Nucleic acid lateral flow dipstick assay for the duplex detection of Gambierdiscus australes and Gambierdiscus excentricus

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/μL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.info:eu-repo/semantics/publishedVersio

Grape seed Proanthocyanidins target the Enteroendocrine system in cafeteria‐diet‐fed rats

Scope The effects on the enteroendocrine system of three different grape seed proanthocyanidin extract (GSPE) treatments are analyzed in rats on a cafeteria diet for 17 weeks. Methods and results GSPE is administered in a corrective manner (15 last days of the cafeteria diet) at two doses, 100 and 500 mg GSPE per kg bw. A third, longer treatment in which GSPE (500 mg kg–1 bw) is administered daily every other week during the 17 weeks of the cafeteria diet is also tested. Most GSPE treatments lead to ghrelin accumulation in the stomach, limited CCK secretion in the duodenum, and increased GLP‐1 and PYY mRNA in colon. GSPE also increases cecal hypertrophy and reduces butyrate content. When the treatment is administered daily every other week during 17 weeks, there is also an increase in colon size. These effects are accompanied by a reduced food intake at the end of the experiment when GSPE is administered at 500 mg GSPE kg–1 during the last 15 days, but not on the other treatments, despite an observed reduction in body weight in the longer treatment. Conclusion GSPE modulates the enteroendocrine system in models in which it also reduces food intake or body weight.info:eu-repo/semantics/acceptedVersio

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Long-Lasting Effects of GSPE on Ileal GLP-1R Gene Expression Are Associated with a Hypomethylation of the GLP-1R Promoter in Female Wistar Rats

Flavonoids have been shown to modulate GLP-1 in obesity. GLP-1 induces some of its effects through the intestinal GLP-1 receptor (GLP-1R), though no data exist on how flavonoids affect this receptor. Here, we examine how a dose of grape seed proanthocyanidin extract (GSPE) with anti-obesity activity affects intestinal GLP-1R and analyze whether epigenetics play a role in the long-lasting effects of GSPE. We found that 10-day GSPE administration prior to the cafeteria diet upregulated GLP-1R mRNA in the ileum 17 weeks after the GSPE treatment. This was associated with a hypomethylation of the GLP-1R promoter near the region where the SP1 transcription factor binds. In the colon, the cafeteria diet upregulated GLP-1R without showing any GSPE effect. In conclusion, we have identified long-lasting GSPE effects on GLP-1R gene expression in the ileum that are partly mediated by hypomethylation at the gene promoter and may affect the SP1 binding factor

Bioactivity of flavanols on the mucosa of the intestinal wall: enteroendocrine effects for preventing diet-induced obesity and associated pathologies

Les proantocianidines han demostrat ser efectives estimulant el sistema enteroendocrí en condicions saludables, però els efectes sota un context d’obesitat encara estan per determinar. Donat això, aquesta tesi va ser dissenyada per estudiar els efectes d'un extracte de proantocianidines de llavor de raïm (GSPE) sobre el sistema enteroendocrí en rates alimentades amb una dieta de cafeteria a llarg termini. Mostrem que uns tractaments preventiu, intermitent sincrònic i correctiu son capaços de modular el sistema enteroendocrí de diferent manera depenent del tractament. A més, cada tractament amb GSPE va mostrar diferents perfils enteroendocrins associats a canvis de pes corporal i/o d’ingesta. No obstant, trobàrem dificultats quantificant les secrecions d'enterohormones i ens va portar al desenvolupament d'una nova metodologia ex vivo, que ens va permetre estimular diferents segments del tracte gastrointestinal i quantificar la seva resposta de secreció enteroendocrina, mantenint la seva vectorialitat. També trobàrem una regulació de llarg termini sobre l'expressió gènica de GLP-1 a ili, induïda pel pre-tractament de 10 dies amb GSPE, mediada parcialment per una hipometilació sobre el promotor de GLP-1. Aquests efectes es mantenen quan el GSPE s’administra cada dues setmanes durant les disset setmanes de la dieta de la cafeteria. A més, atès que el el tractament preventiu de GSPE presentava un quocient respiratori disminuït i tendia a reduir l'augment de pes corporal, vam avaluar si el GSPE induïa efectes de llarg termini sobre la gestió de lípids als teixits perifèrics. Observàrem una limitació d’emmagatzematge al teixit adipós i un augment d’oxidació lipídica al fetge i múscul esquelètic que romania set setmanes després de l'última dosi de GSPE. En resum, aquesta tesi mostra que el GSPE és capaç de modular el sistema enteroendocrí i millorar l'estat energètic, alterat per una dieta de cafeteria, demostrant ser un bon agent per tractar les alteracions metabòliques induïdes per l'obesitat.Las proantocianidinas han demostrado ser efectivas estimulando el sistema enteroendocrino en condiciones saludables, pero sus efectos bajo un contexto de obesidad aún están por determinar. Por ello, esta tesis fue diseñada para estudiar los efectos de un extracto de proantocianidinas de semilla de uva (GSPE) sobre el sistema enteroendocrino en ratas alimentadas con una dieta de cafetería a largo plazo. Mostramos que unos tratamientos preventivo, intermitente sincrónico y correctivo modulan el sistema enteroendocrino de diferente forma dependiendo del tratamiento. Además, cada tratamiento con GSPE mostró diferentes perfiles enteroendocrinos asociados a cambios de peso corporal y/o de ingesta. Sin embargo, encontramos algunas dificultades en la cuantificación de las secreciones de enterohormonas que nos condujeron al desarrollo de una metodología ex vivo, que nos permitió estimular diferentes segmentos del tracto gastrointestinal y cuantificar su respuesta de secreción enteroendocrina, manteniendo su vectorialidad. También encontramos una regulación de largo plazo sobre la expresión génica de GLP-1 en íleon, inducida por un pre-tratamiento de 10 días con GSPE, mediada parcialmente por una hipometilación sobre el promotor de GLP-1. Estos efectos se mantuvieron cuando el GSPE se administró cada dos semanas durante toda la cafetería. Además, dado que el tratamiento preventivo de GSPE presentaba un cociente respiratorio disminuido y tendía a reducir el aumento de peso corporal, evaluamos si el GSPE inducía efectos de largo plazo sobre la gestión de lípidos en tejidos periféricos. Observamos una limitación de almacenamiento en el tejido adiposo y un aumento de la oxidación lipídica en el hígado y músculo esquelético que duró siete semanas después de la última dosis de GSPE. Resumiendo, esta tesis muestra que el GSPE es capaz de modular el sistema enteroendocrino y mejorar el estado energético, alterado por una dieta de cafetería, demostrando ser un buen agente para tratar las alteraciones metabólicas inducidas por la obesidad.Proanthocyanidins have already proved to be effective at stimulating the enteroendocrine system in healthy conditions, but their effects under an obesogenic challenge have still to be determined. For this reason, this thesis was designed to study the effects of a grape seed proanthocyanidin extract (GSPE) on the enteroendocrine system in rats fed with a long-term cafeteria diet. Our results showed that a preventive treatment, a synchronic intermittent treatment and a corrective treatment were all capable of modulating the enteroendocrine system differently. Furthermore, each GSPE treatment showed different enteroendocrine profiles associated with changes in body weight and/or food intake. However, we had certain difficulties regarding the quantification of enterohormone secretions, which led us to develop a new ex vivo methodology that stimulated different segments of the gastrointestinal tract and quantified their enterohormone secretion response, thus keeping their vectoriality. We also found that a 10-day pre-treatment with GSPE induced a long-term upregulation of GLP-1 gene expression in the ileum that was partly mediated by the hypomethylation of its GLP-1 promoter. Moreover, these effects were maintained when GSPE was administered every other week during the seventeen weeks of cafeteria diet. In addition, since this preventive GSPE treatment presented a decreased respiratory quotient and tended to reduce the body weight gain, we evaluated if there were also long-lasting GSPE effects on lipid management in the peripheric tissues. The results showed a limitation on adipose storage and an increase in lipid oxidation in the liver and skeletal muscle that lasted seven weeks after the last GSPE dose. To sum up, this thesis revealed that grape seed proanthocyanidins are capable of modulating the enteroendocrine system and improving the energetic state altered by a cafeteria diet, thus demonstrating that they are good agents for treating metabolic alterations induced by obesity

Bioactivity of flavanols on the mucosa of the intestinal wall: enteroendocrine effects for preventing diet-induced obesity and associated pathologies

