Analysis of the Promoters Involved in Enterocin AS-48 Expression

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.This work was supported in part by the Ministerio de Ciencia e Innovación project BIO2008-01708, the Plan Propio from the University of Granada (Spain) and by the Research Plan Group (BIO 160)

Stem cell‐derived enteroid cultures as a tool for dissecting host‐parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments

Historical Isolation versus Recent Long-Distance Connections between Europe and Africa in Bifid Toadflaxes (Linaria sect. Versicolores)

Background: Due to its complex, dynamic and well-known paleogeography, the Mediterranean region provides an ideal framework to study the colonization history of plant lineages. The genus Linaria has its diversity centre in the Mediterranean region, both in Europe and Africa. The last land connection between both continental plates occurred during the Messinian Salinity Crisis, in the late Miocene (5.96 to 5.33 Ma). Methodology/Principal Findings: We analyzed the colonization history of Linaria sect. Versicolores (bifid toadflaxes), which includes c. 22 species distributed across the Mediterranean, including Europe and Africa. Two cpDNA regions (rpl32-trnL UAG and trnK-matK) were sequenced from 66 samples of Linaria. We conducted phylogenetic, dating, biogeographic and phylogeographic analyses to reconstruct colonization patterns in space and time. Four major clades were found: two of them exclusively contain Iberian samples, while the other two include northern African samples together with some European samples. The bifid toadflaxes have been split in African and European clades since the late Miocene, and most lineage and speciation differentiation occurred during the Pliocene and Quaternary. We have strongly inferred four events of post-Messinian colonization following long-distance dispersal from northern Africa to the Iberian Peninsula, Sicily and Greece. Conclusions/Significance: The current distribution of Linaria sect. Versicolores lineages is explained by both ancien

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

HT29 Cell Line

This chapter is distributed under the terms of the Creative Commons Attribution Noncommercial License.The human colon adenocarcinoma cell line HT29, is not only used to study the biology of human colon cancers, but it is receiving special interest in studies focused on food digestion and bioavailability due to the ability to express characteristics of mature intestinal cells. In the differentiated phenotype, they are able to form a monolayer with tight junctions between cells and a typical apical brush border. In addition, these differentiated cells express brush-border-associated hydrolases typical of the small intestine although the enzymatic activity is lower than that found in vivo. Although they represent a valuable model due to their similarities with enterocytes of the small intestine, their limitations and the relevance to the in vivo situation are also considered in this chapter. The application of this cell line to transport studies of drugs and food compounds is illustrated, especially when the effect of the mucus layer is considered or used as co-culture in combination with Caco-2 cells. They have also been frequently used to study the intestinal immune response to bacterial infection, and microorganism survival, adhesion or invasion. Finally, the use of these cells to evaluate the effect of several food compounds and mucin secretion is summarized.This work was supported by projects AGL2011-24643 from Ministerio de Economía y Competitividad and FP7-SME-2012-315349 (FOFIND). The authors are participants in the FA1005 COST Action INFOGEST on food digestion.Peer Reviewe

Food peptides with biological activities related to instentinal functions

Memoria presentada por el Licenciado en Ciencias Químicas Daniel Martínez Maqueda, para optar al grado de Doctor en Ciencia y Tecnología de Alimentos con Mención de Doctorado Europeo en el Departamento de Química-Física Aplicada de la Universidad Autónoma de Madrid y realizada en el Instituto de Investigación en Ciencias de la Alimentación CSIC-UAM.[ES]: El epitelio gastrointestinal está protegido por una capa, denominada mucus, cuya producción puede estar regulada por diferentes componentes alimentarios. Un péptido derivado de la caseína, la β-casomorfina 7, ha demostrado influencia en la producción del mucus intestinal a través de la estimulación de la secreción y la expresión génica de las mucinas, principales constituyentes macromoleculares, mediante un mecanismo opioide. En esta Tesis Doctoral, se estudió el efecto sobre la producción de mucinas, en células caliciformes intestinales humanas HT29-MTX, de distintos péptidos alimentarios con probada o probable afinidad hacia receptores µ y δ-opioides, algunos de ellos identificados en simulaciones gastrointestinales. Adicionalmente, se ensayaron un hidrolizado obtenido a partir de un concentrado de proteínas de suero (WPC) enriquecido en β-lactoglobulina y un hidrolizado de caseínas y, que contenían algunos de los péptidos seleccionados. El hidrolizado de WPC mostró una actividad significativa sobre la producción de mucinas, incrementando su secreción y sobreexpresando MUC5AC, el gen de la principal mucina secretada en las células HT29-MTX. Este efecto fue parcialmente justificado por la presencia en el hidrolizado del péptido opioide β-lactorfina (YLLF), el cual estimuló la secreción de mucinas pero no modificó la expresión del gen MUC5AC. El hidrolizado de caseínas mostró una potente actividad estimulante sobre la producción de mucinas, que puede provenir de los fragmentos 143-149 (AYFYPEL) y 144-149 (YFYPEL) de la αs1-caseína, aunque ambos péptidos inducían un menor efecto cuando se ensayaron separadamente. Por tanto, otros péptidos o componentes de los hidrolizados podrían participar en sus actividades observadas sobre la síntesis de mucinas. Con el objetivo de elucidar si los péptidos con efecto estimulante sobre la producción de mucinas, pero sin actividad opioide descrita, podrían interaccionar con los receptores opioides, se llevaron a cabo experimentos con íleon de cobayo. Como resultado se demostró, por primera vez, que los fragmentos 144-149 (YFYPEL) y 144-148 (YFYPE) de la αs1-caseína presentan actividad opioide. No obstante, el péptido YFYPE no provocó sobreexpresión de MUC5AC en las células HT29-MTX, por lo que podría considerarse la mediación de mecanismos adicionales. El fortalecimiento del mucus también desempeña un papel crucial en la defensa de la mucosa gástrica. Por ello, se evaluaron las propiedades antiulcerogénicas de los hidrolizados de caseína y WPC en un modelo de úlceras inducidas por etanol en ratas. Ambos hidrolizados exhibieron una eficaz protección frente a las lesiones gástricas inducidas por etanol. La actividad antiulcerogénica del hidrolizado de WPC se encontró mediada por la acción de los grupos sulfhidrilos (SH) activos, ya que disminuía el efecto del hidrolizado al tratar el animal con el agente bloqueante de los grupos sulfidrilo N-etilmaleimida. Por otra parte, se está estudiando el suplemento de la dieta con proteínas de suero para el control de la diabetes tipo 2, cuya actividad podría estar mediada por la inhibición de la enzima dipeptidil-peptidasa (DPP-IV).Por ello, se evaluó la β-lactoglobulina como una fuente de péptidos con capacidad inhibitoria de esta enzima. El hidrolizado de WPC enriquecido en β-lactoglobulina produjo una importante inhibición de la actividad de la DPP-IV y se identificaron por primera vez dos secuencias de la β-lactoglobulina, concretamente los fragmentos 78-82 (IPAVF) y 78-83 (IPAVFK), como potentes inhibidores de la enzima DPP-IV. El péptido IPAVF demostró una notable actividad inhibitoria, con un valor de IC50 de 44.7 µM. En conclusión, dos hidrolizados de proteínas lácteas y una serie de péptidos derivados podrían ser prometedores en el desarrollo de alimentos funcionales para la mejora de la protección del epitelio en enfermedades gastrointestinales y para el control de la diabetes tipo 2.[EN]: The gastrointestinal lumen is strategically covered by a protective layer, known as mucus, and its production can be modulated by different food components. β- Casomorphin 7, a casein-derived peptide, has been shown to modify the dynamics of intestinal mucus by increasing the secretion and expression of mucins, the major macromolecular constituent, through an opioid mechanism. In this Thesis, several milkderived peptides with proved or probable ability to bind µ- and δ-opioid receptors, some of them identified in simulated gastrointestinal digestions, besides two hydrolysates from casein and a whey protein concentrate (WPC) rich in β-lactoglobulin that contain some of the selected peptides, were investigated in HT29-MTX human intestinal goblet-like cells. The WPC hydrolysate exhibited a significant activity with increased mucin secretion and stimulated expression of MUC5AC, the major secreted mucin gene in HT29-MTX cells. This effect was partially supported by the presence in the hydrolysate of the opioid β-lactorphin (YLLF), which demonstrated influence on mucin secretion although not in gene expression. The casein hydrolysate showed a remarkable activity that may result from the αs1-casein fragments 143-149 (AYFYPEL) and 144-149 (YFYPEL), although both peptides induced a lower effect separately. Therefore, other peptides or components of the hydrolysates could participate in the observed activities. In order to elucidate if peptides with effect on mucin production, but not reported opioid activity, could bind opioid receptors, experiments with guinea-pig ileum preparations were performed. Interestingly, αs1-casein fragments 144-149 (YFYPEL) and 144-148 (YFYPE) showed opioid activity for the first time. However, YFYPE exhibited no significant effect on MUC5AC expression, so the mediation of additional pathways should be considered. Mucus strengthening also plays an important role in the gastric mucosa defence. Thus, antiulcerative properties of the WPC and casein hydrolysates were evaluated in an ethanol-induced ulcer model in rats. Both hydrolysates showed effective gastric protection against the induced lesions. The antiulcerative activity of the WPC hydrolysate was mediatedby the action of sulfhydryl (SH) groups, as revealed the lack of protective effect after in vivo SH blockage by N-ethylmaleimide. Moreover, diet supplementation with whey proteins is currently considered an alternative and promising treatment of type 2 diabetes, in part due to the observed inhibition of dipeptidyl peptidase IV enzyme (DPP-IV). β- lactoglobulin was evaluated as source of DPP-IV inhibitory peptides. The WPC hydrolysate showed an important inhibition of DPP-IV activity and two β-lactoglobulin fragments, 78-82 (IPAVF) and 78-83 (IPAVFK), were identified for the first time as potent DPP-IV inhibitory peptides, especially IPAVF with an IC50 of 44.7 µM. In conclusion, milk protein hydrolysates and derived peptides could be promising for the development of functional foods to improve epithelium protection in gastrointestinal diseases and the management of type 2 diabetes.Agradezco al CSIC su financiación con una beca JAE Predoc.Peer Reviewe

In silico and in vitro analysis of multifunctionality of animal food-derived peptides

This article belongs to the Special Issue New Advances in the Research of Antioxidant Food Peptides.Currently, the associations between oxidative stress, inflammation, hypertension, and metabolic disturbances and non-communicable diseases are very well known. Since these risk factors show a preventable character, the searching of food peptides acting against them has become a promising strategy for the design and development of new multifunctional foods or nutraceuticals. In the present study, an integrated approach combining an in silico study and in vitro assays was used to confirm the multifunctionality of milk and meat protein-derived peptides that were similar to or shared amino acids with previously described opioid peptides. By the in silico analysis, 15 of the 27 assayed peptides were found to exert two or more activities, with Angiotensin-converting enzyme (ACE) inhibitory, antioxidant, and opioid being the most commonly found. The in vitro study confirmed ACE-inhibitory and antioxidant activities in 15 and 26 of the 27 synthetic peptides, respectively. Four fragments, RYLGYLE, YLGYLE, YFYPEL, and YPWT, also demonstrated the ability to protect Caco-2 and macrophages RAW264.7 cells from the oxidative damage caused by chemicals. The multifunctionality of these peptides makes them promising agents against oxidative stress-associated diseases.This research was funded by project AGL2015- 66886-R from the Spanish Ministry of Science and Innovation (MICIU).Peer reviewe

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways

The membrane-bound mucin MUC4 is a high molecular weight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-ras mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-ras in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the post-transcriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.Romain Vasseur is a recipient of a doctoral fellowship of Lille2 University. Nicolas Skrypek is a recipient of a PhD fellowship from the Centre Hospitalier Régional et Universitaire (CHRU) de Lille/région Nord-Pas de Calais. Audrey Vincent is the recipient of a postdoctoral fellowship from the Fondation ARC and Région Nord-Pas de Calais. Isabelle Van Seuningen is the recipient of a “Contrat Hospitalier de Recherche Translationnelle”/CHRT 2010, AVIESAN. This work is supported by grants from la Ligue Nationale Contre le Cancer (Equipe Labellisée Ligue 2010, IVS), from SIRIC ONCOLille, Grant INCaDGOSInserm 6041 (IVS, NJ) and from “Contrat de Plan Etat Région” CPER Cancer 2007–2013 (IVS).Peer Reviewe

Relationship between iron status markers and insulin resistance: an exploratory study in subjects with excess body weight

[Background]: Controversy exists on the relationship between iron metabolism and cardiometabolic risk. The aim of this study was to determine if there is a link between dysmetabolic iron and cardiometabolic markers in subjects with excess body weight[Methods]: Cross-sectional study with fifty participants presenting overweight or obesity and at least another metabolic syndrome factor. Determinations: anthropometry, body composition, blood pressure, lipids, glucose, insulin, leptin, areas under the curve (AUC) for glucose and insulin after an oral glucose tolerance test, hs-C reactive protein (hs-CRP), blood count, ferritin, transferrin, transferrin saturation (TSAT), soluble transferrin receptor (sTfR). Gender-adjusted linear correlations and two independent samples t tests were used.[Results]: Ferritin was positively correlated with insulin-AUC (r = 0.547, p = 0.008) and TSAT was negatively correlated with waist-hip ratio (r = −0.385, p = 0.008), insulin (r = −0.551, p 20%. In conclusion, the observed results suggest that iron transport and storage are altered in subjects with overweight/obesity, at the same time that they exhibit the characteristic features of insulin resistance. Nevertheless, this occurs without iron overload or deficiency. These results should be validated in wider cohorts since they suggest that iron transport and storage should be assessed when performing the clinical evaluation of subjects with excess body weight.This study was funded by the Spanish Ministry of Economy and Competitiveness (MINNECO-FEDER, grant: AGL2014 55102-JIN). Angélica Gallego-Narbón was funded by the Youth Employment Initiative (YEI) from the European Social Fund (ESF).Support for the publication fee was provided by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe