RESUMEN
Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA), and moniliformin (MON) mycotoxins are common food and feed contaminants produced by Fusarium spp. However, while they are usually found to co-occur in a large range of commodities, only few data are available on mycotoxin co-exposure effects and cellular response mechanisms. In this study, the individual and combined toxic effects of these fusariotoxins were evaluated on the THP-1 human immune cell line as major fusariotoxins are mostly potent immunomodulators. In particular, four relevant fusariotoxin mixtures, namely DON-MON, DON-FB1, DON-ZEA, and NIV-T2, were studied using several parameters including cell viability as well as the expression of cell surface markers and the main mitogen-activated protein kinases (MAPKs). After 48 h exposure, a reduction of cell viability in a dose-dependent manner was observed for T2, the most cytotoxic mycotoxin, followed by NIV, DON, MON, FB1, and ZEA. Regarding mycotoxin mixtures, they mainly showed antagonism on cell viability reduction. Interestingly, at concentrations inhibiting 50% of cell viability, most viable cells exhibited surface marker loss and thus became potentially non-functional. In addition, during the first 18 h of exposure, the effects of mycotoxin mixtures on early cell apoptosis and necrosis were found to be different from those induced by the toxins alone. At the molecular level, after 1 h exposure of individual and combined mycotoxins, the three main MAPK signaling pathways (p38, SAPK/JNK, and ERK1/2) were activated, highlighting a fast reaction of the exposed cells even at low cytotoxicity levels.
Asunto(s)
Monocitos/efectos de los fármacos , Toxina T-2/toxicidad , Biomarcadores/metabolismo , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células THP-1RESUMEN
OBJECTIVE: Smoking is a recognized cardiovascular risk factor. Perivascular visceral adipose tissue (PVAT) is a source of inflammatory molecules, thus contributing to atherosclerosis progression. The P2X7 receptor (P2X7 R)-inflammasome complex, crucial in determining IL-1ß and IL-18 release, participates in this scenario. We evaluated whether smoking might affect the PVAT inflammatory phenotype and explored the putative role of the axis P2X7 R-inflammasome in this picture. SUBJECTS AND METHODS: TNFα, IL-6, RBP4, MCP-1, as well as P2X7 R and inflammasome components NLRP3, ASC, caspase-1 and IL-1ß and IL-18 expression was determined in adipocytes isolated by PVAT of healthy smokers (Smok) and nonsmokers (No-Smok) subjects. Plasma and culture medium levels of these cytokines were also determined. RESULTS: Perivascular adipose tissue of Smok had a higher expression of P2X7 R and inflammasome components; via P2X7 R activation, it released more IL-1ß and IL-18, whose serum levels were also higher in Smok than in No-Smok. Linear correlations of NLRP3 with P2X7 R and IL-18 expression and release emerged. Smok also had a higher PVAT expression of the chemotactic factor MCP-1. However, no difference was observed in the PVAT expression of genes more strictly related to insulin resistance, like TNFα, RBP4, IL-6; this was coupled with similar plasma levels of TNFα and RBP4 in the two groups. CONCLUSION: Smoking contributes to the pro-inflammatory status of the PVAT by enhancing expression and activity of the P2X7 R-inflammasome complex; the effect on adipocytokines more related to insulin resistance and metabolic abnormalities appears trivial.
Asunto(s)
Adipocitos/metabolismo , Inflamasomas/genética , Grasa Intraabdominal/citología , Arterias Mesentéricas , Receptores Purinérgicos P2X7/genética , Fumar/genética , Adipocitos/inmunología , Adulto , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Estudios de Casos y Controles , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Grasa Intraabdominal/inmunología , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Proteínas Plasmáticas de Unión al Retinol/genética , Proteínas Plasmáticas de Unión al Retinol/inmunología , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Índice de Severidad de la Enfermedad , Fumar/inmunología , Fumar/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The Pacific oyster Crassostrea gigas is established in the marine intertidal zone, experiencing rapid and highly dynamic environmental changes throughout the tidal cycle. Depending on the bathymetry, oysters face oxygen deprivation, lack of nutrients, and high changes in temperature during alternation of the cycles of emersion/immersion. Here we showed that intertidal oysters at a bathymetry level of 3 and 5 m delayed by ten days the onset of mortality associated with Pacific Oyster Mortality Syndrome (POMS) as compared to subtidal oysters. Intertidal oysters presented a lower growth but similar energetic reserves to subtidal oysters but induced proteomic changes indicative of a boost in metabolism, inflammation, and innate immunity that may have improved their resistance during infection with the Ostreid herpes virus. Our work highlights that intertidal harsh environmental conditions modify host-pathogen interaction and improve oyster health. This study opens new perspectives on oyster farming for mitigation strategies based on tidal height.
