notamment de la vitesse de refroidissement en fin de cuisson et des conditions dans lesquelles sont réalisés les essais mécaniques, comme la vitesse de déformation. This paper sums up the current state-of-the-art on epoxy resins crosslinked with polyamines, and more particularly on:- the crosslinking reactions;- the structure and morphology of crosslinked systems and the parameters that influence them;- the relationships between structure and properties over a wide range of temperatures. In spite of numerous studies devoted to these polymers, many points remain controversial, notably concerning the relationships between structure and properties in the temperature range of interest to most users, which is between the ß transition temperature and the alpha transition temperature (glass transition). If it is now widely accepted that the modulus in the glassy state is mainly controlled by the amplitude of the motions of hydroxypropylether groups created during the crosslinking reactions, the results regarding the ultimate mechanical properties (stress and deformation at break, crack propagation energy) are much more debated. The trends obtained appear to depend upon either the way the crosslink density has been modified (cure cycle, mixture composition, chain extenders, prepolymers of different lengths and flexibilities), or the polymers studied or even the authors. The discrepancies between authors could be due to the sample preparation, particularly the cooling rate at the end of the cure cycle, or the testing conditions, e. g. the strain rate.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Ca v 1.2 and Ca v 1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Ca v 1.2 -mediated L-type calcium currents (I CaL ) at concentrations leaving Ca v 1.3-mediated I CaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Ca v 1.2 and Ca v 1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Ca v 1.2- and Ca v 1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Ca v 1.2 Ca 2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms., (© 2024. The Author(s).)

Background: Acute myocardial infarction (AMI) is the major cause of cardiovascular mortality worldwide. Most ischemic episodes are triggered by an increase in heart rate, which induces an imbalance between myocardial oxygen delivery and consumption. Developing drugs that selectively reduce heart rate by inhibiting ion channels involved in heart rate control could provide more clinical benefits. The Ca v 1.3-mediated L-type Ca 2+ current ( I Cav1.3 ) play important roles in the generation of heart rate. Therefore, they can constitute relevant targets for selective control of heart rate and cardioprotection during AMI., Objective: We aimed to investigate the relationship between heart rate and infarct size using mouse strains knockout for Ca v 1.3 ( Ca v 1.3 -/- ) L-type calcium channel and of the cardiac G protein gated potassium channel ( Girk4 -/- ) in association with the funny (f)-channel inhibitor ivabradine., Methods: Wild-type (WT), Ca v 1.3 +/- , Ca v 1.3 -/- and Girk4 -/- mice were used as models of respectively normal heart rate, moderate heart rate reduction, bradycardia, and mild tachycardia, respectively. Mice underwent a surgical protocol of myocardial IR (40 min ischemia and 60 min reperfusion). Heart rate was recorded by one-lead surface ECG recording, and infarct size measured by triphenyl tetrazolium chloride staining. In addition, Ca v 1.3 -/- and WT hearts perfused on a Langendorff system were subjected to the same ischemia-reperfusion protocol ex vivo , without or with atrial pacing, and the coronary flow was recorded., Results: Ca v 1.3 -/- mice presented reduced infarct size (-29%), while Girk4 -/- displayed increased infarct size (+30%) compared to WT mice. Consistently, heart rate reduction in Ca v 1.3 +/- or by the f-channel blocker ivabradine was associated with significant decrease in infarct size (-27% and -32%, respectively) in comparison to WT mice., Conclusion: Our results show that decreasing heart rate allows to protect the myocardium against IR injury in vivo and reveal a close relationship between basal heart rate and IR injury. In addition, this study suggests that targeting Ca v 1.3 channels could constitute a relevant target for reducing infarct size, since maximal heart rate dependent cardioprotective effect is already observed in Ca v 1.3 +/- mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Delgado-Betancourt, Chinda, Mesirca, Barrere, Covinhes, Gallot, Vincent, Bidaud, Kumphune, Nargeot, Piot, Wickman, Mangoni and Barrère-Lemaire.)

Reperfusion therapy during myocardial infarction (MI) leads to side effects called ischemia-reperfusion (IR) injury for which no treatment exists. While most studies have targeted the intrinsic apoptotic pathway to prevent IR injury with no successful clinical translation, we evidenced recently the potent cardioprotective effect of the anti-apoptotic Tat-DAXXp (TD) peptide targeting the FAS-dependent extrinsic pathway. The aim of the present study was to evaluate TD long term cardioprotective effects against IR injury in a MI mouse model. TD peptide (1 mg/kg) was administered in mice subjected to MI (TD; n = 21), 5 min prior to reperfusion, and were clinically followed-up during 6 months after surgery. Plasma cTnI concentration evaluated 24 h post-MI was 70%-decreased in TD (n = 16) versus Ctrl (n = 20) mice (p***). Strain echocardiography highlighted a 24%-increase (p****) in the ejection fraction mean value in TD-treated (n = 12) versus Ctrl mice (n = 17) during the 6 month-period. Improved cardiac performance was associated to a 54%-decrease (p**) in left ventricular fibrosis at 6 months in TD (n = 16) versus Ctrl (n = 20). In conclusion, targeting the extrinsic pathway with TD peptide at the onset of reperfusion provided long-term cardioprotection in a mouse model of myocardial IR injury by improving post-MI cardiac performance and preventing cardiac remodeling.

Aims: Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction., Methods and Results: An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments., Conclusions: Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

The energetic costs of behavioral chronic stress are unlikely to be sustainable without neuronal plasticity. Mitochondria have the capacity to handle synaptic activity up to a limit before energetic depletion occurs. Protective mechanisms driven by the induction of neuronal genes likely evolved to buffer the consequences of chronic stress on excitatory neurons in prefrontal cortex (PFC), as this circuitry is vulnerable to excitotoxic insults. Little is known about the genes involved in mitochondrial adaptation to the buildup of chronic stress. Using combinations of genetic manipulations and stress for analyzing structural, transcriptional, mitochondrial, and behavioral outcomes, we characterized NR4A1 as a stress-inducible modifier of mitochondrial energetic competence and dendritic spine number in PFC. NR4A1 acted as a transcription factor for changing the expression of target genes previously involved in mitochondrial uncoupling, AMP-activated protein kinase activation, and synaptic growth. Maintenance of NR4A1 activity by chronic stress played a critical role in the regressive synaptic organization in PFC of mouse models of stress (male only). Knockdown, dominant-negative approach, and knockout of Nr4a1 in mice and rats (male only) protected pyramidal neurons against the adverse effects of chronic stress. In human PFC tissues of men and women, high levels of the transcriptionally active NR4A1 correlated with measures of synaptic loss and cognitive impairment. In the context of chronic stress, prolonged expression and activity of NR4A1 may lead to responses of mitochondria and synaptic connectivity that do not match environmental demand, resulting in circuit malfunction between PFC and other brain regions, constituting a pathological feature across disorders. SIGNIFICANCE STATEMENT The bioenergetic cost of chronic stress is too high to be sustainable by pyramidal prefrontal neurons. Cellular checkpoints have evolved to adjust the responses of mitochondria and synapses to the buildup of chronic stress. NR4A1 plays such a role by controlling the energetic competence of mitochondria with respect to synapse number. As an immediate-early gene, Nr4a1 promotes neuronal plasticity, but sustained expression or activity can be detrimental. NR4A1 expression and activity is sustained by chronic stress in animal models and in human studies of neuropathologies sensitive to the buildup of chronic stress. Therefore, antagonism of NR4A1 is a promising avenue for preventing the regressive synaptic reorganization in cortical systems in the context of chronic stress., (Copyright © 2018 Wu, Lee et al.)

MLC901, a traditional Chinese medicine containing a cocktail of active molecules, both reduces cerebral infarction and improves recovery in patients with ischemic stroke. The aim of this study was to evaluate the acute and long-term benefits of MLC901 in ischemic and reperfused mouse hearts. Ex vivo, under physiological conditions, MLC901 did not show any modification in heart rate and contraction amplitude. However, upon an ischemic insult, MLC901 administration during reperfusion, improved coronary flow in perfused hearts. In vivo, MLC901 (4 µg/kg) intravenous injection 5 minutes before reperfusion provided a decrease in both infarct size (49.8%) and apoptosis (49.9%) after 1 hour of reperfusion. Akt and ERK1/2 survival pathways were significantly activated in the myocardium of those mice. In the 4-month clinical follow-up upon an additional continuous per os administration, MLC901 treatment decreased cardiac injury as revealed by a 45%-decrease in cTnI plasmatic concentrations and an improved cardiac performance assessed by echocardiography. A histological analysis revealed a 64%-decreased residual scar fibrosis and a 44%-increased vascular density in the infarct region. This paper demonstrates that MLC901 treatment was able to provide acute and long-term cardioprotective effects in a murine model of myocardial ischemia-reperfusion injury in vivo.

Aims: In a previous study using a genome-wide microarray strategy, we identified metabotropic glutamate receptor 1 (mGluR1) as a putative cardioprotective candidate in ischaemic postconditioning (PostC). In the present study, we investigated the role of cardiac mGluR1 receptors during cardioprotection against myocardial ischaemia-reperfusion injury in the mouse myocardium., Methods and Results: mGluR1 activation by glutamate administered 5 min before reperfusion in C57Bl/6 mice subjected to a myocardial ischaemia protocol strongly decreased both infarct size and DNA fragmentation measured at 24 h reperfusion. This cardioprotective effect was mimicked by the mGluR1 agonist, DHPG (10 μM), and abolished when glutamate was coinjected with the mGluR1 antagonist YM298198 (100 nM). Wortmannin (100 nM), an inhibitor of PI3-kinase, was able to prevent glutamate-induced cardioprotection. A glutamate bolus at the onset of reperfusion failed to protect the heart of mGluR1 knockout mice subjected to a myocardial ischaemia-reperfusion protocol, although PostC still protected the mGluR1 KO mice. Glutamate-treatment improved post-infarction functional recovery as evidenced by an echocardiographic study performed 15 days after treatment and by a histological evaluation of fibrosis 21 days post-treatment. Interestingly, restoration of functional mGluR1s by a PostC stimulus was evidenced at the transcriptional level. Since mGluR1s were localized at the surface membrane of cardiomyocytes, they might contribute to the cardioprotective effect of ischaemic PostC as other Gq-coupled receptors., Conclusion: This study provides the first demonstration that mGluR1 activation at the onset of reperfusion induces cardioprotection and might represent a putative strategy to prevent ischaemia-reperfusion injury., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Rationale: Polyamides (PA) are among the most used classes of polymers because of their attractive properties. Depending on the nature and proportion of the co-monomers used for their synthesis, they can exhibit a very large range of melting temperatures (Tm ). This study aims at the correlation of data from mass spectrometry (MS) with differential scanning calorimetry (DSC) and X-ray diffraction analyses to relate molecular structure to physical properties such as melting temperature, enthalpy change and crystallinity rate., Methods: Six different PA copolymers with molecular weights around 3500 g mol(-1) were synthesized with varying proportions of different co-monomers (amino-acid AB/di-amine AA/di-acid BB). Their melting temperature, enthalpy change and crystallinity rate were measured by DSC and X-ray diffraction. Their structural characterization was carried out by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Because of the poor solubility of PA, a solvent-free sample preparation strategy was used with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix and sodium iodide as the cationizing agent., Results: The different proportions of the repeating unit types led to the formation of PA with melting temperatures ranging from 115°C to 185°C. The structural characterization of these samples by MALDI-TOF-MS revealed a collection of different ion distributions with different sequences of repeating units (AA, BB; AB/AA, BB and AB) in different proportions according to the mixture of monomers used in the synthesis. The relative intensities of these ion distributions were related to sample complexity and structure. They were correlated to DSC and X-ray results, to explain the observed physical properties., Conclusions: The structural information obtained by MALDI-TOF-MS provided a better understanding of the variation of the PA melting temperature and established a structure-properties relationship. This work will allow future PA designs to be monitored., (Copyright © 2014 John Wiley & Sons, Ltd.)

Formulated lubricants are complex mixtures composed of base oil(s) and additives with various functions (detergents, corrosion inhibiter, antioxidant, viscosity modifiers, etc.). Because of the aliphatic nature of base oil and the chemical diversity of additives, the characterization of lubricant is currently a long and complex process. The comprehensive analysis of lubricant samples involves several techniques such as nuclear magnetic resonance, mass spectrometry, chromatography and infrared spectroscopy. The coupling of atmospheric solid analysis probe (ASAP) with ion mobility-mass spectrometry (IM-MS) has been shown to be an efficient tool for the characterization of complex mixture containing vaporizable polar to non-polar compounds. This approach affords the coupling of a direct ionization technique that does not require sample preparation, with a bi-dimensional separation method with high peak capacity. In this work, we show that ASAP-IM-MS is a suitable method for rapid and direct characterization of lubricant samples. Indeed, base oil and additives yielded, by ASAP, ions series which could be separated by IM-MS. Molecular additives such as Zn-dithiocarbamate, phosphite, thiophosphate and Alkyl diphenylamine were ionized as molecular ions [M](+•) or protonated molecules [M + H](+), depending of their polarity. In some cases, fragment ions were observed, confirming the additive identification. In addition, high molecular weight polymeric additives such as poly(alkyl methacrylate) (PAM) were pyrolized in the ASAP source leading to characteristic fragment ions. ASAP-IM-MS is shown to be a powerful tool for studying complex mixtures, allowing the first comprehensive analysis of lubricants in just a few minutes., (Copyright © 2014 John Wiley & Sons, Ltd.)

The structural characterization of polyamides (PA) was achieved by tandem mass spectrometry (MS/MS) with a laser induced dissociation (LID) strategy. Because of interferences for precursor ions selection, two chemical modifications of the polymer end groups were proposed as derivatization strategies. The first approach, based on the addition of a trifluoroacetic acid (TFA) molecule, yields principally to complementary bn and yn product ions. This fragmentation types, analogous to those obtained with peptides or other PA, give only poor characterization of polymer end-groups [1]. A second approach, based on the addition of a basic diethylamine (DEA), permits to fix the charge and favorably direct the fragmentation. In this case, bn ions were not observed. The full characterization of ω end group structure was obtained, in addition to the expected yn and consecutive fragment ions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polyolefin, including polypropylene (PP), constitutes an important class of materials. In particular, the recent interest in recycling plastic wastes necessitates their characterization as well as their degradation mechanism being understood. PP materials characterization by mass spectrometry, including polymer and additives parts, is not direct and generally involves a pyrolysis step to produce ionizable species. In this study, we extended the use of atmospheric solid analysis probe (ASAP) in combination with traveling wave ion mobility mass spectrometry (TWIM-MS) for the characterization of PP materials, including polymer as well as additives. Different commercial PP samples, from polymer standard to plastic item, were studied. The use of ASAP allow analysis to be done without any sample preparation, while TWIM-MS permitted a clear separation of polymer ions and additive signals. Several series of polymer pyrolysis residues, similar to those produced by classic pyrolysis, were obtained. Moreover, additive characterization has been done and supported by accurate mass measurements and tandem mass spectrometry experiments. Finally, this strategy put in evidence the role of additives in polymer degradation.

A sample pretreatment was evaluated to enable the production of intact cationic species of synthetic polymers holding a labile end-group using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. More specifically, polymers obtained by nitroxide-mediated polymerization involving the MAMA-SG1 alkoxyamine were stirred for a few hours in trifluoroacetic acid (TFA) to induce the substitution of a tert-butyl group on the nitrogen of nitroxide end-group by a hydrogen atom. Nuclear magnetic resonance, electrospray ionization tandem mass spectrometry, and theoretical calculations were combined to scrutinize this sample pretreatment from both mechanistic and energetic points of view. The substitution reaction was found to increase the dissociation energy of the fragile C-ON bond to a sufficient extent to prevent this bond to be spontaneously cleaved during MALDI analysis. This TFA treatment is shown to be very efficient regardless of the nature of the polymer, as evidenced by reliable MALDI mass spectrometric data obtained for poly(ethylene oxide), polystyrene and poly(butylacrylate)., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationale: Polyamides (PA) belong to the most used classes of polymers because of their attractive chemical and mechanical properties. In order to monitor original PA design, it is essential to develop analytical methods for the characterization of these compounds that are mostly insoluble in usual solvents., Methods: A low molecular weight polyamide (PA11), synthesized with a chain limiter, has been used as a model compound and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In the solvent-based approach, specific solvents for PA, i.e. trifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP), were tested. Solvent-based sample preparation methods, dried-droplet and thin layer, were optimized through the choice of matrix and salt. Solvent-based (thin layer) and solvent-free methods were then compared for this low solubility polymer. Ultra-high-performance liquid chromatography/electrospray ionization (UHPLC/ESI)-TOF-MS analyses were then used to confirm elemental compositions through accurate mass measurement., Results: Sodium iodide (NaI) and 2,5-dihydroxybenzoic acid (2,5-DHB) are, respectively, the best cationizing agent and matrix. The dried-droplet sample preparation method led to inhomogeneous deposits, but the thin-layer method could overcome this problem. Moreover, the solvent-free approach was the easiest and safest sample preparation method giving equivalent results to solvent-based methods. Linear as well as cyclic oligomers were observed. Although the PA molecular weights obtained by MALDI-TOF-MS were lower than those obtained by (1)H NMR and acido-basic titration, this technique allowed us to determine the presence of cyclic and linear species, not differentiated by the other techniques. TFA was shown to induce modification of linear oligomers that permitted cyclic and linear oligomers to be clearly highlighted in spectra., Conclusions: Optimal sample preparation conditions were determined for the MALDI-TOF-MS analysis of PA11, a model of polyamide analogues. The advantages of the solvent-free and solvent-based approaches were shown. Molecular weight determination using MALDI was discussed., (Copyright © 2012 John Wiley & Sons, Ltd.)

Aims: Myocardial infarction leads to heart failure and death. Ischaemic preconditioning (PreC) and postconditioning (PostC) reduce infarct size in animal models and human. Zac1 was identified as the only gene related to apoptosis and jointly down-regulated upon PreC and PostC. The aim of our study was to investigate the role of Zac1 down-regulation during ischaemia-reperfusion (I/R) in vivo., Methods and Results: C57BL/6 mice were submitted to myocardial I/R injury, PreC, or PostC protocols. QPCR and immunochemistry showed that Zac1 expression was down-regulated both at the transcriptional and the protein levels upon PreC and PostC. Zac1(-/-) Knockout mice (n = 7) developed smaller infarcts (54%) than Zac1(+/+) littermates (n = 8) and decreased apoptosis (61.7%) in the ischaemic part of the left ventricle during I/R (Zac1(-/-), n = 6 vs. Zac1(+/+), n = 7; P = 0.0012). Mutants showed under control conditions a decrease of 53.9% in mRNA of Daxx, a pro-apoptotic protein playing a key role in I/R injuries (4.81 ± 0.77, n = 4 Zac1(-/-) mice vs. 10.44 ± 3.5, n = 7 Zac1(+/+) mice; P = 0.0121)., Conclusion: Our study shows for the first time that Zac1 is down-regulated both at the transcriptional and protein levels upon PreC and PostC in wild-type mice. Moreover, inactivation of Zac1 in vivo is associated with a decreased amount of Daxx transcripts and, upon I/R injury, decreased infarct size and apoptosis. Altogether, our results show that Zac1 down-regulation plays a key role during cardioprotection against I/R injury and support the concept that cardioprotection regulates a network of interacting pro-apoptotic genes including Zac1 and Daxx.

Pulsed Gradient Spin Echo (PGSE) diffusion NMR experiments constitute a powerful tool for analyzing complex mixtures because they can in principle separate the NMR spectra of each mixture component. However, because these experiments intrinsically rely on spin echoes, they are traditionally regarded as non-quantitative, due to the signal attenuation caused by longitudinal (T(1)) and transverse (T(2)) nuclear magnetic relaxation during the rather long delays of the pulse sequence. Alternatively to the quantitative Direct Exponential Curve Resolution Algorithm (qDECRA) approach proposed by Antalek (J. Am. Chem. Soc. 128 (2006) 8402-8403), this work presents an acquisition strategy that renormalizes this relaxation attenuation using estimates of the T(1) and T(2) relaxation times for all the nuclei in the mixture, as obtained directly with the pulse sequence used to record the PGSE experiment. More specifically, it is shown that only three distinct PGSE experiments need to be recorded, each with a specific set of acquisition parameters. For small- and medium-sized molecules, only T(1) is required for obtaining accurate quantification. For larger molecular weight species, which typically exhibit short T(2) values, estimates of T(2) must also be included but only a rough estimation is required. This appears fortunate because these data are especially hard to obtain with good accuracy when analyzing homonuclear scalar-coupled systems. Overall, the proposed methodology is shown to yield a quantification accuracy of ±5%, both in the absence and in the presence of spectral overlap, giving rise--at least, in our hands--to results that superseded those achieved by qDECRA, while requiring substantially less experimental time., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A new solution-state NMR method is proposed to determine apparent transverse NMR relaxation rates in both weakly and strongly scalar coupled two-spin systems.

The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyrate-mediated rodent model of IBS. Both the selective genetic inhibition of Ca(V)3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of Ca(V)3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant Ca(V)3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest Ca(V)3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.

Solid-state Nuclear magnetic resonance (NMR) was used here to explore structural characteristics of samples to be subjected to matrix-assisted laser desorption/ionization (MALDI) and prepared without the use of any solvent. The analytical systems scrutinized in NMR were mixtures of a 2,5-dihydroxybenzoic acid (2,5-DHB) matrix and caesium fluoride (CsF), used as the cationization agent in synthetic polymer MALDI mass analysis, at different molar ratios (1:1, 5:1, and 10:1). Complementary information could be obtained from 13C, 133Cs, and 19F NMR spectra. Grinding the matrix together with the salt in the solid state was shown to induce a strong modification in the molecular organization within the MALDI sample. The evidenced mechano-induced reactions allow strong interactions between the matrix and the cation, up to the formation of a salt, and only occur in the presence of some water molecules. Addition of a poly(ethylene oxide) polymer as the analyte did not further modify the observed molecular organizations. Although relative matrix and salt concentrations in the scrutinized samples were unusual for MALDI analysis, mass spectra of good quality could be obtained and revealed that cation attachment on polymers during the MALDI process is not a matrix-independent event since a lower ionization efficiency was obtained from highly organized solid samples, mostly consisting of 2,5-DHB caesium salt species.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is the technique of choice to achieve molecular weight data for synthetic polymers. Because the success of a MALDI-MS analysis critically depends on a proper matrix and cation selection, which in turn relates closely to the polymer chemical nature and size, prior estimation of the polymer size range strongly helps in rationalizing MALDI sample preparation. We recently showed how pulsed gradient spin echo (PGSE) nuclear magnetic resonance could be used as an advantageous alternative to size exclusion chromatography, to rationalize MALDI sample preparation and confidently interpret MALDI mass spectra for homopolymers. Our aim here is to extend this methodology to the demanding case of amphiphilic block copolymers, for which obtaining prior estimates on the Mw values appears as an even more stringent prerequisite. Specifically, by studying poly(ethylene oxide) polystyrene block copolymers of distinct molecular weights and relative block weight fractions, we show how PGSE data can be used to derive the block Mw values. In contrast to homopolymers, such determination requires not only properly recorded calibration curves for each of the polymers constituting the block copolymers but also an appropriate hydrodynamic model to correctly interpret the diffusion data.

Channelopathies are often linked to defective protein folding and trafficking. Among them, the calcium channelopathy episodic ataxia type-2 (EA2) is an autosomal dominant disorder related to mutations in the pore-forming Ca(v)2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca(v)2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (D(I-II)) of the Ca(v)3.2 subunit of T-type calcium channel are misfolded, retained in the endoplasmic reticulum, and subject to proteasomal degradation. Pulse-chase experiments revealed that misfolded mutants bind to nascent wild-type Ca(v) subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca(v) mutants is likely to occur in EA2 and in other inherited dominant channelopathies.

Background: Apoptosis has been described extensively in acute myocardial infarction and chronic heart failure. Because Daxx (death-associated protein) appears to be essential for stress-induced cell death and acts as an antisurvival molecule, we tested the hypothesis that Daxx is involved in myocardial ischemia/reperfusion-induced cell death in vivo., Methods and Results: Transgenic mice overexpressing a dominant-negative form of Daxx (Daxx-DN) under the control of the beta-actin promoter and control wild-type mice underwent an ischemia/reperfusion protocol: 40 minutes of left coronary artery occlusion and 60 minutes of reperfusion. Area at risk and infarct size were measured after dual staining by triphenyltetrazolium chloride and phthalocyanine blue dye. Apoptosis was measured in the ischemic versus the nonischemic part of the left ventricle by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining, enzyme-linked immunosorbent assay, and Western blotting of caspase-3, caspase-8, and poly(ADP-ribose) polymerase. The mitogen-activated protein kinase status was investigated by Western blot analysis. Comparison between groups was assessed by ANOVA or Student t test (statistical significance: P<0.05). Left ventricle tissues from transgenic mice expressed Daxx-DN at the protein level. Area at risk/left ventricle values were comparable among groups. Infarct size/area at risk was 45% reduced in Daxx-DN versus wild-type mice (P<0.001). This cardioprotection was maintained for a 4-hour reperfusion. Ischemia/reperfusion-induced apoptosis was significantly decreased and ERK1/2 prosurvival pathway was activated in ischemic Daxx-DN hearts., Conclusions: Our study clearly indicates that Daxx participates in myocardial ischemia/reperfusion proapoptotic signaling in vivo.

Modulation of low voltage-activated Ca(V)3 T-type calcium channels remains poorly characterized compared with high voltage-activated Ca(V)1 and Ca(V)2 calcium channels. Notably, it is yet unresolved whether Ca(V)3 channels are modulated by protein kinases in mammalian cells. In this study, we demonstrate that protein kinase A (PKA) and PKC (but not PKG) activation induces a potent increase in Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 currents in various mammalian cell lines. Notably, we show that protein kinase effects occur at physiological temperature ( approximately 30-37 degrees C) but not at room temperature ( approximately 22-27 degrees C). This temperature dependence could involve kinase translocation, which is impaired at room temperature. A similar temperature dependence was observed for PKC-mediated increase in high voltage-activated Ca(V)2.3 currents. We also report that neither Ca(V)3 surface expression nor T-current macroscopic properties are modified upon kinase activation. In addition, we provide evidence for the direct phosphorylation of Ca(V)3.2 channels by PKA in in vitro assays. Overall, our results clearly establish the role of PKA and PKC in the modulation of Ca(V)3 T-channels and further highlight the key role of the physiological temperature in the effects described.

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (CaV3.1, CaV3.2, and CaV3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting CaV3.2 induced a knockdown of the CaV3.2 mRNA and protein expression as well as a large reduction of 'CaV3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that CaV3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking CaV3.2 T-type channels to pain perception and suggest that CaV3.2 may offer a specific molecular target for the treatment of pain.

Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.

Reaming of piriform orifices is a simple, short procedure easily integrated into functional surgery of nasal fossae. Results in 27 cases were rated as very good in 20 patients. Results are compared with those of three series reported in the literature, and the rare side effects noted are discussed.