Individual variability in patterns and dynamics of fecal gluten immunogenic peptides excretion after low gluten intake

This work was supported by grants from Ministerio de Ciencia e Innovacion (DI-16-08943), Junta de Andalucia, Consejeria de Economia, Conocimiento, Empresas y Universidad and FEDER funds (AT17_5489_USE), Centro para el Desarrollo Tecnologico Industrial (IDI-20180051) and Corporacion Tecnologica de Andalucia (17/957).Purpose Determination of Gluten Immunogenic Peptides (GIP) in feces is a direct tool for gluten exposure detection. The sensitivity of GIP detection methods for cases of unintentional low gluten intakes is unknown. We studied the interindividual variability in the kinetic of excretion under homogeneously controlled dietary conditions, and the sensitivity of fecal GIP tests after low amounts of punctual gluten ingestions. Methods Participants (n = 20) followed the same gluten-free menu for 12 days in which two separated doses of gluten (50 mg and 2 g) were ingested and all the depositions were collected. GIP from stool samples were analyzed by ELISA and lateral flow immunoassay (LFIA) tests. Results Most participants had detectable GIP after 50 mg and 2 g gluten ingestions using ELISA test (72.2% and 95%, respectively), whereas the LFIA test showed less sensitivity (22.2% and 80%, respectively). GIP were detected at higher either frequency or concentration in the range of 12–36 h after 50 mg intake, and 12–84 h after 2 g consumption. Considering this period, diagnostic sensitivity of GIP detection after a single 50 mg ingestion may be significatively increased analyzing three stool samples per individual. High variability among participants was found in the time and amount of GIP excretion; however, some individuals showed common patterns for both gluten intakes. Conclusion Sporadic gluten exposure detection may require several fecal samples to achieve level of sensitivity above 90%. Interindividual variability in the dynamic of GIP excretion may suggest patterns of gluten metabolism.Instituto de Salud Carlos III Spanish Government European Commission DI-16-08943Junta de Andalucia Consejeria de Economia, Conocimiento, Empresas y Universidad European Commission AT17_5489_USECentro para el Desarrollo Tecnologico Industrial IDI-20180051Corporacion Tecnologica de Andalucia 17/95

Gluten Immunogenic Peptides as Standard for the Evaluation of Potential Harmful Prolamin Content in Food and Human Specimen

Gluten is a complex mixture of storage proteins in cereals like wheat, barley, and rye. Prolamins are the main components of gluten. Their high content in proline and glutamine makes them water-insoluble and difficult to digest in the gastrointestinal tract. Partial digestion generates peptide sequences which trigger immune responses in celiac and gluten-sensitive patients. Gluten detection in food is challenging because of the diversity, in various food matrices, of protein proportions or modifications and the huge number of immunogenic sequences with differential potential immunoactivity. Attempts to develop standard reference materials have been unsuccessful. Recent studies have reported the detection of a limited number of dominant Gluten Immunogenic Peptides (GIP) that share similarities to epitopes presented in the α-gliadin 33-mer, which showed to be highly proteolytic resistant and is considered to be the most immunodominant peptide within gluten in celiac disease (CD). GIP were detectable and quantifiable in very different kind of difficult to analyze food, revealing the potential immunogenicity by detecting T-cell activity of celiac patients. But GIP were also found in stool and urine of celiac patients on a supposedly gluten-free diet (GFD), showing the capacity to resist and be absorbed and excreted from the body, providing the first simple and objective means to assess adherence to the GFD. Methods to specifically and sensitively detect the most active GIP in food and biological fluids are rational candidates may use similar analytical standard references for determination of the immunopathological risk of gluten exposure in gluten-related diseases.España, MINECO AGL2013-48946-CEspaña, Ministerio de Ciencia, Innovación y Universidades y Corporación Tecnológica de Andalucía (CTA

Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reactionbased saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent Ks * values 5–100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met116, Arg132, Asn169, and Arg248 are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoter

Current Trends in the GFD Follow-Up

A poor adherence to a gluten-free diet (GFD) have a negative impact on people with celiac disease (CD). However, committing to a gluten-free lifelong carries social and economic burden and, a high degree of knowledge, motivation and a continuous effort. It is essential that the patient understands its disease, how to perform a GFD and the consequences that entail if the patient is not followed in the long term. However, a large percentage of patients does not still achieve a complete mucosal healing, likely due to a poor adherence to the GFD. We describe the current tools for the control of adherence to a GFD, with a special focus on the detection of gluten immunogenic peptides (GIP) in feces and urine, as GIP detection allows direct evidence that the gluten that has been ingested. GIP are becoming useful biomarkers for this aim. Here, we summarize the current information about the main applications and limitations of the use of the GIP determinations in the follow up of celiac disease

Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153- YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd21 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusion

An improved system for estradiol-dependent regulation of gene expression in yeast

BACKGROUND: Saccharomyces cerevisiae is widely utilized in basic research as a model eukaryotic organism and in biotechnology as a host for heterologous protein production. Both activities demand the use of highly regulated systems, able to provide accurate control of gene expression in functional analysis, and timely recombinant protein synthesis during fermentative production. The tightly regulated GAL1-10 promoter is commonly used. However, induction of the GAL system requires the presence of the rather expensive inducer galactose and the absence of glucose in the culture media. An alternative to regulate transcription driven by GAL promoters, free of general metabolic changes, is the incorporation of the hybrid Gal4-ER-VP16 protein developed by D. Picard. This chimeric protein provides galactose-independent activation of transcription from GAL promoters in response to β-estradiol, even in the presence of glucose. However, constitutive expression of this transactivator results in relatively high basal activity of the GAL promoters, therefore limiting the gene expression capacity that is required for a number of applications. RESULTS: In order to improve this expression tool, we have introduced additional regulatory elements allowing a simultaneous control of both the abundance and the intrinsic activity of the Gal4-ER-VP16 chimeric transactivator. The most efficient combination was obtained by placing the coding sequence of the hybrid activator under the control of the GAL1 promoter. This configuration results in an amplification feedback loop that is triggered by the hormone, and ultimately leads to the enhanced regulation of recombinant genes when these are also driven by a GAL1 promoter. The basal expression level of this system is as low as that of native GAL-driven genes in glucose-containing media. CONCLUSION: The feedback regulatory loop that we have engineered allows a 250-fold induction of the regulated gene, without increasing the basal activity of the target promoter, and achieving a 12-fold higher regulation efficiency than the previous configuration

El sonido y su influencia en la mente del consumidor

Las empresas innovan constantemente para intentar influir en la decisión de compra del consumidor. El Neuromarketing es una de las técnicas con más potencial y de mayor interés para las compañías dado que se consigue conocer la respuesta del cliente ante diferentes estímulos. El Marketing auditivo o “Audiomarketing” es un conjunto de técnicas de neuromarketing que influyen sobre el sentido del oído. El presente Trabajo Fin de Grado enlaza los tres aspectos fundamentales para un análisis completo del Marketing auditivo.- Primero se investiga sobre la influencia de la música en la mente analizando qué recorrido sigue y qué zonas activa en nuestro cerebro, investigando qué estructuras melódicas son las mas recordadas y el por qué. Seguidamente se analizan las técnicas de Audiomarketing más utilizadas por las empresas y los diferentes objetivos que plantean, proporcionando así una visión global de los beneficios que estas técnicas pueden ofrecer. Por último, se realiza una investigación de mercados sobre el poder de recuerdo que tiene el Audiobranding y su efectividad respecto al esloga

Determination of gluten immunogenic peptides for the management of the treatment adherence of celiac disease: A systematic review

BACKGROUND Gluten is a complex mixture of proteins with immunogenic peptide sequences triggering the autoimmune activity in patients with celiac disease (CeD). Gluten immunogenic peptides (GIP) are resistant to gastrointestinal digestion and are then excreted via the stool and urine. Most common detection methods applied in the follow-up visits for CeD patients such as serology tests, dietetic interviews, questionnaires, and duodenal biopsy have been proved to be inefficient, invasive, or inaccurate for evaluating gluten-free diet (GFD) compliance. Determination of excreted GIP in stool and urine has been developed as a non-invasive, direct, and specific test for GFD monitoring. AIM To summarize published literature about the clinical utility of GIP determination in comparison to the tools employed for GFD monitoring. METHODS PubMed and Web of Science searches were performed using the keywords “gluten immunogenic peptides” or “gluten immunogenic peptide” and a combination of the previous terms with “feces”, “stools”, “urine”, “celiac disease”, “gluten-free diet”, and “adherence” to identify relevant clinical studies published in English and Spanish between 2012 to January 2021. Reference lists from the articles were reviewed to identify additional pertinent articles. Published articles and abstracts reporting the clinical use of GIP determination in stool and/or urine for the follow-up of patients with CeD in comparison with other tools in use were included. Case reports, commentaries, reviews, conference papers, letters, and publications that did not focus on the aims of this review were excluded. RESULTS Total of 15 publications were found that involved the use of GIP determination in stool and/or urine to monitor the adherence to the GFD in comparison to other tools. Studies included both children and adults diagnosed with CeD and healthy volunteers. Overall, these preliminary studies indicated that this novel technique was highly sensitive for the detection of GFD transgressions and therefore could facilitate the follow-up of patients with CeD. Tools identified in this work included the CeD-specific serology, dietetic questionnaires, symptomatology, and the duodenal biopsy. Review of the literature revealed that the rates of GFD adherence may vary between 30%-93% using either stool or urine GIP determination, 49%-96% by the serology, 59%-94% using the dietetic questionnaires, 56%-95% by the reported symptoms and 44%-76% with the duodenal biopsy. In addition, the association between the different methods and histological abnormalities (Marsh II-III) was found to be 33%-100% for GIP determination (stool and urine), 25%-39% for CeD-specific serology, 3%-50% for dietetic questionnaires, and 22%-28% for the symptomatology. CONCLUSION Excreted GIP detection is the precise approach for determining voluntary or involuntary gluten consumption in CeD patients preventing future complications arising from gluten exposure

Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

We present a novel procedure for affinity partitioning of recombinant proteins fused to the cholinebinding module C-LytA in aqueous two-phase systems containing poly(ethylene glycol) (PEG). Proteins tagged with the C-LytA module and exposed to the two-phase systems are quantitatively localized in the PEG-rich phase, whereas subsequent addition of the natural ligand choline specifically shifts their localization to the PEG-poor phase by displacement of the polymer from the binding sites. The described procedure is simple, scalable and reproducible, and has been successfully applied to the purification of four diverse proteins, resulting in high yields and purity

El contrato en prácticas

El presente trabajo recoge un estudio sobre las peculiaridades que presenta el contrato en prácticas, además de las especialidades que podemos encontrar en diferentes ámbitos laborales.<br /