Asunto(s)
Crassostrea , Herpesviridae , Animales , Interacciones Huésped-Patógeno , Inmunidad Innata , ProteómicaRESUMEN
Fatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-¹4C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules.
Asunto(s)
Diferenciación Celular/fisiología , Sistema Enzimático del Citocromo P-450/biosíntesis , Ácidos Grasos/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Empalme Alternativo , Línea Celular , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Ácidos Docosahexaenoicos/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Hepatocitos/enzimología , Humanos , Hidroxilación , Metabolismo de los Lípidos , Lipogénesis , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Isoformas de Proteínas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
A family history of type 2 diabetes (T2D) confers a high risk of developing the disease, independent of that due to other common risk factors. Postprandial state is a pro-inflammatory condition associated with a transiently impaired endothelial function; an increased oxidative stress is considered as a mediator of such effects in T2D. We evaluated the short-term effect of a lipid meal on markers of early vascular damage in subjects at risk of developing T2D. A total of thirty-two healthy volunteers, divided according to the presence (FHD+) or absence (FHD - ) of a family history of T2D, underwent a fatty meal test. We measured the monocyte mRNA expressions of IL-6, IL-8 and IL-1ß, and IL-6, soluble CD40 ligand (sCD40L), vascular cellular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and nitrotyrosine plasma concentrations at baseline and in the post-meal phase, relating them to the lipid profile and other biochemical parameters. The basal expression of the cytokines did not differ in FHD - and FHD+ subjects; neither was it modified by the meal ingestion. IL-6 and sCD40L plasma levels, similar in the two groups in the fasting state, did not vary after the meal. VCAM-1 and ICAM-1 increased in FHD+ subjects but not in FHD - subjects. Nitrotyrosine, similar between the FHD - and FHD+ subjects at baseline, increased more in FHD+ subjects than in FHD - subjects after the meal. In conclusion, the presence of a familial history of T2D confers an abnormal endothelial activation after an oral lipid meal, coupled with an increased oxidative stress, supporting the hypothesis of an early endothelial dysfunction already present in healthy individuals prone to develop T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Grasas de la Dieta/farmacología , Endotelio Vascular/metabolismo , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/metabolismo , Lípidos/farmacología , Biomarcadores/sangre , Biomarcadores/metabolismo , Ligando de CD40/genética , Ligando de CD40/metabolismo , Estudios de Casos y Controles , Citocinas/sangre , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Familia , Ayuno , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Periodo Posprandial , ARN Mensajero/metabolismo , Valores de Referencia , Factores de Riesgo , Tirosina/análogos & derivados , Tirosina/sangre , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.
