Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine- protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu++-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake. that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

Phospholipid bilayer perturbing-properties underlying lysis induced by pH-sensitive cationic lysine-based surfactants in biomembranes

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

Cationic and charge segregation in La2/3Ca1/3MnO3 thin films grown on (001) and (110) SrTiO3.

Electron energy-loss spectroscopy is used to map composition and electronic states in epitaxial La2/3Ca1/3MnO3 films grown on SrTiO3 001 and 110 substrates. It is found that in partially relaxed 110 films cationic composition and valence state of Mn3+/4+ ions are preserved across the film thickness. In contrast, in fully strained 001 films, the Ca/La ratio gradually changes across the film, being La rich at film/substrate interface and La depleted at free surface; Mn valence state changes accordingly. These observations suggest that a strongly orientation-dependent adaptative composition mechanism dominates stress accommodation in manganite films and provides microscopic understanding of their dissimilar magnetic properties.

New cationic nanovesicular systems containing lysine-based surfactants for topical administration: Toxicity assessment using representative skin cell lines

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Cationic nanovesicles have attracted considerable interest as effective carriers to improve the delivery of biologically active molecules into and through the skin. In this study, lipid-based nanovesicles containing three different cationic lysine-based surfactants were designed for topical administration. We used representative skin cell lines and in vitro assays to assess whether the cationic compounds modulate the toxic responses of these nanocarriers. The nanovesicles were characterized in both water and cell culture medium. In general, significant agglomeration occurred after 24 h incubation under cell culture conditions. We found different cytotoxic responses among the formulations, which depended on the surfactant,cell line (3T3, HaCaT, and THP-1) and endpoint assayed (MTT, NRU, and LDH). Moreover, no potential phototoxicity was detected in fibroblast or keratinocyte cells, whereas only a slight inflammatory response was induced, as detected by IL-1a and IL-8 production in HaCaT and THP-1 cell lines, respectively. A key finding of our research was that the cationic charge position and the alkyl chain length of the surfactants determine the nanovesicles resulting toxicity. The charge on the a-amino group of lysine increased the depletion of cell metabolic activity, as determined by the MTT assay, while a higher hydrophobicity tends to enhance the toxic responses of the nanovesicles. The insights provided here using different cell lines and assays offer a comprehensive toxicological evaluation of this group of new nanomaterials.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Epigenetic Activation of SOX11 in Lymphoid Neoplasms by Histone Modifications

Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.

Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8, and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the AP and consequently modify nociceptive transmission, a process that is observed in persistent pain conditions. Post-translational modification (PTM) of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG) sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e., peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B, and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological tools to alleviate pain.

Heart Rate Variability (HRV) modifications in adult hemiplegic patients after botulinum toxin type A (nt-201) injection.

BACKGROUND: The most important adverse effect of BoNT-A is the systemic diffusion of the toxin. There is some evidence that the administration of high doses can increase the risk of systemic diffusion and the development of clinically evident adverse effects, however an international consensus does not exist about its maximum dose. AIM: The aim of this study was to evaluate changes in autonomic heart drive induced by high doses (higher than 600 units) of incobotulinumtoxinA injection in spastic stroke patients. Moreover, the treatment safety by monitoring adverse events occurrence was assessed. DESIGN: Case control study. POPULATION: Eleven stroke survivors with spastic hemiplegia. METHODS: Patients were treated with intramuscular focal injections of IncobotulinumtoxinA (NT 201; Xeomin®, Merz Pharmaceuticals GmbH, Frankfurt, Germany). Doses were below 12 units/Kg. Each patient underwent an ECG recording before injection and 10 days after treatment. Linear and non-linear Heart Rate variability (HRV) measures were derived from ECGs with a dedicated software. RESULTS: None of the variable considered showed statistically significant changes after BoNT-A injection. CONCLUSION: The use of incobotulinumtoxinA in adult patients at doses up to 12 units/kg seems to be safe regarding autonomic heart drive. CLINICAL REHABILITATION IMPACT: The use of IncobotulinumtoxinA up to 600 units could be a safe therapeutic option in spastic hemiplegic stroke survivors.