Intracisternal delivery of NFκB-inducible scAAV2/9 reveals locoregional neuroinflammation induced by systemic kainic acid treatment.

We have previously demonstrated disease-dependent gene delivery in the brain using an AAV vector responding to NFκB activation as a probe for inflammatory responses. This vector, injected focally in the parenchyma prior to a systemic kainic acid (KA) injection mediated inducible transgene expression in the hippocampus but not in the cerebellum, regions, respectively, known to be affected or not by the pathology. However, such a focal approach relies on previous knowledge of the model parameters and does not allow to predict the whole brain response to the disease. Global brain gene delivery would allow to predict the regional distribution of the pathology as well as to deliver therapeutic factors in all affected brain regions. We show that self-complementary AAV2/9 (scAAV2/9) delivery in the adult rat cisterna magna allows a widespread but not homogenous transduction of the brain. Indeed, superficial regions, i.e., cortex, hippocampus, and cerebellum were more efficiently transduced than deeper regions, such as striatum, and substantia nigra. These data suggest that viral particles penetration from the cerebrospinal fluid (CSF) into the brain is a limiting factor. Interestingly, AAV2/9-2YF a rationally designed capsid mutant (affecting surface tyrosines) increased gene transfer efficiency approximately fivefold. Neurons, astrocytes, and oligodendrocytes, but not microglia, were transduced in varying proportions depending on the brain region and the type of capsid. Finally, after a single intracisternal injection of scAAV2/9-2YF using the NFκB-inducible promoter, KA treatment induced transgene expression in the hippocampus and cortex but not in the cerebellum, corresponding to the expression of the CD11b marker of microglial activation. These data support the use of disease-inducible vectors administered in the cisterna magna as a tool to characterize the brain pathology in systemic drug-induced or transgenic disease models. However, further improvements are required to enhance viral particles penetration into the brain.

Subtype-specific Modulation of Acid-sensing Ion Channel (ASIC) Function by 2-Guanidine-4-methylquinazoline.

Acid-sensing ion channels (ASICs) are neuronal Na(+)-selective channels that are transiently activated by extracellular acidification. ASICs are involved in fear and anxiety, learning, neurodegeneration after ischemic stroke, and pain sensation. The small molecule 2-guanidine-4-methylquinazoline (GMQ) was recently shown to open ASIC3 at physiological pH. We have investigated the mechanisms underlying this effect and the possibility that GMQ may alter the function of other ASICs besides ASIC3. GMQ shifts the pH dependence of activation to more acidic pH in ASIC1a and ASIC1b, whereas in ASIC3 this shift goes in the opposite direction and is accompanied by a decrease in its steepness. GMQ also induces an acidic shift of the pH dependence of inactivation of ASIC1a, -1b, -2a, and -3. As a consequence, the activation and inactivation curves of ASIC3 but not other ASICs overlap in the presence of GMQ at pH 7.4, thereby creating a window current. At concentrations >1 mm, GMQ decreases maximal peak currents by reducing the unitary current amplitude. Mutation of residue Glu-79 in the palm domain of ASIC3, previously shown to be critical for channel opening by GMQ, disrupted the GMQ effects on inactivation but not activation. This suggests that this residue is involved in the consequences of GMQ binding rather than in the binding interaction itself. This study describes the mechanisms underlying the effects of a novel class of ligands that modulate the function of all ASICs as well as activate ASIC3 at physiological pH.

Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system.

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.

Low estimates of dietary acid load are positively associated with bone ultrasound in women older than 75 years of age with a lifetime fracture

Dietary acid load from Western diets may be a risk factor for osteoporosis. It can be estimated by net endogenous acid production (NEAP). No data currently exists for NEAP estimates and bone indices in the very elderly (i.e. > or = 75 y). The aim of this study was to determine the association between NEAP estimates by using the potential renal acid load (PRAL) equation and quantitative bone ultrasound (QUS) measurements at the heel [broadband ultrasound attenuation (BUA)] in Caucasian women. We assessed NEAP and QUS in 401 very elderly Swiss ambulatory women. We evaluated dietary intake and NEAP estimates with a validated FFQ. QUS was measured using Achilles (Lunar). We identified 2 subgroups: 256 women (80.6 y +/- 3; BUA, 96.8 dB/MHz) with a fracture history and the remaining 145 (79.9 y SD 2.9; BUA, 101.7 dB/MHz) without. Women who reported having suffered a fracture had lower BUA (P < 0.001) than nonfractured women but did not differ in nutrient intakes and NEAP. Lower NEAP (P = 0.023) and higher potassium intake (P = 0.033) were correlated with higher BUA, which remained significant even after adjustment for age, BMI, and osteoporosis treatment. BUA was positively correlated with calcium (P = 0.016) and BMI (P < 0.001). Women who reported no fractures had no significant correlations between nutrient intake, NEAP, and BUA. Low nutritional acid load was correlated with higher BUA in very elderly women with a fracture history. Although relatively weak compared with age and BMI, this association was significant and may be an important additional risk factor that might be particularly relevant in frail patients with an already high fracture risk.

Role of glucagon in disposal of an amino acid load.

Amino acids stimulate the release of glucagon and insulin. To assess the role of aminogenic hyperglucagonemia, we have studied, in healthy young males, the effects of basal (less than 100 pg/ml) and high (200-400 pg/ml) plasma glucagon concentrations on amino acid metabolism during intravenous infusion (0.5 g.h-1.4 h) of a mixture of 15 amino acids. Basal plasma glucagon concentrations were obtained by infusion of somatostatin (0.5 mg/h) plus glucagon (0.25 ng.kg-1.min-1) and high plasma glucagon concentrations by infusion of somatostatin plus glucagon (3.0 ng.kg-1.min-1) or by infusion of amino acids alone. All studies were performed under conditions of euglycemic (83-91 mg/dl) hyperinsulinemia (50-80 microU/ml). Hyperglucagonemia significantly increased 1) net amino acid transport from the extracellular into the intracellular space (by approximately 4%), 2) net degradation of amino acids entering the intracellular space (by approximately 40%), and 3) conversion of degraded amino acids into glucose from 0-10% (basal glucagon) to 70-100% (high glucagon). Hyperglucagonemia did not affect the amount of amino acids excreted in the urine (approximately 4%). We conclude that glucagon plays an important role in the disposition of amino acids by increasing their inward transport, their degradation, and their conversion into glucose.

The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism.

The peroxisome proliferator-activated receptor alpha is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. PPARalpha mediates the effects of fibrates, which are potent hypolipidemic drugs, on gene expression. To better understand the biological effects of fibrates and PPARalpha, we searched for genes regulated by PPARalpha using oligonucleotide microarray and subtractive hybridization. By comparing liver RNA from wild-type and PPARalpha null mice, it was found that PPARalpha decreases the mRNA expression of enzymes involved in the metabolism of amino acids. Further analysis by Northern blot revealed that PPARalpha influences the expression of several genes involved in trans- and deamination of amino acids, and urea synthesis. Direct activation of PPARalpha using the synthetic PPARalpha ligand WY14643 decreased mRNA levels of these genes, suggesting that PPARalpha is directly implicated in the regulation of their expression. Consistent with these data, plasma urea concentrations are modulated by PPARalpha in vivo. It is concluded that in addition to oxidation of fatty acids, PPARalpha also regulates metabolism of amino acids in liver, indicating that PPARalpha is a key controller of intermediary metabolism during fasting.

Correction: Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women.

This corrects the article on p. e73445 in vol. 8.]. This corrects the article "Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Non-Obese Women" , e73445. There was an error in the title of the article. The correct version of the title in the article is: Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women The correct citation is: Martin F-PJ, Montoliu I, Collino S, Scherer M, Guy P, et al. (2013) Topographical Body Fat Distribution Links to Amino Acid and Lipid Metabolism in Healthy Obese Women. PLoS ONE 8(9): e73445. doi:10.1371/journal.pone.0073445

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii.

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

Velocity estimates for signal propagation leading to systemic jasmonic acid accumulation in wounded Arabidopsis.

The wound response prohormone jasmonic acid (JA) accumulates rapidly in tissues both proximal and distal to injury sites in plants. Using quantitative liquid chromatography-mass spectrometry after flash freezing of tissues, we found that JA accumulated within 30 s of injury in wounded Arabidopsis leaves (p = 3.5 e(-7)). JA augmentation distal to wounds was strongest in unwounded leaves with direct vascular connections to wounded leaves wherein JA levels increased significantly within 120 s of wounding (p = 0.00027). This gave conservative and statistically robust temporal boundaries for the average velocity of the long distance signal leading to distal JA accumulation in unwounded leaves of 3.4-4.5 cm min(-1). Like JA, transcripts of the JA synthesis gene LIPOXYGENASE2 (LOX2) and the jasmonate response gene JAZ10.3 also accumulated to higher levels in directly interconnected leaves than in indirectly connected leaves. JA accumulation in a lox2-1 mutant plant was initiated rapidly after wounding then slowed progressively compared with the wild type (WT). Despite this, JAZ10.3 expression in the two genotypes was similar. Free cyclopentenone jasmonate levels were similar in both resting WT and lox2-1. In contrast, bound cyclopentenone jasmonates (arabidopsides) were far lower in lox2-1 than in the WT. The major roles of LOX2 are to generate arabidopsides and the large levels of JA that accumulate proximal to the wound. LOX2 is not essential for some of the most rapid events elicited by wounding.

Molecular and cellular analysis of genodermatoses

Summary Skin is the essential interface between our body and its environment; not only does it prevent water loss and protect us from external insults it also plays an essential role in the central nervous system acting as a major sense organ primarily for touch and pain. The main cell type present in skin, keratinocyte, undergoes a differentiation process leading to the formation of this protecting barrier. This work is intended to contribute to the understanding of how keratinocyte differentiates and skin functions. To do this, we studied two genetic skin diseases: Erythrokeratodermia variabilis and Mal de Meleda. Our approach was to examine the expression and localization of proteins implicated in these two pathologies in normal and diseased tissues and to determine the influence of mutant proteins at the molecular and cellular levels. Connexins are major components of gap junctions, channels allowing direct communication between cells. Our laboratory has identified mutations in both connexin 30.3 (Cx30.3) and 31 (Cx31) to be causally involved in erythrokeratodermia variabilis (EKV), an autosomal dominant disorder of keratinization. In the first chapter, we show a new mutation of Cx31, L209P-Cx31, in 3 EKV patients, extending the field of EKV-causing mutations although the mechanism by which connexin mutations lead to the disease is unclear. In the second chapter, we studied the effect of F137L-Cx30.3 on expression, trafficking and localization of cotransfected Cx31 and Cx30.3 in connexin-deficient HeLa cells. The F137 amino acid, highly conserved in connexin family, is oriented towards the channel pore and F137L mutation in either Cx30.3 or Cx31 lead to EKV. As two genes can lead to EKV when mutated, our hypothesis was that Cx31 and Cx30.3 might cooperate at a molecular level. We were able to demonstrate a physical interaction between Cx31 and Cx30.3. The presence of F137L-Cx30.3 disturbed the trafficking of both connexins, less connexins were integrated into gap junctions and thus, the coupling between cell was diminished. Connexins formed in the presence of F137L-Cx30.3 are degraded at their exit from the endoplasmic reticulum. In conclusion, our results indicate that the genetic heterogeneity of EKV is due to mutations in two interacting proteins. F137L-Cx30.3 has a dominant negative effect and affects Cx31, disturbing cellular communication in epidermal cells. Mal de Meleda is an autosomal recessive inflammatory and a keratotic palmoplantar skin disorder due to mutations in SLURP1 (secreted LY6/PLAUR-related protein 1). SLURP1 belongs to the LY6/PLAUR family of proteins and has the particularity of being secreted instead of being GPI-anchored. The high degree of structural similarity between SLURP1 and the three fingers motif of snake neurotoxins and LYNX 1-C suggests that this protein could interact with the neuronal acetylcholine receptors. In the third chapter, we show that SLURP1 potentiates responses of the a7 nicotinic acetylcholine receptor (nAchR) to acetylcholine. These results identify SLURP1 as a secreted epidermal neuromodulator that is likely to be essential for palmoplantar skin. In the fourth chapter, we show that SLURP1 is expressed in the granular layer of the epidermis but is absent from skin biopsies of Mal de Meleda patients. SLURP1 is also present in secretions such as sweat, tears or saliva. An in vitro analysis on two mutant of SLURP-I demonstrates that W15R-SLURP1 is absent in cells while G86R-SLURP1 is expressed and secreted, suggesting that SLURP1 can lead to the disease by either an absent or an abnormal protein. Finally, in the fifth chapter, we analyse the expression and biological properties of other LY6/PLAUR members, clustered around SLURP] on chromosome 8. Their GPI-anchored or secreted status were analysed in vitro. SLURP1, LYNX1-A and -B are secreted while LYPDC2 and LYNX 1-C are GPI anchored. Three of these proteins are expressed in the epidermis and in cultured keratinocytes. These results suggest that these LY6/PLAUR members may have an important role in skin homeostasis. Résumé Résumé La peau est la barrière essentielle entre notre corps et l'environnement, nous protégeant des agressions extérieures, de la déshydratation et assurant aussi un rôle dans le système nerveux central en tant qu'organe du toucher et de la douleur. Le principal type de cellules présent dans la peau est le kératinocyte qui suit un processus de différenciation aboutissant à la formation de cette barrière protectrice. Ce travail est destiné à comprendre la différenciation des kératinocytes et le fonctionnement de la peau. Pour cela, nous avons étudié deux maladies génodermatoses : l'Erthrokeratodermia Variabilis (EKV) et le Mal de Meleda. Nous avons examiné l'expression et la localisation des protéines impliquées dans ces deux pathologies dans des tissus normaux et malades puis déterminé l'influence des protéines mutantes aux niveaux moléculaires et cellulaires. Les connexines (Cx) sont les composants majeurs des jonctions communicantes, canaux permettant la communication directe entre les cellules. Notre laboratoire a identifié des mutations dans les Cx30.3 et Cx31 comme responsables de l'EKV, génodermatose de transmission autosomique dominante. Dans le ler chapitre, nous décrivons une nouvelle mutation de Cx31, L209-Cx31, et contribuons à l'établissement du catalogue des mutations de Cx31 entraînant cette maladie. Cependant, le mécanisme par lequel les mutations de Cx31 et C3x0.3 provoquent l'EKV est inconnu. Dans le 2ème chapitre, nous étudions les effets de la mutation F137L-Cx30.3 sur l'expression, le trafic et la localisation des Cx31 et Cx30.3 transfectées dans des cellules HeLa, déficientes en connexines. Comme deux gènes peuvent causer une EKV quand ils sont mutés, notre hypothèse était que Cx31 et Cx30.3 pourraient coopérer au niveau moléculaire. Nous avons montré l'existence d'une interaction physique entre ces deux connexines. La présence de la mutation F137L-Cx30.3 perturbe le trafic des deux connexines, moins de connexines sont intégrées dans les jonctions communicantes et donc le couplage entre les cellules est diminué. Les connexons formés en présence de cette mutation sont dégradés à leur sortie du réticulum endoplasmique. En conclusion, nos résultats indiquent que l'hétérogénéité génétique de EKV est due à des mutations dans deux protéines qui interagissent. F137L-Cx30.3 a un effet dominant négatif et affecte Cx31, perturbant la communication entre les cellules épidermiques. Le Mal de Meleda est une maladie récessive de la peau palmoplantaire due à des mutations dans SLURP1. SLURP1 appartient à la famille des protéines contenant un domaine LY6/PLAUR et a la particularité d'être sécrétée. La grande homologie de structure existant entre SLURP1, les neurotoxines de serpent et LYNX1-C suggère que la protéine pourrait interagir avec des récepteurs à acétylcholine (Ach). Dans le 3ème chapitre, nous montrons que SLURP1 module la réponse à l'Ach du récepteur nicotinique α7. Ces résultats identifient SLURP1 comme un neuromodulateur épidermique sécrété, probablement essentiel pour la peau palmoplantaire. Dans le 4ème chapitre, nous montrons que SLURP1 est exprimé dans la couche granuleuse de l'épiderme et qu'il est absent des biopsies des patients. SLURP1 a aussi été détecté dans des sécrétions telles que la sueur, les lamies et la salive. Une analyse in vitro de deux mutants de SLURP1 a montré que W15R-SLURP1 est absent des cellules tandis que G86R-SLURP1 est exprimé et sécrété, suggérant qu'une absence ou une anomalie de SLURP1 peuvent causer la maladie. Finalement, dans le 5ème chapitre, nous analysons l'expression et les propriétés biologiques d'autres membres de la famille LY6/PLAUR localisés autour de SLURP1 sur le chromosome 8. Leur statut de protéines sécrétées ou liées à la membrane par une ancre GPI est analysé in vitro. SLURP1, LYNXI-A et -B sont sécrétées alors que LYPDC2 et LYNX1-C sont liés à la membrane. Trois de ces protéines sont exprimées dans l'épiderme et dans des kératinocytes cultivés. Ces résultats suggèrent que la famille LY6/PLAUR pourrait avoir un rôle important dans l'homéostasie de la peau.

Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues.

γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem(®), were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ(13)C values, spreading from -24.810/00 to -25.060/00, was observed, whilst mean δ(13)C value of Xyrem(®) corresponded to -24.990/00. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ(13)C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programmes.

Uric acid transport and disease.

Uric acid is the metabolic end product of purine metabolism in humans. It has antioxidant properties that may be protective but can also be pro-oxidant, depending on its chemical microenvironment. Hyperuricemia predisposes to disease through the formation of urate crystals that cause gout, but hyperuricemia, independent of crystal formation, has also been linked with hypertension, atherosclerosis, insulin resistance, and diabetes. We discuss here the biology of urate metabolism and its role in disease. We also cover the genetics of urate transport, including URAT1, and recent studies identifying SLC2A9, which encodes the glucose transporter family isoform Glut9, as a major determinant of plasma uric acid levels and of gout development.

Identification of the biosynthetic gene cluster for the Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid.

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.

Weights in the balance: jasmonic acid and salicylic acid signaling in root-biotroph interactions.

Work on the interaction of aerial plant parts with pathogens has identified the signaling molecules jasmonic acid (JA) and salicylic acid (SA) as important players in induced defense of the plant against invading organisms. Much less is known about the role of JA and SA signaling in root infection. Recent progress has been made in research on plant interactions with biotrophic mutualists and parasites that exclusively associate with roots, namely arbuscular mycorrhizal and rhizobial symbioses on one hand and nematode and parasitic plant interactions on the other hand. Here, we review these recent advances relating JA and SA signaling to specific stages of root colonization and discuss how both signaling molecules contribute to a balance between compatibility and defense in mutualistic as well as parasitic biotroph-root interactions.