RESUMEN
Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Biomarcadores de Tumor/fisiología , Carcinoma Papilar/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiología , Neoplasias de la Tiroides/genética , Adenosina Trifosfato/análisis , Adenosina Trifosfato/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/fisiopatología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Glándula Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/fisiopatología , Regulación hacia ArribaRESUMEN
Deoxynivalenol (DON) and zearalenone (ZEA) are mycotoxins primarily produced by Fusarium species and commonly co-occur in European grains. Some in vitro studies reported synergistic combined effects on cell viability reduction for these two natural food contaminants. However, most of these studies were carried out on conventional cell culture systems involving only one cell type and thus did not include cell-cell communication that is closer to in vivo conditions. In this context, we developed easy bi- and tri-culture systems using the Caco-2 (intestinal epithelial cells), THP-1 (monocytes) and HepaRG (hepatic cells) human cell lines in a proliferating state. Individual and combined cytotoxic effects of DON and ZEA were then assessed using co-cultures during 48 h. In bi-culture systems, results showed that only the highest tested dose of ZEA (IC30) induced a significant reduction in THP-1 viability with both Caco-2 and HepaRG cells cultured in transwells above. On the contrary, only the highest tested dose of DON (IC30) significantly affected HepaRG cell viability located under the Caco-2 cell monolayer. In addition, the DON + ZEA combination seemed to induce higher cytotoxicity than each toxin alone. Mycotoxin quantification in the abluminal compartment by Q-TOF LC-MS suggested uptake of both mycotoxins by the different cell lines. According to the co-culturing cell type, possible cell-cell interactions were also observed. Finally, in the tri-culture system, no cytotoxic effects were observed, regardless of the treatment. These findings highlighted the importance of the proposed models to better decipher toxicological impacts of mycotoxins on more complex cellular systems.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Micotoxinas/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Técnicas de Cocultivo , Humanos , Micotoxinas/análisis , Espectrometría de Masa por Ionización de Electrospray , Pruebas de Toxicidad Aguda , Tricotecenos/toxicidad , Zearalenona/toxicidadRESUMEN
Numerous surveys have highlighted the natural co-occurrence of deoxynivalenol (DON) and zearalenone (ZEA) mycotoxins in food and feed. Nevertheless, data regarding cellular mechanisms involved in response to their individual and simultaneous exposures are lacking. In this study, in order to analyze how low mycotoxin doses could impact cellular physiology and homeostasis, proteomic profiles of proliferating human hepatic cells (HepaRG) exposed for 1h and 24h to low DON and ZEA cytotoxicity levels (0.2 and 20µM respectively), alone or in combination, were analyzed by LC-MS/MS. Proteome analyses of mycotoxin-treated cells identified 4000 proteins with about 1.4% and 3.7% of these proteins exhibiting a significantly modified abundance compared to controls after 1h or 24h, respectively. Analysis of the Gene Ontology biological process annotations showed that cell cycle, proliferation and/or development as well as on DNA metabolic processes were affected for most treatments. Overall, different proteins, and thus biological processes, were impacted depending on the considered mycotoxin and exposure duration. Finally, despite the important proteome changes observed following 24h exposure to both mycotoxins, only the uptake of ZEA by the cells was suggested by the mycotoxin quantification in cell supernatants. BIOLOGICAL SIGNIFICANCE: This study investigated the proteomic changes that occurred after DON and ZEA (individually and in combination) short exposures at low cytotoxicity levels in proliferating HepaRG cells using LC-MS/MS. The obtained results showed that the cellular response is time- and mycotoxin or mixture-dependent. In particular, after 1h exposure, the DON+ZEA combination led to more proteomic changes than DON or ZEA alone, whereas the opposite was observed after 24h. In addition, the significant cellular response to stress induced by ZEA after 24h exposure seemed to be reduced when combined with DON. Thus, these results supported a possible mitigation by the hepatocytes when exposed to the mycotoxin mixture for a long duration. These findings represent an essential step to further explore adaptive cell response to mycotoxin exposure using with more complex incubation kinetics and combining different "omics" tools. Moreover, as mycotoxin quantification in cell supernatants showed different behaviors for DON and ZEA, this also raises the question about how mycotoxins actually trigger the cell response.
Asunto(s)
Hepatocitos/química , Proteoma/efectos de los fármacos , Tricotecenos/farmacología , Zearalenona/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Interacciones Farmacológicas , Exposición a Riesgos Ambientales , Humanos , Micotoxinas/farmacologíaRESUMEN
While numerous surveys highlighted the natural co-occurrence of mycotoxins in food, data about their toxicological combined effects is still limited. This is especially the case for chronic exposure conditions, although the latter are more representative of the mycotoxin risk associated with food consumption than acute exposure. In the present study, cell viability and gene expression levels of relevant hepatocyte-specific functions were evaluated for the HepaRG human liver cell line exposed to deoxynivalenol (DON) and/or zearalenone (ZEA) during 14, 28 and 42days at three subtoxic concentrations corresponding to i) the determined average exposure dose of French adult population, ii) the tolerable daily intake established by the Joint FAO/WHO Expert Committee and iii) the maximum level permitted by the European regulation in cereals intended for direct human consumption. For the latter, DON and DON+ZEA induced 90% cell mortality after 14days. In addition, depending on the considered toxin or mixture, doses and exposure periods, important variations of gene expression levels were observed. Despite the fact that in vitro conditions differ from the in vivo situation, the obtained results clearly highlighted that long-term toxicological effects of chronic exposure to mycotoxin combinations should be further investigated and, if necessary, taken into consideration at the regulatory level.
Asunto(s)
Hepatocitos/efectos de los fármacos , Tricotecenos/toxicidad , Zearalenona/toxicidad , Línea Celular , Supervivencia Celular , Esquema de Medicación , Quimioterapia Combinada , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Tricotecenos/administración & dosificación , Zearalenona/administración & dosificaciónRESUMEN
The data presented are associated with the "Proteomic analysis of the adaptative response of Mucor spp. to cheese environment" (Morin-Sardin et al., 2016) article [1]. Mucor metabolism is poorly documented in the literature and while morphology and growth behavior suggest potential adaptation to cheese for some strains, no adaptation markers to cheese environment have been identified for this genus. To establish the possible existence of metabolic functions related to cheese adaptation, we used a gel based 2-DE proteomic approach coupled to LC-MS/MS to analyze three strains from species known or proposed to have a positive or negative role in cheese production as well as a strain from a non-related cheese-species.
RESUMEN
While the reality of mycotoxin co-occurrence in food commodities is now established, their effects in mixtures are not well studied. The present study investigated the individual and combined effects of deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA) and moniliformin (MON) fusariotoxins on cell viability and cell death mechanisms in proliferating HepaRG cells, a human derived liver cell line. In addition, DON-ZEA being one of the most widespread mycotoxin mixtures in grains worldwide, its effect on the expression levels of genes encoding for sets of hepatocyte-specific functions was studied. After 48 h, T2 appeared to be the most cytotoxic tested fusariotoxins, followed by NIV, DON and ZEA. Furthermore, at low cytotoxic doses, all tested fusariotoxin mixtures (DON-MON, DON-FB1, DON-ZEA and NIV-T2) acted synergistically on cell death. Interestingly, during the first 18 h of exposure, only FB1 and ZEA alone and in combination with DON seemed to induce cell apoptosis and necrosis. At the gene level, after only 1 h, DON-ZEA combination induced expression of drug-metabolizing enzymes contrary to individual exposures. Thus, the observed synergy of fusariotoxin mixtures suggested that their simultaneous presence in food commodities can induce a toxic risk that should be better taken into consideration.
Asunto(s)
Hepatocitos/efectos de los fármacos , Toxina T-2/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Hepatocitos/citología , Humanos , Hígado/citología , Hígado/efectos de los fármacosRESUMEN
In the cheese industry context, Mucor species exhibit an ambivalent behavior as some species are essential "technological" organisms of some cheeses while others can be spoiling agents. Previously, we observed that cheese "technological" species exhibited higher optimal growth rates on cheese related matrices than on synthetic media. This growth pattern combined with morphological differences raise the question of their adaptation to cheese. In this study, using a comparative proteomic approach, we described the metabolic pathways of three Mucor strains considered as "technological" or "contaminant" in the cheese environment (M. lanceolatus UBOCC-A-109153, M. racemosus UBOCC-A-109155, M. circinelloides CBS 277-49) as well as a non-cheese related strain (M. endophyticus CBS 385-95). Overall, 15.8 to 19.0% of the proteomes showed a fold change ≥1.6 in Potato Dextrose Agar (PDA) versus Cheese Agar (CA), a cheese mimicking-medium. The 289 differentially expressed proteins identified by LC MS-MS analysis were mostly assigned to energy and amino-acid metabolisms in PDA whereas a higher diversity of biological processes was observed for cheese related strains in CA. Surprisingly, the vast majority (72.9%) of the over-accumulated proteins were different according to the considered medium and strain. These results strongly suggest that the observed better adaptative response of "technological" strains to cheese environment is mediated by species-specific proteins. BIOLOGICAL SIGNIFICANCE: The Mucor genus consists of a multitude of poorly known species. In the food context, few species are known for their positive role in the production of various food products, including cheese, while others are spoiling agents. The present study focused on the analysis of morphological and proteome differences of various Mucor spp. representative strains known as either positively (hereafter referred as "technological") or negatively (hereafter referred as "contaminant") associated with cheese or non-related to cheese (endophyte) on two different media, a synthetic medium and a cheese-mimicking medium. The main goal was to assess if adaptative traits of "technological" strains to the cheese environment could be identified. This work was based on observations we did in a recently published physiological study (Morin-Sardin et al., 2016). One of the important innovative aspects lies in the use for the first time of an extensive 2-DE approach to compare proteome variations for 4 strains on two different media. Results obtained offered an insight in the metabolic mechanisms associated with growth on a given medium and showed that adaptation to cheese environment is probably supported by species-specific proteins. The obtained data represent an essential step point for more targeted studies at the genomic and transcriptomic levels.