Les proantocianidines han demostrat ser efectives estimulant el sistema enteroendocrí en condicions saludables, però els efectes sota un context d’obesitat encara estan per determinar. Donat això, aquesta tesi va ser dissenyada per estudiar els efectes d'un extracte de proantocianidines de llavor de raïm (GSPE) sobre el sistema enteroendocrí en rates alimentades amb una dieta de cafeteria a llarg termini. Mostrem que uns tractaments preventiu, intermitent sincrònic i correctiu son capaços de modular el sistema enteroendocrí de diferent manera depenent del tractament. A més, cada tractament amb GSPE va mostrar diferents perfils enteroendocrins associats a canvis de pes corporal i/o d’ingesta. No obstant, trobàrem dificultats quantificant les secrecions d'enterohormones i ens va portar al desenvolupament d'una nova metodologia ex vivo, que ens va permetre estimular diferents segments del tracte gastrointestinal i quantificar la seva resposta de secreció enteroendocrina, mantenint la seva vectorialitat. També trobàrem una regulació de llarg termini sobre l'expressió gènica de GLP-1 a ili, induïda pel pre-tractament de 10 dies amb GSPE, mediada parcialment per una hipometilació sobre el promotor de GLP-1. Aquests efectes es mantenen quan el GSPE s’administra cada dues setmanes durant les disset setmanes de la dieta de la cafeteria. A més, atès que el el tractament preventiu de GSPE presentava un quocient respiratori disminuït i tendia a reduir l'augment de pes corporal, vam avaluar si el GSPE induïa efectes de llarg termini sobre la gestió de lípids als teixits perifèrics. Observàrem una limitació d’emmagatzematge al teixit adipós i un augment d’oxidació lipídica al fetge i múscul esquelètic que romania set setmanes després de l'última dosi de GSPE. En resum, aquesta tesi mostra que el GSPE és capaç de modular el sistema enteroendocrí i millorar l'estat energètic, alterat per una dieta de cafeteria, demostrant ser un bon agent per tractar les alteracions metabòliques induïdes per l'obesitat.Las proantocianidinas han demostrado ser efectivas estimulando el sistema enteroendocrino en condiciones saludables, pero sus efectos bajo un contexto de obesidad aún están por determinar. Por ello, esta tesis fue diseñada para estudiar los efectos de un extracto de proantocianidinas de semilla de uva (GSPE) sobre el sistema enteroendocrino en ratas alimentadas con una dieta de cafetería a largo plazo. Mostramos que unos tratamientos preventivo, intermitente sincrónico y correctivo modulan el sistema enteroendocrino de diferente forma dependiendo del tratamiento. Además, cada tratamiento con GSPE mostró diferentes perfiles enteroendocrinos asociados a cambios de peso corporal y/o de ingesta. Sin embargo, encontramos algunas dificultades en la cuantificación de las secreciones de enterohormonas que nos condujeron al desarrollo de una metodología ex vivo, que nos permitió estimular diferentes segmentos del tracto gastrointestinal y cuantificar su respuesta de secreción enteroendocrina, manteniendo su vectorialidad. También encontramos una regulación de largo plazo sobre la expresión génica de GLP-1 en íleon, inducida por un pre-tratamiento de 10 días con GSPE, mediada parcialmente por una hipometilación sobre el promotor de GLP-1. Estos efectos se mantuvieron cuando el GSPE se administró cada dos semanas durante toda la cafetería. Además, dado que el tratamiento preventivo de GSPE presentaba un cociente respiratorio disminuido y tendía a reducir el aumento de peso corporal, evaluamos si el GSPE inducía efectos de largo plazo sobre la gestión de lípidos en tejidos periféricos. Observamos una limitación de almacenamiento en el tejido adiposo y un aumento de la oxidación lipídica en el hígado y músculo esquelético que duró siete semanas después de la última dosis de GSPE. Resumiendo, esta tesis muestra que el GSPE es capaz de modular el sistema enteroendocrino y mejorar el estado energético, alterado por una dieta de cafetería, demostrando ser un buen agente para tratar las alteraciones metabólicas inducidas por la obesidad.Proanthocyanidins have already proved to be effective at stimulating the enteroendocrine system in healthy conditions, but their effects under an obesogenic challenge have still to be determined. For this reason, this thesis was designed to study the effects of a grape seed proanthocyanidin extract (GSPE) on the enteroendocrine system in rats fed with a long-term cafeteria diet. Our results showed that a preventive treatment, a synchronic intermittent treatment and a corrective treatment were all capable of modulating the enteroendocrine system differently. Furthermore, each GSPE treatment showed different enteroendocrine profiles associated with changes in body weight and/or food intake. However, we had certain difficulties regarding the quantification of enterohormone secretions, which led us to develop a new ex vivo methodology that stimulated different segments of the gastrointestinal tract and quantified their enterohormone secretion response, thus keeping their vectoriality. We also found that a 10-day pre-treatment with GSPE induced a long-term upregulation of GLP-1 gene expression in the ileum that was partly mediated by the hypomethylation of its GLP-1 promoter. Moreover, these effects were maintained when GSPE was administered every other week during the seventeen weeks of cafeteria diet. In addition, since this preventive GSPE treatment presented a decreased respiratory quotient and tended to reduce the body weight gain, we evaluated if there were also long-lasting GSPE effects on lipid management in the peripheric tissues. The results showed a limitation on adipose storage and an increase in lipid oxidation in the liver and skeletal muscle that lasted seven weeks after the last GSPE dose. To sum up, this thesis revealed that grape seed proanthocyanidins are capable of modulating the enteroendocrine system and improving the energetic state altered by a cafeteria diet, thus demonstrating that they are good agents for treating metabolic alterations induced by obesity

AP-TO-BAS, AN INTESTINAL DISPOSITIVE TO MEASURE PIG MUCOSA ENTEROENDOCRINE SECRETORY ACTIVITY IN RESPONSE TO APICAL STIMULATION

The enteroendocrine system helps to coordinate the processes occurring at the gastrointestinal (GI) tract and the functionality of the whole organism. However, the low abundance and scattered distribution of the enteroendocrine cells makes the study of the mechanisms underlying their function difficult. To overcome the ethical limitations of animal studies and the limitations of the available cell lines, ex vivo approaches seem to be the most simplified physiological alternative. In its turn, ex vivo models, such as Ussing Chambers still have the drawback of the limited number of samples that can be analyzed or the number of cells in each section. Here we provide an ex vivo system to assay vectorial transepithelial processes without the main limitations of the Ussing Chambers methodology.We worked with different segments of the GI wall of a pig, from which muscular layers were removed, and mounted it in a dispositive that we called `Ap-to-Bas`. We glued the samples to a silicon tube and placed it inside of a cell culture insert without bottom membrane. This allowed us to work with plenty of replicates of each intestinal segment. Tissue viability was checked by measuring lactate dehydrogenase activity in culture media, which after 60 minutes was negligible. The efficiency of the gluing process was evaluated using a 70-kDa fluorescein isothionate (FITC)-dextran. Barrier properties, measured by the crossing of Lucifer Yellow from apical to basolateral side, were maintained similar as in segments mounted in Ussing Chambers. Finally, to check the enteroendocrine functionality, tissues were apically treated with a Grape Seed Proanthocyanidin Extract (GSPE) and basolateral enterohormone secretions were measured. GSPE modulated entehormone (acyl-ghrelin, PYY and CCK) secretion at the duodenum. It also stimulated the secretion of total GLP-1 in the right colon.In conclusion, the Ap-to-Bas dispositive allows performing entoerohormone studies while keeping the apical and basolateral zones of the intestine separated

Effects of an Intermittent Grape-Seed Proanthocyanidin (GSPE) Treatment on a Cafeteria Diet Obesogenic Challenge in Rats

Obesity is highly associated with the pathologies included in the concept of the Metabolic Syndrome. Grape-seed proanthocyanins (GSPE) have showed very positive effects against all these metabolic disruptions; however, there is, as yet, no consensus about their effectiveness against an obesogenic challenge, such as a cafeteria diet. We determined the effectiveness of a dose of 500 mg GSPE/kg b.w. (body weight) against the obesogenic effects of a 17-week cafeteria diet, administered as a sub-chronic treatment, 10–15 days before, intermittently and at the end of the diet, in Wistar rats. Body weight, adiposity, indirect calorimetry and plasma parameters were analyzed. GSPE pre-treatment showed a long-lasting effect on body weight and adiposity that was maintained for seven weeks after the last dose. A corrective treatment was administered for the last two weeks of the cafeteria diet intervention; however, it did not effectively correct any of the parameters assessed. The most effective treatment was an intermittent GSPE dosage, administered every second week during the cafeteria diet. This limited body weight gain, adiposity and most lipotoxic effects. Our results support the administration of this GSPE dose, keeping an intermittent interval between dosages longer than every second week, to improve obesogenic disruptions produced by a cafeteria diet

Grape-Seed Proanthocyanidins are Able to Reverse Intestinal Dysfunction and Metabolic Endotoxemia Induced by a Cafeteria Diet in Wistar Rats

We evaluated the effectiveness of pharmacological doses of grape-seed proanthocyanidin extract (GSPE) in reversing intestinal barrier alterations and local inflammation in female Wistar rats fed a long-term obesogenic diet. Animals were fed a 17-week cafeteria diet (CAF diet), supplemented with daily GSPE doses (100 or 500 mg kg−1 body weight) during the final two weeks. CAF diet enhanced the intestinal permeation of an orally administered marker (ovalbumin, OVA) and increased the plasma levels of tumor necrosis factor-α (TNF-α) and lipopolysaccharides (LPS) in 2−3-fold. Ex vivo Ussing chamber assays showed a 55−70% reduction in transepithelial electrical resistance (TEER) and increased the TNF-α secretions in both small and large intestinal sections with a 25-fold increment in the ileum. Ileal tissues also presented a 4-fold increase of myeloperoxidase (MPO) activity. Both GSPE-treatments were able to restitute TEER values in the ileum and colon and to reduce plasma LPS to basal levels without a dose-dependent effect. However, effects on the OVA permeation and TNF-α secretion were dose and section-specific. GSPE also reduced ileal MPO activity and upregulated claudin 1 gene expression. This study provides evidence of the efficacy of GSPE-supplementation ameliorating diet-induced intestinal dysfunction and metabolic endotoxemia when administered at the end of a long-term obesogenic diet

Proanthocyanidins Limit Adipose Accrual Induced by a Cafeteria Diet, Several Weeks after the End of the Treatment

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, muscle showed a higher fatty oxidation (Oxct1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle