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Stool collection devices minimizing the exposure of gut bacteria to oxygen are critical for the standardization of further microbiota-based studies, analysis and developments. The aim of this work was to evidence that keeping anaerobiosis has a deep impact on the viability and diversity of the fecal microbiota that is recovered in the laboratory. Recovering certain microbial populations, such as obligate anaerobic bacteria, is particularly critical if the purpose of the study is to envisage personalized therapeutic purposes, such as autologous Fecal Microbiota Transplant. In this study the same fecal specimens were sampled in conventional stool containers and GutAlive, a disposable device that minimizes exposure of the gut microbiota to oxygen. Samples from five healthy donors were analysed and 150 differential colonies were recovered and identified by 16S rRNA gene sequencing. Globally, GutAlive maintained extremely oxygen sensitive (EOS) populations that were lost in conventional stool containers, and thus viability of species such as as Akkermansia muciniphila, Faecalibacterium prausnitzii and a novel member of the Clostridiales order was kept. These obligate anaerobes were not recovered using the conventional stool collection device. In conclusion, the use of GutAlive for stool collection and transport optimized the viability and recovery of EOS bacteria in the lab by diminishing oxygen toxicity., Tis work was funded by the project IDE/2016/000282 from IDEPA (“Instituto de Desarrollo Empresarial of the Principality of Asturias”), which is partially supported by FEDER funds, SME-2017-0946-10 from MEIC (Ministry of Economy, Industry and competitiveness), SNEO 20171062 from CDTI (Centre for the Development of Industrial Technology) and a collaboration contract between Microviable Terapeutics SL. and the Consejo Superior de Investigaciones Científcas (Spanish National Research Council - CSIC).

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2020.103969., This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohns patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides., Our work is supported by the Spanish “Programa Estatal de Investigación. Desarrollo e Innovación Orientada a los Retos de la Sociedad” (grants AGL2013-44761-P and AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016), by the Asturias Regional Plan I + D + i for research groups (FYCYT-IDI/2018/000236) and by the Autonomic “Investigadores Emerxentes do Sistema Universitario de Galicia” (Grant EM2014/046). This work was partially supported by the Consellería de Educación. Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group. Finally, the authors wish to thank Jaume Morales and Rubén García form Agilent Technologies for technical support., info:eu-repo/semantics/publishedVersion

COVID-2019 has progressed in around 10-15% of patients to an acute respiratory distress syndrome characterized by extensive pulmonary inflammation and elevated production of pro-inflammatory cytokines. Neutrophil activation seems to be crucial in the initiation and perpetuation of this exacerbated lung inflammation. However, the precise mechanisms by which this activation occurs remain yet elusive. To this end, this in silico study tried to identify potential proinflammatory inducing peptides (PIPs) produced by the action of the elastase released in neutrophil-extracellular traps over SARS-CoV-2 particles. We found nine potential PIPs exclusive from the SARS-CoV-2, showing homology against T cell recognition epitopes. Moreover, 78 percent of these exclusive PIPs were found produced by the enzymatic cleavage on the spike glycoproteins, suggesting that high PIP concentrations might be released following SARS-CoV-2 huge replication rate. Therefore, these PIPs might play a role in the exacerbated inflammatory response observed in some patients.

Lactobacillus genus includes both probiotic and representative strains of the human gut microbiota. Independent studies have reported on the anti-inflammatory properties of different Lactobacillus strains, although we are far from understanding the underlying molecular interplay. In this work we show that a daily administration of Lactobacillus acidophilus DSM20079T (DSM20079) to healthy piglets resulted in plasmatic increases of the anti-inflammatory IL10, whilst IL12 and the pro-inflammatory ratio IL12+TNF/IL10 decreased. The extracellular protein fraction of DSM20079 was identified as the responsible for the crosstalk interaction that elicited these tolerogenic effects. This strain was able to activate innate immune pathways in dendritic cells and to decrease the production of pro-inflammatory cytokines in both CD4+/CD8+ T cell subsets in healthy donors and in Crohns Disease patients. The tolerogenic effect exerted by the extracellular proteins of this strain suggests their potential use as coadjutant for therapeutic applications targeting chronic inflammatory illnesses., Our work is supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2016-78311-R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer ColoRectal a partir de secuencias genéticas de microbiomas intestinales”, Grant PS-2016) and by the Asturias Regional Plan I+D+i for research groups (FYCYT-IDI/2018/000236). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure. LJR was supported by the Principado de Asturias, PCTI 2018–2020 (GRUPIN: IDI2018-000237) and FEDER. This work was partially supported by the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group., info:eu-repo/semantics/publishedVersion

Background Some patients complain that eating lettuce, gives them gas and abdominal distention. Our aim was to determine to what extent the patients' assertion is sustained by evidence. Methods An in vitro study measured the amount of gas produced during the process of fermentation by a preparation of human colonic microbiota (n = 3) of predigested lettuce, as compared to beans, a high gas‐releasing substrate, to meat, a low gas‐releasing substrate, and to a nutrient‐free negative control. A clinical study in patients complaining of abdominal distention after eating lettuce (n = 12) measured the amount of intestinal gas and the morphometric configuration of the abdominal cavity in abdominal CT scans during an episode of lettuce‐induced distension as compared to basal conditions. Key Results Gas production by microbiota fermentation of lettuce in vitro was similar to that of meat (P = .44), lower than that of beans (by 78 ± 15%; P, In some patients lettuce produces abdominal distension, which is not related to gas, but rather to an uncontrolled reaction of the abdominal muscles and the diaphragm. Hence, learning to control the abdominal muscles may prevent distension with no need of dietary restrictions.

Trabajo presentado en la 13ª Reunión de la Red Española de Bacterias Lácticas (RedBAL), celebrada en Madrid (España) del 17 al 18 de junio de 2019, El uso de probióticos o compuestos bacterianos se ha propuesto como un mecanismo para modular la respuesta inmune del hospedador y remodelar el microbioma humano. Sin embargo, actualmente existe una falta de información sobre las vías moleculares inducidas por las bacterias probióticas, o sobre la interacción entre los probióticos y el sistema inmunológico. Lactobacillus es un género del filo Firmicutes que incluye cepas probióticas y representativas de la microbiota gastrointestinal humana. Estudios independientes han demostrado las propiedades antiinflamatorias de diferentes cepas de Lactobacillus, aunque estamos lejos de comprender la interacción molecular subyacente. En este trabajo, demostramos que una administración diaria de 5e10 células viables de Lactobacillus acidophilus DSM 20079T (DSM20079) a lechones sanos produjo un aumento plasmático de la interleucina antinflamatoria IL10, disminuyendo, a su vez, la interleucina IL12 y la ratio proinflamatorio IL12 + TNF α / IL10. Además, las células mononucleares de sangre preriféricas (PBMCs) extraídas de estos lechones se volvieron menos reactivas contra el lipopolisacárido de Escherichia coli (LPS) y la fitohemaglutinina de Phaseolus vulgaris (PHA), cuando se ven afectadas por la cepa DSM20079. Se identificó la fracción proteica extracelular de DSM20079 como la responsable de esta modulación. El efecto tolerogénico ejercido por las proteínas extracelulares de esta cepa sugiere su uso potencial como coadyuvante para aplicaciones terapéuticas dirigidas a enfermedades inflamatorias crónicas., Este trabajo fue respaldado por el >Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad> (AGL2016-78311- R); la Asociación Española Contra el Cáncer (>Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales>, PS-2016).

This work presents a novel in silico approach to the prediction and characterisation of the glycolytic capacities of the beneficial intestinal bacterium Faecalibacterium prausnitzii. Available F. prausnitzii genomes were explored taking the glycolytic capacities of F. prausnitzii SL3/3 and F. prausnitzii L2-6 as reference. The comparison of the generated glycolytic profiles offered insights into the particular capabilities of F. prausnitzii SL3/3 and F. prausnitzii L2-6 as well as the potential of the rest of strains. Glycoside hydrolases were mostly detected in the pathways responsible for the starch and sucrose metabolism and the biosynthesis of secondary metabolites, but this analysis also identified some other potentially interesting, but still uncharacterised activities, such as several hexosyltransferases and some hydrolases. Gene neighbourhood maps offered additional understanding of the genes coding for relevant glycoside hydrolases. Although information about the carbohydrate preferences of F. prausnitzii is scarce, the in silico metabolic predictions were consistent with previous knowledge about the impact of fermentable sugars on the growth promotion and metabolism of F. prausnitzii. So, while the predictions still need to be validated using culturing methods, the approach holds the potential to be reproduced and scaled to accommodate the analysis of other strains (or even families and genus) as well as other metabolic activities. This will allow the exploration of novel methodologies to design or obtain targeted probiotics for F. prausnitzii and other strains of interest., This work was supported by the Spanish "Programa Estatal de Investigacion, Desarrollo e Innovacion Orientada a los Retos de la Sociedad" (Grant AGL2016-78311-R); and, the Asociacion Espanola Contra el Cancer (" Obtencion de peptidos bioactivos contra el Cancer Colo-Rectal a partir de secuencias geneticas de microbiomas intestinales," Grant PS-2016). This work was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). This work was partially supported by the Conselleria de Educacion, Universidades e Formacion Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group., info:eu-repo/semantics/publishedVersion

Bile is a biological fluid synthesized in the liver, mainly constituted by bile acids and cholesterol, which functions as a biological detergent that emulsifies and solubilizes lipids, thereby playing an essential role in fat digestion. Besides, bile acids are important signaling molecules that regulate key functions at intestinal and systemic levels in the human body, affecting glucose and lipid metabolism, and immune homeostasis. Apart from this, due to their amphipathic nature, bile acids are toxic for bacterial cells and, thus, exert a strong selective pressure on the microbial populations inhabiting the human gut, decisively shaping the microbial profiles of our gut microbiota, which has been recognized as a metabolic organ playing a pivotal role in host health. Remarkably, bacteria in our gut also display a range of enzymatic activities capable of acting on bile acids and, to a lesser extent, cholesterol. These activities can have a direct impact on host physiology as they influence the composition of the intestinal and circulating bile acid pool in the host, affecting bile homeostasis. Given that bile acids are important signaling molecules in the human body, changes in the microbiota-residing bile biotransformation ability can significantly impact host physiology and health status. Elucidating ways to fine-tune microbiota-bile acids-host interplay are promising strategies to act on bile and cholesterol-related disorders. This manuscript summarizes the current knowledge on bile and cholesterol metabolism by intestinal bacteria, as well as its influence on host physiology, identifying knowledge gaps and opportunities to guide further advances in the field., This study was supported by MINECO under grant number AGL2013-44761-P. LR is a postdoctoral researcher supported by the Juan de la Cierva Postdoctoral Trainee Program (MINECO, JCI-2015-23196) and NM is the recipient of an FPI Predoctoral Grant (BES-2014-068736).

[Background] Transcriptomics profiling aims to identify and quantify all transcripts present within a cell type or tissue at a particular state, and thus provide information on the genes expressed in specific experimental settings, differentiation or disease conditions. RNA-Seq technology is becoming the standard approach for such studies, but available analysis tools are often hard to install, configure and use by users without advanced bioinformatics skills. [Methods] Within reason, DEWE aims to make RNA-Seq analysis as easy for non-proficient users as for experienced bioinformaticians. DEWE supports two well-established and widely used differential expression analysis workflows: using Bowtie2 or HISAT2 for sequence alignment; and, both applying StringTie for quantification, and Ballgown and edgeR for differential expression analysis. Also, it enables the tailored execution of individual tools as well as helps with the management and visualisation of differential expression results. [Results] DEWE provides a user-friendly interface designed to reduce the learning curve of less knowledgeable users while enabling analysis customisation and software extension by advanced users. Docker technology helps overcome installation and configuration hurdles. In addition, DEWE produces high quality and publication-ready outputs in the form of tab-delimited files and figures, as well as helps researchers with further analyses, such as pathway enrichment analysis. [Conclusions] The abilities of DEWE are exemplified here by practical application to a comparative analysis of monocytes and monocyte-derived dendritic cells, a study of clinical relevance. DEWE installers and documentation are freely available at https://www.sing-group.org/dewe., This work was supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2013-44039R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, grant PS-2016); the Consellería de Educación, Universidades e Formación Profesional (Xunta de Galicia) under the scope of the strategic funding of ED431C2018/55-GRC Competitive Reference Group; the Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2019 unit; and the Asturias Regional Plan I + D + i for research groups (FYCYT-IDI/2018/000236). H. López-Fernández is supported by a post-doctoral fellowship from Xunta de Galicia (ED481B 2016/068-0).

Advances in the field of genome sequencing have enabled a comprehensive analysis and annotation of the dynamics of the protein inventory of cells. This has been proven particularly rewarding for microbial cells, for which the majority of proteins are already accessible to analysis through automatic metagenome annotation. The large-scale in silico screening of proteomes and metaproteomes is key to uncover bioactivities of translational, clinical and biotechnological interest, and to help assign functions to certain proteins, such as those predicted as hypothetical. This work introduces a new method for the prediction of the bioactivity potential of proteomes/metaproteomes, supporting the discovery of functionally relevant proteins based on prior knowledge. This methodology complements functional annotation enrichment methods by allowing the assignment of functions to proteins annotated as hypothetical/putative/uncharacterised, as well as and enabling the detection of specific bioactivities and the recovery of proteins from defined taxa. This work shows how the new method can be applied to screen proteome and metaproteome sets to obtain predictions of clinical or biotechnological interest based on reference datasets. Notably, with this methodology, the large information files obtained after DNA sequencing or protein identification experiments can be associated for translational purposes that, in cases such as antibiotic-resistance pathogens or foodborne diseases, may represent changes in how these important and global health burdens are approached in the clinical practice. Finally, the Sequence-based Expert-driven pRoteome bioactivity Prediction EnvironmENT, a public Web service implemented in Scala functional programming style, is introduced as means to ensure broad access to the method as well as to discuss main implementation issues, such as modularity, extensibility and interoperability., This work was supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2013-44039R); the Asociación Española Contra el Cancer (“Obtención de péptidos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales”, grant PS-2016). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). SING group thanks CITI (Centro de Investigación, Transferencia e Innovación) from University of Vigo for hosting its IT infrastructure.

There are strong evidences that probiotics influence the immune status of the host, in a strain-specific manner, acting in the gastrointestinal tract. On the hypothesis that certain extracellular proteins and peptides from gut bacteria may mediate part of this immunomodulation and assuming they are able to diffuse through the mucus layer and interact with immune cells we have developed this work. Our study attempts to understand the immunomodulatory mechanisms of (i) Pext, the extracellular protein fraction of Lactobacillus acidophilus DSM20079T, (ii) HM14, a peptide encrypted in an extracellular glycoside hydrolase from Bifidobacterium longum NCIMB 8809 and (iii) Escherichia coli O111:B4 lipopolysaccharide (LPS), a well-known pro-inflammatory molecule, over human peripheral blood mononuclear cells (PBMCs). An untargeted LC-ESI-QTOF-MS metabolomics approach was applied to reveal intracellular changes in treated-PBMCs isolated from healthy donors. Differences in NADH arrest, NAD+ concentration reduction, as well as increases in palmitic acid and methanephrin were observed in HM14 and Pext treated-cells compared to those stimulated with LPS. This would support an anti-inflammatory molecular mechanism of action of such proteinaceous molecules. Moreover, this methodology has confirms the importance of metabolomics approaches to better understanding immune cell responses to gut bacterial-derived molecules., This work was funded by the Autonomic Investigadores Emerxentes do Sistema Universitario de Galicia (Grant EM2014/046) and the Spanish Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (Grant AGL2013-44039R). This work has received also financial support from the Xunta de Galicia (Plan de mellora do Centro de Investigacións Agroalimentarias CIA3 do Campus de Ourense).

Introduction: The role of the human intestinal microbiota in the maintenance of a healthy physiological condition, as well as its relation to the development of disease, remains to be clarified. Current evidence suggests that intestinal microbes could be involved in the initiation and amplification of autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus (SLE). Despite recent progress in understanding how these microbes influence the pathophysiology of lupus, studies are still limited. Areas covered: In this review, we have tried to summarize the most relevant findings that have contributed to our understanding of the links between the human intestinal microbiota and the development of lupus. We also describe the potential role of individual microbial players in the physiology of lupus, and how they can shape relevant immune responses. Expert commentary: Culture-independent techniques based on massive sequencing represent a powerful tool to unravel the biological activity of gut microbes. Current data demonstrates that, depending on the pattern of intestinal microorganisms or the presence of specific bacteria, different responses related to lupus physiology can be triggered. Fecal microbiota transplantation, live biotherapeutics, or dietary interventions targeting the microbiota will likely become a treatment for SLE.

In a recent excellent Review1 (Bile acid–microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat. Rev. Gastroenterol. Hepatol. 15, 111–128 (2018)), the connection between human and microbial bile acid (BA) metabolism was described. In this Review, the authors identified possible therapeutic targets from data published in the context of gastrointestinal inflammation and carcinogenesis. From the microbial point of view, BAs are one of the main factors determining probiotic survival through the human gastrointestinal tract, which indeed was the focus of my PhD work

Despite the existing evidence on the impact of olive oil and red wine on the intestinal microbiota, the effect of the global Mediterranean Diet (MD) has not been sufficiently studied. We explored the association between the adherence to a Mediterranean dietary pattern, and its components, with faecal microbiota in a cohort of adults with non-declared pathology. This transversal study involved 31 adults without a previous diagnosis of cancer, autoimmune or digestive diseases. Based on the data obtained by means of an annual food frequency questionnaire (FFQ), and the information existing in the literature, a Mediterranean Diet Score (MDS) was calculated. Dietary fibre was obtained from Marlett et al. tables and Phenol-Explorer Database was used for phenolic compounds intake. Quantification of microbial groups was performed by Ion Torrent 16S rRNA gene-based analysis and quantification of short-chain fatty acids (SCFAs) was performed using gas chromatography-mass spectrometry (MS). MDS was associated with a higher abundance of Bacteroidetes (p = 0.001), Prevotellacea (p = 0.002) and Prevotella (p = 0.003) and a lower concentration of Firmicutes (p = 0.003) and Lachnospiraceae (p = 0.045). Also, in subjects with MDS ≥ 4, higher concentrations of faecal propionate (p = 0.034) and butyrate (p = 0.018) were detected. These results confirm the complexity of the diet-microbiota interrelationship., Supported by a grant AGL2010-14952 from the Spanish Ministry of Science and Innovation and in part by a grant from the European Regional Development Fund (GRUPIN14-043; to I. Gutiérrez-Díaz). B. Sánchez was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness.

[Background] The wide range of potential applications has made the Basic Local Alignment Search Tool (BLAST) a ubiquitous tool in the field of Molecular Biology. Within this context, it is increasingly appealing to embed BLAST services within larger Web applications. [Results] This work introduces BlasterJS viewer, a new JavaScript library for the lightweight development of Web-based applications supporting the visualisation of BLAST outputs. BlasterJS detaches from similar data viewers by focusing on the visual and interactive display of sequence similarity results and being completely independent of BLAST services. BlasterJS is compatible with the text outputs generated by the BLAST family of programs, namely BLASTp, BLASTn, BLASTx, tBLASTn, and tBLASTx, and works in all major Web browsers. Furthermore, BlasterJS is available through the EBI’s BioJS registry 5, which extends its potential use to a wider scope of bioinformatics applications. [Conclusions] BlasterJS is new Javascript library that enables easy and seamless integration of visual and interactive representations of BLAST outputs in Web-based applications supporting sequence similarity search. BlasterJS is free accessible at http://sing-group.org/blasterjs/., This work was supported by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (grant AGL2013-44039R to BS and AL); and the Asociación Española Contra el Cancer ("Obtención de pépti-dos bioactivos contra el Cáncer Colo-Rectal a partir de secuencias genéticas de microbiomas intestinales", grant PS-2016 to BS and ABM). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit (AL) and COMPETE 2020 (POCI-01-0145-FEDER-006684 to AL). SING group thanks CITI (Centro de Investigación, Trans-ferencia e Innovación) from University of Vigo for hosting its IT infrastructure

Se sabe que las bacterias de la microbiota intestinal humana poseen efectos beneficiosos en el hospedador, entre ellos estimular el sistema inmunológico y proteger contra la actividad carcinogénica de elementos de la dieta. Por ello, se ha propuesto que los probióticos podrían tener un papel en la prevención del desarrollo del cáncer colorrectal. El objetivo de este trabajo fue estudiar el efecto de diferentes péptidos procedentes de bacterias de la microbiota humana sobre la proliferación de células de adenocarcinoma colónico humano. Para ello, se seleccionaron 41 péptidos del metaproteoma del tracto digestivo humano por presentar alta similitud (mayor del 60%) con péptidos de actividad antiproliferativa o inmunomoduladora ya conocida. Se incubaron células de adenocarcinoma colónico humano HT-29 en presencia de dichos péptidos cuantificando la proliferación mediante el dispositivo xCELLigence RTCA. Se compararon los resultados con controles negativos sin péptido y controles positivos con el quimioterápico 5-fluorouracilo. Los péptidos que mostraron mayor actividad antiproliferativa se ensayaron a distintas concentraciones para determinar su concentración inhibitoria del 50%. Como conclusión, 21 de los 41 péptidos estudiados presentaron actividad antiproliferativa, siendo más relevante el efecto observado con cuatro de ellos (DW15, GR16, TS13 y VV15). Este efecto fue de menor magnitud que utilizando un agente quimioterápico clásico y requirió un tiempo de incubación más largo.

[ES] El estudio de los probióticos ha cobrado una gran importancia en los últimos años debido a su papel en el mantenimiento de una microbiota intestinal equilibrada, así como en el tratamiento de ciertas enfermedades, incluidos ciertos tipos de cáncer. En este trabajo, se ha analizado el efecto anti-proliferativo de un extracto bacteriano obtenido a partir de la cepa probiótica Lactobacillus acidophilus sobre la línea celular HT29. Este efecto se ha detectado con una técnica de impedancia no invasiva que permite cuantificar la proliferación celular. Además, se ha realizado un análisis del ciclo celular mediante citometría de flujo, ya que una posible hipótesis era que este extracto afectase a la división celular. Finalmente, se observó un efecto sobre la línea celular dependiente de la concentración. Aunque dicho efecto se atribuyó inicialmente a un descenso en la proliferación, los resultados de citometría no parecen reflejar esta idea y dan pie a nuevas hipótesis, incluidas una alteración en la morfología/adhesión celular o un efecto anti-proliferativo que no se detectaría debido a la aparición de mecanismos de resistencia. Además, un segundo análisis con citometría de flujo descarta su efecto sobre la viabilidad celular. Por otro lado, la elevada complejidad del extracto requiere de un fraccionamiento previo, por ejemplo mediante cromatografía líquida, para poder identificar qué moléculas son responsables del efecto observado. Antes de llevar a cabo esta operación, es necesaria una optimización de las condiciones de separación, lo cual se presenta en este trabajo utilizando la técnica de cromatografía en capa fina. De esta forma, se consiguió establecer condiciones óptimas tanto para cromatografía en fase reversa con mezclas de acetonitrilo y agua, como para fase normal con mezclas de hexano y acetato de etilo, acetato de etilo y metanol, y cloroformo y metanol., [EN] The global interest in probiotics has gained a great relevance over the last few years due to its importance in both the maintenance of a balanced gut microbiota, and the treatment of several diseases, including certain type of cancers. In the present study, the anti-proliferative effect from a bacterial extract which has been obtained from probiotic strain, Lactobacillus acidophilus, has been tested on the HT29 cell line, derived from a colon adenocarcinome. This effect could be observed thanks to a non-invasive impedance assay, which allows for cell proliferation quantification. Furthermore, a cell cycle analysis through flow cytometry has been also carried out in order to get a better idea about the action mechanism of the extract, as one of the main hypothesis would be its possible impact on the cell division. Finally it could be observed an effect over the cell line dependent on concentration. Although this effect had been previously associated with a decrease in cell proliferation, it seems that flow cytometry results do not reflect this idea and thus, several hypothesis have been purposed, including changes in cell morphology/adhesion or an anti-proliferative effect which was not detected because of resistance mechanisms. Additionally, a second flow cytometry analysis showed no effect on cell viability. On the other hand, the huge complexity of the extract requires a previous separation through liquid chromatography, in order to be able to identify which molecule or molecules are responsible for the observed effect. Before performing this technique, it is necessary to carry out an optimization through thin layer chromatography, which is included in this study. At the end, several optimal conditions have been reached for both reverse phase with mixtures of acetonitrile and water, and normal phase with mixtures of hexane and ethyl acetate, ethyl acetate

Probiotics are used to improve human health due to their capability to induce beneficial effects in gastrointestinal diseases and modulate the immune response. However, scarce information is known about the bacterial active compounds and the metabolic pathways of interaction with immune cells. In the present study, we analyzed the immune response elicited by surface-associated proteins, the whole surface extract (WSE) and the genomic DNA of three strains from the main representative probiotic species for human consumption, L. acidophilus DSM20079T, L. rhamnosus GG, B. longum NCIMB 8809, using an in vitro model of human PBMCs from healthy donors. Eighteen cytokines were quantified using Luminex Technology to understand the immune pathways modulate by bacterial extracts. Overall, the bacterial fractions elicited a different immune response in PBMCs, whereas the isolated DNA was able to induce a higher Th17-like response that could lead to a protective effect in the epithelial barrier function., This work was funded by the Spanish “Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad” (Grant AGL2013-44039R) and the “Fundación Científica Asociación Española contra el Cáncer” (Grant PS-2016).

Bifidobacterium represents a genus within the phylum Actinobacteria which is one of the major phyla in the healthy intestinal tract of humans. Bifidobacterium is one of the most abundant genera in adults, but its predominance is even more pronounced in infants, especially during lactation, when they can constitute the majority of the total bacterial population. They are one of the pioneering colonizers of the early gut microbiota, and they are known to play important roles in the metabolism of dietary components, otherwise indigestible in the upper parts of the intestine, and in the maturation of the immune system. Bifidobacteria have been shown to interact with human immune cells and to modulate specific pathways, involving innate and adaptive immune processes. In this mini-review, we provide an overview of the current knowledge on the immunomodulatory properties of bifidobacteria and the mechanisms and molecular players underlying these processes, focusing on the corresponding implications for human health. We deal with in vitro models suitable for studying strain-specific immunomodulatory activities. These include peripheral blood mononuclear cells and T cell-mediated immune responses, both effector and regulatory cell responses, as well as the modulation of the phenotype of dendritic cells, among others. Furthermore, preclinical studies, mainly germ-free, gnotobiotic, and conventional murine models, and human clinical trials, are also discussed. Finally, we highlight evidence supporting the immunomodulatory effects of bifidobacterial molecules (proteins and peptides, exopolysaccharides, metabolites, and DNA), as well as the role of bifidobacterial metabolism in maintaining immune homeostasis through cross-feeding mechanisms., LR is a postdoctoral researcher supported by the Juan de la Cierva Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (MINECO; IJCI-2015-23196). BS and AM thanks MINECO for the funding of the project AGL2016-78311-R and PR-M for the project AGL2015-64901-R. SD is supported by a research contract associated to the project BIO2014-55019-JIN from the Spanish “Plan Estatal de I+D+i”.

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies, This study was funded by start-up funds from North Carolina State University and the North Carolina Ag Foundation

Scientific studies focused on the role of the human microbiome over human health have generated billions of gigabits of genetic information during the last decade. Nowadays integration of all this information in public databases and development of pipelines allowing us to biotechnologically exploit this information are urgently needed. Prediction of the potential bioactivity of the products encoded by the human gut microbiome, or metaproteome, is the first step for identifying proteins responsible for the molecular interaction between microorganisms and the immune system. We have recently published the Mechanism of Action of the Human Microbiome (MAHMI) database (http://www.mahmi.org), conceived as a resource compiling peptide sequences with a potential immunomodulatory activity. Fifteen out of the 300 hundred million peptides contained in the MAHMI database were synthesized. These peptides were identified as being encrypted in proteins produced by gut microbiota members, they do not contain cleavage points for the major intestinal endoproteases and displayed high probability to have immunomodulatory bioactivity. The bacterial peptides FR-16 and LR-17 encrypted in proteins from Bifidobacterium longum DJ010A and Bifidobacterium fragilis YCH46 respectively, showed the higher immune modulation capability over human peripheral blood mononuclear cells. Both peptides modulated the immune response toward increases in the Th17 and decreases in the Th1 cell response, together with an induction of IL-22 production. These results strongly suggest the combined use of bioinformatics and in vitro tools as a first stage in the screening of bioactive peptides encrypted in the human gut metaproteome., This work was financed by the Spanish “Programa Estatal de Investigación, Desarrollo e Inovación Orientada a los Retos de la Sociedad” (Grant AGL2013-44039R). Research in our laboratory is funded by the “Fundación Científica Asociación Española Contra el Cáncer” (Grant agreement PS-2016)

Peptidome similarity analysis enables researchers to gain insights into differential peptide profiles, providing a robust tool to discriminate strain-specific peptides, true intra-species differences among biological replicates or even microorganism-phenotype variations. However, no in silico peptide fingerprinting software existed to facilitate such phylogeny inference. Hence, we developed the Peptidomes for Phylogenies (P4P) web tool, which enables the survey of similarities between microbial proteomes and simplifies the process of obtaining new biological insights into their phylogeny. P4P can be used to analyze different peptide datasets, i.e. bacteria, viruses, eukaryotic species or even metaproteomes. Also, it is able to work with whole proteome datasets and experimental mass-to-charge lists originated from mass spectrometers. The ultimate aim is to generate a valid and manageable list of peptides that have phylogenetic signal and are potentially sample-specific. Sample-to-sample comparison is based on a consensus peak set matrix, which can be further submitted to phylogenetic analysis. P4P holds great potential for improving phylogenetic analyses in challenging taxonomic groups, biomarker identification or epidemiologic studies. Notably, P4P can be of interest for applications handling large proteomic datasets, which it is able to reduce to small matrices while maintaining high phylogenetic resolution. The web server is available at http://sing-group.org/p4p., Spanish ‘Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad’ [AGL2013-44039R]; Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020[POCI-01-0145-FEDER-006684];INOU16-05project from the University of Vigo; Fundación AECC. Funding for open access charge: Spanish ‘Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad’ [AGL2013-44039R]., info:eu-repo/semantics/publishedVersion

Bifidobacteria are members of the intestinal microbiota of mammals and other animals, and some strains are able to exert health-promoting effects. The genus Bifidobacterium belongs to the Actinobacteria phylum. Firmicutes, Bacteroidetes, and Actinobacteria constitute the most abundant phyla in the human intestinal microbiota, Firmicutes and Bacteroidetes being predominant in adults, and Actinobacteria in breast-fed infants, where bifidobacteria can reach levels higher than 90% of the total bacterial population. They are among the first microbial colonizers of the intestines of newborns, and play key roles in the development of their physiology, including maturation of the immune system and use of dietary components. Indeed, some nutrients, such as human milk oligosaccharides, are important drivers of bifidobacterial development. Some Bifidobacterium strains are considered probiotic microorganisms because of their beneficial effects, and they have been included as bioactive ingredients in functional foods, mainly dairy products, as well as in food supplements and pharma products, alone, or together with, other microbes or microbial substrates. Well-documented scientific evidence of their activities is currently available for bifidobacteria-containing preparations in some intestinal and extraintestinal pathologies. In this review, we focus on the role of bifidobacteria as members of the human intestinal microbiota and their use as probiotics in the prevention and treatment of disease., Research in our group is supported by grants AGL2015- 64901-R, BIO2014-55019-JIN, AGL2013-44761-P, and AGL2013-44039-R from the Spanish Ministry of Economy and Competitiveness

Probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts. Toxicity induced by molecular oxygen significantly affects the viability of probiotic bacteria during biomass generation, product manufacturing and storage also constitute a major challenge for their inclusion in dairy products. Oxygen toxicity is particularly critical in the case of bifidobacteria, which are strict anaerobes. This mini-review aims at compiling the knowledge on the response mechanisms, mainly in this bacterial group, focused on counteracting the deleterious effects of oxygen on probiotic cells. The effect of other technological and microbiological ways to attenuate oxygen toxicity, such as milk deaeration, microencapsulation or co-culture is reviewed. Potential uses of the isolation of better adapted strains of probiotic Bifidobacterium strains, as well as the next generation probiotics such as Faecalibacterium prausnitzii, are discussed., This work was funded by Grant AGL2013-44039-R from the Spanish ‘Plan Estatal de I+D+I’. Borja Sánchez was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness.

The Mechanism of Action of the Human Microbiome (MAHMI) database is a unique resource that provides comprehensive information about the sequence of potential immunomodulatory and antiproliferative peptides encrypted in the proteins produced by the human gut microbiota. Currently, MAHMI database contains over 300 hundred million peptide entries, with detailed information about peptide sequence, sources and potential bioactivity. The reference peptide data section is curated manually by domain experts. The in silico peptide data section is populated automatically through the systematic processing of publicly available exoproteomes of the human microbiome. Bioactivity prediction is based on the global alignment of the automatically processed peptides with experimentally validated immunomodulatory and antiproliferative peptides, in the reference section. MAHMI provides researchers with a comparative tool for inspecting the potential immunomodulatory or antiproliferative bioactivity of new amino acidic sequences and identifying promising peptides to be further investigated. Moreover, researchers are welcome to submit new experimental evidence on peptide bioactivity, namely, empiric and structural data, as a proactive, expert means to keep the database updated and improve the implemented bioactivity prediction method. Bioactive peptides identified by MAHMI have a huge biotechnological potential, including the manipulation of aberrant immune responses and the design of new functional ingredients/foods based on the genetic sequences of the human microbiome. Hopefully, the resources provided by MAHMI will be useful to those researching gastrointestinal disorders of autoimmune and inflammatory nature, such as Inflammatory Bowel Diseases. MAHMI database is routinely updated and is available free of charge., This work was funded by the Grant AGL2013-44039-R from the Spanish “Plan Estatal de I + D+I”, and the Grant EM2014/046 from the “Plan Galego de investigación, innovación e crecemento 2011-2015”. Borja Sánchez was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness. This work was also partially funded by the [14VI05] Contract-Programme from the University of Vigo and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273) and the European Union’s Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement n° 316265, BIOCAPS.

Introducción: el lupus eritematoso sistémico es una enfermedad inflamatoria crónica en la que está implicado el estrés oxidativo.Objetivo: evaluar la concentración de antioxidantes de la dieta y sanguíneos, así como de la microbiota sobre las concentraciones de malondialdehído y proteína C reactiva en 21 pacientes de lupus y 21 controles pareados por edad y sexo.Métodos: los parámetros bioquímicos de rutina y proteína C reactiva se determinaron a través de métodos enzimáticos: cobre, zinc y selenio por espectrometría de masas, malondialdehído y capacidad antioxidante total por métodos espectrofotométricos, la microbiota fecal por técnicas metagenómicas y la dieta a través de cuestionarios de frecuencia de consumo.Resultados: no se han observado diferencias en la dieta en los pacientes con lupus respecto al grupo control, excepto en la ingesta de ácidos grasos trans, siendo mayor en el grupo de lupus. En estas pacientes se observaron mayores niveles circulantes de cobre y menores de zinc. La concentración de cobre en suero se relacionó directamente con los niveles de proteína C reactiva y esta proteína, a su vez, con la proporción de Lentisphaerae, Proteobacteria y Verrucomicrobia en heces. Además, mientras que los niveles de malondialdehído se asociaban inversamente con la proporción de Cyanobacteria y Firmicutes, con Actinobacteria se encontró una correlación positiva. La presencia de anti-SSA/Ro en lúpicas se relaciona con mayores concentraciones de zinc.Conclusión: estos resultados podrían ser útiles para profundizar en el futuro conocimiento de esta compleja enfermedad. Received

The gastrointestinal tract of mammals hosts a high and diverse number of different microorganisms, known as intestinal microbiota. Many probiotics were originally isolated from the gastrointestinal tract, and they were defined by the Food and Agriculture Organization of the United Nations (FAO)/WHO as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.” Probiotics exert their beneficial effects on the host through four main mechanisms: interference with potential pathogens, improvement of barrier function, immunomodulation and production of neurotransmitters, and their host targets vary from the resident microbiota to cellular components of the gut–brain axis. However, in spite of the wide array of beneficial mechanisms deployed by probiotic bacteria, relatively few effects have been supported by clinical data. In this regard, different probiotic strains have been effective in antibiotic‐associated diarrhea or inflammatory bowel disease for instance. The aim of this review was to compile the molecular mechanisms underlying the beneficial effects of probiotics, mainly through their interaction with the intestinal microbiota and with the intestinal mucosa. The specific benefits discussed in this paper include among others those elicited directly through dietary modulation of the human gut microbiota., Research in our lab is funded by Grants AGL2013‐44039‐R and AGL2013‐44761‐P from the Spanish “Plan Estatal de I+D+I.” Part of the authors is also partially funded by the [15VI013] Contract‐Programme from the University of Vigo and the Agrupamento INBIOMED from DXPCTSUG‐FEDER unha maneira de facer Europa (2012/273). B. S. was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness.

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Oviedo.--2016-06-03 Trabajo disponible en: http://hdl.handle.net/10651/38993, La microbiota intestinal es el conjunto de microorganismos que habita en inmunológicoel intestino humano de manera natural, estableciendo una relación estrecha con el hospedador que la alberga, al que le proporciona una serie de beneficios entre los que cabe destacar la función barrera contra bacterias potencialmente patógenas, el aprovechamiento de energía de alimentos no digeribles, y la maduración y desarrollo del sistema inmunológico. Hasta el momento, tanto estudios en humanos como en animales han demostrado que cambios en las poblaciones microbianas intestinales pueden tener consecuencias para la salud, y existen numerosos trabajos científicos que relacionan a una microbiota intestinal alterada (disbiosis intestinal), con procesos inflamatorios, asma alérgicos, enfermedades autoinmunes e incluso cáncer. El objetivo general de esta Tesis Doctoral es generar nuevo conocimiento sobre la composición de la microbiota intestinal en el marco de dos trastornos inmunológicos, el lupus eritematoso sistémico (LES) y el asma. A través del uso de técnicas independientes de cultivo, se analizaron muestras fecales y se determinó el perfil microbiano de grupos de individuos que padecían estas enfermedades, comparándolo con el de individuos sanos. De esta manera los resultados de esta Tesis Doctoral han contribuido a entender la relación entre la disbiosis intestinal que presenta la microbiota de estos individuos y su estado inmunológico. Para el caso del LES se estudiaron 20 mujeres de entre 34 y 68 años de edad, y se compararon con un grupo control sano. En asma alérgico se analizaron muestras de 21 pacientes de edades entre 24 y 57 años y se compararon con un grupo de 22 controles sanos de mismo sexo y rango de edad. Durante la primera parte de esta Tesis se optimizaron metodologías y protocolos de tratamiento de muestras fecales humanas.

[Purpose] Short-chain fatty acids (SCFAs) formation by intestinal bacteria is regulated by many different factors, among which dietary fibre is currently receiving most attention. However, since fibre-rich foods are usually good dietary sources of phenolic compounds, which are also known to affect the microbiota, authors hypothesize that the regular intake of these bioactive compounds could be associated with a modulation of faecal SCFA production by the intestinal microbiota. [Methods] In this work, food intake was recorded by means of a validated Food Frequency Questionnaire. Fibres were determined using Marlett food composition tables, and phenolic compounds were obtained from Phenol-Explorer Database. Analysis of SCFA was performed by gas chromatography–flame ionization/mass spectrometry and quantification of microbial populations in faeces by quantitative PCR. [Results] Klason lignin and its food contributors, as predictors of faecal butyrate production, were directly associated with Bacteroides and Bifidobacterium levels, as well as lignans with Bacteroides. Also, anthocyanidins, provided by strawberries, were associated with faecal propionate and inversely related to Lactobacillus group. [Conclusions] These results support the hypothesis we put forward regarding the association between some vegetable foods (strawberries, pasta, lentils, lettuce and olive oil) and faecal SCFA. More studies are needed in order to elucidate whether these associations have been mediated by the bacterial modulatory effect of the bioactive compounds, anthocyanins, lignans or Klason lignin, present in foodstuffs., This study was financed by the Spanish Plan Nacional de I+D (Grant AGL2010-14952) and by Biópolis SL. within the framework of the e-CENIT Project SENIFOOD from the Spanish Ministry of Science and Innovation. The activity of the group is being partly supported through the Grant GRUPIN14-043 from “Plan Regional de Investigación” of Principado de Asturias. Both AGL2010-14952 and GRUPIN14-043 received cofounding from European Union FEDER funds. N. Salazar benefits from a “Clarín” postdoctoral contract (Marie Curie European CoFund Program) co-funded by the “Plan Regional de Investigación” of Principado de Asturias, Spain. B. Sánchez was the recipient of a “Ramón y Cajal” postdoctoral contract from the Spanish Ministry of Economy and Competitiveness. The contract of I. Gutierrez-Diaz was supported by grant GRUPIN14-043. We acknowledge the excellent technical assistance of Lidia Alaez, whose contract was partially supported by the Project AGL2010-16525 and by “Plan Regional de Investigación” of Principado de Asturias, through the Grant COF 13-020. We show our greatest gratitude to all the volunteers participating in the study and to “Alimerka Foundation”.

Bifidobacteria are members of the human gut microbiota, being numerically dominant in the colon of infants, while also being prevalent in the large intestine of adults. In this study, we determined and analyzed the pan-genome of Bifidobacterium adolescentis, which is one of many bacteria found in the human adult gut microbiota. In silico analysis of the genome sequences of eighteen B. adolescentis strains isolated from various environments, such as human milk, human feces and bovine rumen, revealed a high level of genetic variability, resulting in an open pan-genome. Compared to other bifidobacterial taxa such as Bifidobacterium bifidum and Bifidobacterium breve, the more extensive B. adolescentis pan-genome supports the hypothesis that the genetic arsenal of this taxon expanded so as to become more adaptable to the variable and changing ecological niche of the gut. These increased genetic capabilities are particularly evident for genes required for dietary glycan-breakdown, This work was financially supported by a PostDoc fellowship (Fondazione Caritro) to SD. DvS is member of The APC Microbiome Institute funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant number SFI/12/RC/2273).

Trabajo también disponible en: http://hdl.handle.net/10651/39089, Los probióticos están adquiriendo un gran interés en los últimos tiempos tanto por su uso para la elaboración de multitud de productos fermentados, fundamentalmente lácteos, como por su contribución al mantenimiento del estado de salud del consumidor. Las propiedades saludables que se les atribuyen y que sustentan su aplicación en la clínica, se relacionan con algunos mecanismos de acción sobre enfermedades relacionadas con el sistema digestivo y la función inmune. Ha sido evidenciado que afectan a la microbiota intestinal aumentando el número de bacterias beneficiosas y disminuyendo el de patógenas para el mantenimiento del equilibrio intestinal y prevención del desarrollo de enfermedades. Es necesario demostrar su eficacia en la salud humana mediante la realización de ensayos clínicos bien documentados que permitan seleccionar las poblaciones más apropiadas, siendo las bacterias probióticas más habituales las del género Lactobacillus y Bifidobacterium. Para la realización de éste trabajo fueron seleccionadas tres cepas pertenecientes a la colección del IPLA-CSIC: Lactobacillus casei subsp. rhamnosus GG (LGG), Lactobacillus acidophilus DSM 20079T y Bifidobacterium longum subsp. longum NCIMB 8809. Los efectos inmunomoduladores de estas bacterias y la influencia de las mismas sobre la respuesta inmunitaria llevan a estudiar el comportamiento de algunos de sus componentes (proteínas extracelulares, proteínas de superficie, ácido lipoteicoico y ADN) en individuos sanos, siendo el objetivo de este trabajo.

Intestinal dysbiosis, characterized by a reduced Firmicutes/Bacteroidetes ratio, has been reported in systemic lupus erythematosus (SLE) patients. In this study, in vitro cultures revealed that microbiota isolated from SLE patient stool samples (SLE-M) promoted lymphocyte activation and Th17 differentiation from naïve CD4+ lymphocytes to a greater extent than healthy control-microbiota. Enrichment of SLE-M with Treg-inducing bacteria showed that a mixture of two Clostridia strains significantly reduced the Th17/Th1 balance, whereas Bifidobacterium bifidum supplementation prevented CD4+ lymphocyte over-activation, thus supporting a possible therapeutic benefit of probiotics containing Treg-inducer strains in order to restore the Treg/Th17/Th1 imbalance present in SLE. In fact, ex vivo analyses of patient samples showed enlarged Th17 and Foxp3+ IL-17+ populations, suggesting a possible Treg-Th17 trans-differentiation. Moreover, analyses of fecal microbiota revealed a negative correlation between IL-17+ populations and Firmicutes in healthy controls, whereas in SLE this phylum correlated directly with serum levels of IFNγ, a Th1 cytokine slightly reduced in patients. Finally, the frequency of Synergistetes, positively correlated with the Firmicutes/Bacteroidetes ratio in healthy controls, tended to be reduced in patients when anti-dsDNA titers were increased and showed a strong negative correlation with IL-6 serum levels and correlated positively with protective natural IgM antibodies against phosphorylcholine., This work was supported by European Union FEDER funds, Fondo de Investigación Sanitaria (PI12/00523), Spanish Plan Nacional de I+D (AGL2010-14952 and AGL2013-44039-R) and Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (EQUIP09-19). J.R.-C. is a recipient of a FPU grant from the Spanish Ministerio de Educación, Cultura y Deporte. B.J. and A.H. are recipients of a Ramón y Cajal postdoctoral contract and a FPI grant, respectively, both from the Spanish Ministerio deEconomía y Competitividad. We thank SLE patients and ALAS (Asociación Lúpicos de Asturias) for their continuous encouragement.

Los organismos macroscópicos, tales como animales, plantas u hongos, suelen ser fáciles de distinguir gracias a diferencias morfológicas, características de su comportamiento o por el aislamiento reproductivo. Para los microorganismos pertenecientes a los dos dominios Prokaryota de la vida, las Bacteria y las Archaea, la clasificación de especies es una tarea mucho más complicada, dada la similitud genética existente.

Typical bacterial strain differentiation methods are often challenged by high genetic similarity between strains. To address this problem, we introduce a novel in silico peptide fingerprinting method based on conventional wet-lab protocols that enables the identification of potential strain-specific peptides. These can be further investigated using in vitro approaches, laying a foundation for the development of biomarker detection and application-specific methods. This novel method aims at reducing large amounts of comparative peptide data to binary matrices while maintaining a high phylogenetic resolution. The underlying case study concerns the Bacillus cereus group, namely the differentiation of Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus strains. Results show that trees based on cytoplasmic and extracellular peptidomes are only marginally in conflict with those based on whole proteomes, as inferred by the established Genome-BLAST Distance Phylogeny (GBDP) method. Hence, these results indicate that the two approaches can most likely be used complementarily even in other organismal groups. The obtained results confirm previous reports about the misclassification of many strains within the B. cereus group. Moreover, our method was able to separate the B. anthracis strains with high resolution, similarly to the GBDP results as benchmarked via Bayesian inference and both Maximum Likelihood and Maximum Parsimony. In addition to the presented phylogenomic applications, whole-peptide fingerprinting might also become a valuable complementary technique to digital DNA-DNA hybridization, notably for bacterial classification at the species and subspecies level in the future., This research was funded by Grant AGL2013-44039-R from the Spanish “Plan Estatal de I+D+I”, and by Grant EM2014/046 from the “Plan Galego de investigación, innovación e crecemento 2011-2015”. BS was recipient of a Ramón y Cajal postdoctoral contract from the Spanish Ministry of Economy and Competitiveness. This work was also partially funded by the [14VI05] Contract-Programme from the University of Vigo and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273). The research leading to these results has also received funding from the European Union's Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement n° 316265, BIOCAPS

La relación entre la microbiota intestinal y la salud es un campo muy interesante y en el que hoy en día hay mucha investigación. El propósito de este trabajo fue estudiar la actividad antiproliferativa de dos fracciones extracelulares (proteína extracelular y compuestos polares de bajo peso molecular) de cinco Lactobacillus y un Clostridium procedentes de la microbiota intestinal de heces de cerdo, sobre la línea celular CaCo2 de carcinoma colónico humano. Para ello se aislaron e identificaron diferentes cepas, de las cuales se extrajeron las dos fracciones anteriormente mencionadas que se incubaron con las células de carcinoma humano CaCo2. Se obtuvieron los resultados con el software xCELLigence RTCA y se compararon con un control negativo sin fracción bacteriana y dos controles positivos. Los resultados mostraron una tendencia de inhibición de crecimiento celular de CaCo2 por parte de ambas fracciones bacterianas. The relationship between the gut microbiota and health is a very interesting field and in which there is a lot of research today. The purpose of this work was to study the antiproliferative activity of two extracellular fractions (extracellular protein and polar compounds of low molecular weight) of five Lactobacillus and one Clostridium from the intestinal microbiota of pig feces, on the human colon carcinoma cell line CaCo2. To this end, different strains were isolated and identified, from which the two mentioned fractions were extracted, which were incubated with the human carcinoma cells CaCo2. The results were obtained with the xCELLigence RTCA software and they were compared with a negative control without bacterial fraction and two positive controls. The results showed a tendency of inhibition of cellular growth of CaCo2 by both bacterial fractions.

Trabajo presentado en el IX Workshop de la Sociedad Española de Probióticos y Prebióticos (SEPyP), celebrado en Zaragoza (España) el 15 y 16 de febrero de 2018, [Introduction] Bifidobacteria and lactobacilli include both probiotic strains and representative microorganisms of the human gut microbiota, and their presence has been associated to a proper immune development and maturation. In addition, several independent works report the anti-inflammatory properties of several strains of those groups. However, we are far from understanding the molecular interplay behind/supporting these beneficial interactions. [Objective] To study the effect of different fractions of lactobacilli and bifidobacteria in human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors and patients with Inflammatory Bowel Disease (IBD) at the time of diagnosis. [Methods] The fractions were obtained from strains of Bifidobacterium longum NCIMB 8809, Lactobacillus rhamnosus GG and Lactobacillus acidophilus DSM 20079: DNA, surface-associated proteins, whole cell soluble extract and extracellular proteins. Different concentrations of the fractions were incubated with PBMCs, and key T-cell cytokines, T regulatory cell differentiation and RNASeq of CD4+ cells were performed. [Results] Among the extracts, extracellular proteins from L. acidophilus DSM 20079 increased IL-2 production by PBMCs isolated from healthy donors, and were able to increment the proportion of functional T regulatory cells. Data on the signaling mechanisms involved were obtained by RNASeq of CD4+ Th cell subpopulations. Finally, this fraction decreased drastically the production of the pro-inflammatory mediator TNF-alpha and induced increments of IL-12 in PBMCs isolated from IBD patients at the diagnosis. [Conclusions] Extracellular proteins from strain L. acidophilus DSM 20079 had a positive impact on the immunological status of both healthy donors and IBD patients. This fraction will be on the basis of new clinical studies focused in the immunomodulation of the human host targeting pro-inflammatory effectors and the T-cell regulatory response. This fraction may represent a new therapeutic approach for chronic inflammatory illnesses. [Competing interests] Borja Sánchez and Claudio Hidalgo are members of the scientific committee of Microviable Therapeutics SL.

El lupus eritematoso sistémico (LES) es una enfermedad autoinmune sistémica de etiología no precisada, caracterizada por daño de naturaleza inmunológica en los diferentes órganos; también es una enfermedad de distribución mundial, afectando a todas las razas y poblaciones estudiadas, aunque no con la misma frecuencia. Las importantes variaciones interpoblacionales descritas, tanto en la frecuencia de aparición de la enfermedad como en sus principales características clínicas e inmunológicas, están posiblemente relacionadas con la existencia de distintos factores genéticos y ambientales. De acuerdo con los trabajos existentes en la bibliografía, sabemos que la microbiota intestinal puede jugar un papel importante en el LES, aunque no se sabe si su participación es como causa o como consecuencia de la misma. El objetivo de este proyecto es la extracción, separación e identificación de proteínas bacterianas del tracto gastrointestinal que puedan ser seleccionadas como potenciales biomarcadores que permitan diferenciar individuos con LES de individuos sanos. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology, characterized by immunological damage in different organs. It is a worldwide disease, affecting all races and populations, although not with the same frequency. Significant interpopulation variations described in both the frequency of occurrence of the disease and its main clinical and immunological characteristics, are possibly related to the existence of different genetic and environmental factors. According to previous work, we know that the intestinal microbiota could play an important role in SLE, although it is unclear if their participation is as a cause or as a result of it. Therefore, the objective of this project is the extraction, separation and identification of bacterial proteins from the microbiota of the gastrointestinal tract as potential biomarkers allowing the differentiation of individuals with SLE from healthy individuals.

En este trabajo se pretende utilizar los niveles de anticuerpos dirigidos contra bacterias ingeridas con los alimentos, representantes de la microbiota intestinal, como posibles biomarcadores para uso en la Enfermedad Inflamatoria Intestinal con el fin de facilitar el desarrollo de mejores métodos de diagnóstico precoz.