Asunto(s)
Adaptación Fisiológica , Queso/microbiología , Mucor/química , Proteómica/métodos , Microbiología de Alimentos , Proteínas Fúngicas/análisis , Proteínas Fúngicas/metabolismo , Redes y Vías Metabólicas , Mucor/crecimiento & desarrollo , Mucor/fisiología , Especificidad de la EspecieRESUMEN
Some foods and feeds are often contaminated by numerous mycotoxins, but most studies have focused on the occurrence and toxicology of a single mycotoxin. Regulations throughout the world do not consider the combined effects of mycotoxins. However, several surveys have reported the natural co-occurrence of mycotoxins from all over the world. Most of the published data has concerned the major mycotoxins aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), fumonisins (FUM) and trichothecenes (TCTs), especially deoxynivalenol (DON). Concerning cereals and derived cereal product samples, among the 127 mycotoxin combinations described in the literature, AFs+FUM, DON+ZEA, AFs+OTA, and FUM+ZEA are the most observed. However, only a few studies specified the number of co-occurring mycotoxins with the percentage of the co-contaminated samples, as well as the main combinations found. Studies of mycotoxin combination toxicity showed antagonist, additive or synergic effects depending on the tested species, cell model or mixture, and were not necessarily time- or dose-dependent. This review summarizes the findings on mycotoxins and their co-occurrence in various foods and feeds from all over the world as well as in vitro experimental data on their combined toxicity.
Asunto(s)
Alimentación Animal , Contaminación de Alimentos/análisis , Micotoxinas , Alimentación Animal/análisis , Alimentación Animal/toxicidad , Animales , Monitoreo del Ambiente , Contaminación de Alimentos/legislación & jurisprudencia , Humanos , Micotoxinas/análisis , Micotoxinas/toxicidadRESUMEN
In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.
Asunto(s)
Sustitución de Aminoácidos/genética , Antibacterianos/farmacología , Ácido Clavulánico/farmacología , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Inhibidores de beta-Lactamasas , beta-Lactamasas/genética , Alanina/genética , Alanina/metabolismo , Antibacterianos/metabolismo , Ácido Clavulánico/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Glicina/genética , Glicina/metabolismo , Hidrólisis , Concentración 50 Inhibidora , Cinética , Metionina/genética , Metionina/metabolismo , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida/genética , Mutación , Especificidad por Sustrato , beta-Lactamasas/metabolismoRESUMEN
Pacific oyster Crassostrea gigas were inoculated with OsHV-1 at low load (control) or high load (challenged) to better understand the pathogenesis of ostreid herpesvirus 1 (OsHV-1 µVar) and to determine which metabolic pathways might be affected during infection. Animals were sampled for proteomic analysis two days post-injection, at the same time as OsHV-1 initiated an intense replication phase in challenged oysters. Twenty-five abundant protein spots that showed a marked change in accumulated levels were identified using a two-dimensional electrophoresis (2-DE) proteomic approach. Overall, these proteins are involved in cytoskeleton organization, protein turnover, induction of stress signals, signalling pathways and energy metabolism. Challenged oysters exhibited an increased glycolysis and VDAC accumulation, which reflect a "Warburg effect" as initially reported in cancer cells and more recently in shrimp infected with virus. The results presented here should be useful for identifying potential biomarkers of disease resistance and developing antiviral measures. BIOLOGICAL SIGNIFICANCE: This study is the first 2-DE proteomic analysis dedicated to the pathogenesis of ostreid herpesvirus 1 (OsHV-1 µVar) in oyster Crassostrea gigas, the most important bivalve produced in the world. OsHV-1 has affected oysters every year since 2008. All the proteins identified in this paper are key targets involved in OsHV-1 infection processes. We presented evidence that the metabolic changes during infection in oyster somehow resemble the Warburg effect occurring in cancer cells. This work constitutes a real advance in the comprehension of the host metabolic pathways affected during OsHV-1 disease. Overall, this work contributes to a better understanding of disease mortalities in aquatic ecosystems which could guide management actions to mitigate their impacts.
