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61.
Jean-Luc Montillet Nathalie Leonhardt Samuel Mondy Sylvain Tranchimand Dominique Rumeau Marie Boudsocq Ana Victoria Garcia Thierry Douki Jean Bigeard Christiane Laurière Anne Chevalier Carmen Castresana Heribert Hirt 《PLoS biology》2013,11(3)
Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity. 相似文献
62.
Tim Hendrikx Veerle Bieghs Sofie M. A. Walenbergh Patrick J. van Gorp Fons Verheyen Mike L. J. Jeurissen Mandy M. F. Steinbusch Nathalie Vaes Christoph J. Binder Ger H. Koek Rinke Stienstra Mihai G. Netea Marten H. Hofker Ronit Shiri-Sverdlov 《PloS one》2013,8(12)
Background & Aims
While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.Methods
Ldlr -/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11-/- mice were incubated with oxLDL for 24h and autophagy was assessed.Results
In line with our hypothesis, caspase-1/11-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11-/- mice had normal autophagy activity.Conclusion
Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome. 相似文献63.
Karen Page David S. Guttery Nathalie Zahra Lindsay Primrose Shona R. Elshaw J. Howard Pringle Kevin Blighe Stephanie D. Marchese Allison Hills Laura Woodley Justin Stebbing R. Charles Coombes Jacqueline A. Shaw 《PloS one》2013,8(10)
Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA. 相似文献
64.
65.
Simon Lévesque Eric Fournier Nathalie Carrier Eric Frost Robert D. Arbeit Sophie Michaud 《PloS one》2013,8(12)
Campylobacter infection is a leading cause of bacterial gastroenteritis worldwide, and most clinical cases appear as isolated, sporadic infections for which the source is rarely apparent. From July 2005 to December 2007 we conducted a prospective case-case study of sporadic, domestically-acquired Campylobacter enteritis in rural versus urban areas and a prevalence study of Campylobacter in animal and environmental sources in the Eastern Townships, Quebec. Isolates were typed using Multilocus Sequence Typing (MLST) to reinforce the case-case findings and to assign a source probability estimate for each human isolate. The risk of human campylobacteriosis was 1.89-fold higher in rural than urban areas. Unconditional multivariate logistic regression analysis identified two independent risk factors associated with human Campylobacter infections acquired in rural area: occupational exposure to animals (OR = 10.6, 95% CI: 1.2–91, p = 0.032), and household water coming from a private well (OR = 8.3, 95% CI: 3.4–20.4, p<0.0001). A total of 851 C. jejuni isolates (178 human, 257 chicken, 87 bovine, 266 water, 63 wild bird) were typed using MLST. Among human isolates, the incidence rates of clonal complexes (CC) CC-21, CC-45, and CC-61 were higher in rural than urban areas. MLST-based source attribution analysis indicated that 64.5% of human C. jejuni isolates were attributable to chicken, followed by cattle (25.8%), water (7.4%), and wild birds (2.3%). Chicken was the attributable source for the majority of cases, independent of residential area, sex and age. The increased incidence in rural compared to urban areas was associated with occupational exposure to animals, particularly cattle among those aged 15–34 years, and with consumption of private well water. Both bovine and water exposure appeared to contribute to the seasonal variation in campylobacteriosis. These results provide a basis for developing public education and preventive programs targeting the risk factors identified. 相似文献
66.
Nathalie Aulner Anne Danckaert Eline Rouault-Hardoin Julie Desrivot Olivier Helynck Pierre-Henri Commere Hélène Munier-Lehmann Gerald F. Sp?th Spencer L. Shorte Geneviève Milon Eric Prina 《PLoS neglected tropical diseases》2013,7(4)
Background/Objectives
Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis.Methodology/Principal Findings
We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline.Conclusions/Significance
Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential. 相似文献67.
Ingrid Kikkas Roberto Mallone Nadia Tubiana-Rufi Didier Chevenne Jean Claude Carel Christophe Créminon Hervé Volland Christian Boitard Nathalie Morel 《PloS one》2013,8(7)
Type 1 diabetes (T1D) is an autoimmune disease which results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect amongst the four major islet autoantibodies. A non-radioactive IAA bridging ELISA was developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate. In the present study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from non-diabetic individuals were analyzed with our new assay and the results were correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs were detected in 32 out of 50 T1D children, whereas with IAA RIA, 41 out of 50 children with newly diagnosed T1D were scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes. 相似文献
68.
Yann Lamarre Marie-Laure Lalanne-Mistrih Marc Romana Nathalie Lemonne Daniele Mougenel Xavier Waltz Beno?t Tressières Maryse Etienne-Julan Vanessa Tarer Marie-Dominique Hardy-Dessources Philippe Connes 《PloS one》2013,8(6)
Patients with sickle cell anemia (SCA) have usually lower diastolic, systolic and mean blood pressure (BP) than the general population. However, BP values ≥120/70 mmHg considerably increase the risk for acute and chronic complications in SCA. The aim of this study was to identify biological factors associated with relative hypertension in adults with SCA. We compared the hematological, lipid and hemolytic profiles, as well as blood viscosity, between SCA patients with normal BP (<120/70 mmHg, n = 54) and those with relative hypertension (BP≥120/70 mmHg, n = 43). Our results demonstrated that male gender (OR: 3.49; 95%CI 1.20 to 10.16, p<0.05), triglycerides (OR: 9.19; 95% CI 2.29 to 36.95, p<0.01), blood viscosity (OR: 1.35; 95% CI 1.01 to 1.81, p<0.05) and body mass index (OR: 1.37; 95% CI 1.14 to 1.64, p<0.01) were independent risks factors for relative hypertension in SCA. No association was found between the BP status and the positive history of painful vaso-occlusive crisis or acute chest syndrome. An association between triglycerides level and the occurrence of these two major acute complications was detected. Our study suggests that male gender, increased triglycerides level, BMI and blood viscosity could increase the risk for developing relative hypertension in SCA. In addition, our results support a role of moderately elevated triglycerides in the pathophysiology of vaso-occlusive events. 相似文献
69.
Audrey M. Neyrinck Maud Alligier Patrick B. Memvanga Elodie Névraumont Yvan Larondelle Véronique Préat Patrice D. Cani Nathalie M. Delzenne 《PloS one》2013,8(11)
Background
Supra-nutritional doses of curcumin, derived from the spice Curcuma longa, have been proposed as a potential treatment of inflammation and metabolic disorders related to obesity. The aim of the present study was to test whether Curcuma longa extract rich in curcumin and associated with white pepper (Curcuma-P®), at doses compatible with human use, could modulate systemic inflammation in diet-induced obese mice. We questioned the potential relevance of changes in adiposity and gut microbiota in the effect of Curcuma-P® in obesity.Methodology/Principal Findings
Mice were fed either a control diet (CT), a high fat (HF) diet or a HF diet containing Curcuma longa extract (0.1 % of curcumin in the HF diet) associated with white pepper (0.01 %) for four weeks. Curcumin has been usually combined with white pepper, which contain piperine, in order to improve its bioavailability. This combination did not significantly modify body weight gain, glycemia, insulinemia, serum lipids and intestinal inflammatory markers. Tetrahydrocurcumin, but not curcumin accumulated in the subcutaneous adipose tissue. Importantly, the co-supplementation in curcuma extract and white pepper decreased HF-induced pro-inflammatory cytokines expression in the subcutaneous adipose tissue, an effect independent of adiposity, immune cells recruitment, angiogenesis, or modulation of gut bacteria controlling inflammation.Conclusions/Significance
These findings support that nutritional doses of Curcuma longa, associated with white pepper, is able to decrease inflammatory cytokines expression in the adipose tissue and this effect could be rather linked to a direct effect of bioactive metabolites reaching the adipose tissue, than from changes in the gut microbiota composition. 相似文献70.
Iris Marangon Nicole Boggetto Cécilia Ménard-Moyon Nathalie Luciani Claire Wilhelm Alberto Bianco Florence Gazeau 《Journal of visualized experiments : JoVE》2013,(82)
Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light. 相似文献