Asunto(s)
Crassostrea/metabolismo , Infecciones por Herpesviridae/metabolismo , Herpesviridae , Proteoma/metabolismo , Proteómica , Animales , Crassostrea/virologíaRESUMEN
Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs) of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion). Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively). Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease). For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.
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Proteínas Bacterianas/metabolismo , Moluscos/metabolismo , Moluscos/microbiología , Proteómica/métodos , Vibriosis/metabolismo , Vibrio/patogenicidad , Factores de Virulencia/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Hemocitos/metabolismo , Hemocitos/microbiología , Moluscos/inmunología , Fagocitosis , Espectrometría de Masas en Tándem , Vibrio/clasificación , Vibriosis/inmunología , Vibriosis/microbiologíaRESUMEN
The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams.
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Bivalvos/genética , Bivalvos/microbiología , Perfilación de la Expresión Génica , Vibrio , Animales , Citoesqueleto/microbiología , Etiquetas de Secuencia Expresada , Hemocitos/microbiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We used a 2-DE proteomic approach to identify abundant proteins linked to oocyte quality in the Pacific oyster Crassostrea gigas, an economically important bivalve. Oocyte quality of 14 females was estimated by recording fertilisation and early developmental success until D-larval stage under controlled conditions. Proteins that were differentially expressed between females showing high or low oocyte quality were identified by nano-liquid chromatography tandem mass spectrometry. Twelve up-accumulated spots associated with low quality oocytes revealed 10 distinct proteins, including vitellogenin - breakdown products and metabolic enzymes. Eight up-accumulated spots from high quality oocytes revealed 6 distinct proteins, including chaperone molecules and cell-cycle control proteins. This is the first proteomic study dedicated to oocytes in C. gigas. Our results improve current knowledge about protein factors associated with oocyte quality in this species, and our understanding of the proteomic processes involved in their developmental competence.
Asunto(s)
Crassostrea/genética , Oocitos/citología , Proteómica/métodos , Animales , Electroforesis en Gel Bidimensional , Femenino , Proteínas , Espectrometría de Masas en Tándem , Regulación hacia ArribaRESUMEN
OBJECTIVE: Persistent inflammation and oxidative stress influence the progression of diabetic nephropathy. Metalloproteinases (MMPs) participate in extracellular matrix remodeling. Statins show favorable anti-inflammatory effects in chronic kidney disease. We evaluated the effect of rosuvastatin on inflammatory and pro-fibrotic responses due to exposure to different glucose or free fatty acid (FFA) concentrations. METHODS: Human mesangial cells (HMCs) grown at 5.5 (normal glucose) or 22âmmol/l (high glucose) glucose or exposed to FFA were treated with angiotensin-II in the presence or absence of rosuvastatin. We measured MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 expression and activity, and quantified the fibrotic factors transforming growth factor-ß1 (TGF-ß1), fibronectin, and collagen IV. RESULTS: At normal glucose, angiotensin-II induced a dose-dependent downregulation of MMP-2; rosuvastatin reversed this effect. On the contrary, TIMP-2 and MMP-9 were upregulated by angiotensin-II and downregulated by rosuvastatin; the effects on TIMP-1 were negligible. Some of the angiotensin-II effects were potentiated in the presence of high glucose and FFA; under both conditions, rosuvastatin was able to reverse these effects. MMP-2 and MMP-9 activity followed the same trend of expression, with rosuvastatin able to upregulate MMP-2 activity. The modulation of the MMP/TIMP system was paralleled by an increase in TGF-ß1, fibronectin, and collagen-IV; all were reduced by rosuvastatin treatment. Silencing the MMP-2 gene confirmed its role in modulating some of these angiotensin-II effects. CONCLUSION: Angiotensin-II induces a pro-fibrotic response in HMCs mainly via a dysregulation of the MMP-2/TIMP-2 pattern. This effect, partially amplified in the presence of high glucose and FFA, is reversed by rosuvastatin, suggesting another potential therapeutic application for this 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor.