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The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspic, Ministerio de Ciencia, Innovación y Universidades (España), Comunidad de Madrid, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

Dysbiosis of the microbiome has been related to Celiac disease (CeD) progress, an autoimmune disease characterized by gluten intolerance developed in genetically susceptible individuals under certain environmental factors. The microbiome contributes to CeD pathophysiology, modulating the immune response by the action of short-chain fatty acids (SCFA), affecting gut barrier integrity allowing the entrance of gluten-derived proteins, and degrading immunogenic peptides of gluten through endoprolyl peptidase enzymes. Despite the evidence suggesting the implication of gut microbiome over CeD pathogenesis, there is no consensus about the specific microbial changes observed in this pathology. Here, we compiled the largest dataset of 16S prokaryotic ribosomal RNA gene high-throughput sequencing for consensus profiling. We present for the first time an integrative analysis of metataxonomic data from patients with CeD, including samples from different body sites (saliva, pharynx, duodenum, and stool). We found the presence of coordinated changes through the gastrointestinal tract (GIT) characterized by an increase in Actinobacteria species in the upper GIT (pharynx and duodenum) and an increase in Proteobacteria in the lower GIT (duodenum and stool), as well as site-specific changes evidencing a dysbiosis in patients with CeD’ microbiota. Moreover, we described the effect of adherence to a gluten-free diet (GFD) evidenced by an increase in beneficial bacteria and a decrease in some Betaproteobacteriales but not fully restoring CeD-related dysbiosis. Finally, we built a Random Forest model to classify patients based on the lower GIT composition achieving good performance., European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Convocatoria FEDER INNTERCONECTA 2015, Juan de la Cierva, Ministerio de Ciencia, Innovación y Universidades, Depto. de Nutrición y Ciencia de los Alimentos, Fac. de Farmacia, TRUE, pub

HIV Nef is a central auxiliary protein in HIV infection and pathogenesis. Our results indicate that HDAC6 promotes the aggresome/autophagic degradation of the viral polyprotein Pr55Gag to inhibit HIV-1 production. Nef counteracts this antiviral activity of HDAC6 by inducing its degradation and subsequently stabilizing Pr55Gag and Vif viral proteins. Nef appears to neutralize HDAC6 by an acidic/endosomal-lysosomal processing and does not need the downregulation function, since data obtained with the non-associated cell-surface Nef-G2A mutant – the cytoplasmic location of HDAC6 – together with studies with chemical inhibitors and other Nef mutants, point to this direction. Hence, the polyproline rich region P72xxP75 (69–77 aa) and the di-Leucin motif in the Nef-ExxxLL160-165 sequence of Nef, appear to be responsible for HDAC6 clearance and, therefore, required for this novel Nef proviral function. Nef and Nef-G2A co-immunoprecipitate with HDAC6, whereas the Nef-PPAA mutant showed a reduced interaction with the anti-HIV-1 enzyme. Thus, the P72xxP75 motif appears to be responsible, directly or indirectly, for the interaction of Nef with HDAC6. Remarkably, by neutralizing HDAC6, Nef assures Pr55Gag location and aggregation at plasma membrane, as observed by TIRFM, promotes viral egress, and enhances the infectivity of viral particles. Consequently, our results suggest that HDAC6 acts as an anti-HIV-1 restriction factor, limiting viral production and infection by targeting Pr55Gag and Vif. This function is counteracted by functional HIV-1 Nef, in order to assure viral production and infection capacities. The interplay between HIV-1 Nef and cellular HDAC6 may determine viral infection and pathogenesis, representing both molecules as key targets to battling HIV., Julià Blanco5,6 and Agustín Valenzuela-Fernández1, Ministerio de Economía y Competitividad (MINECO)/FEDER, Ministerio de Ciencia, Innovación y Universidades (MICINN)/FEDER, Fundación CajaCanarias/FEDER, Red Española de Investigación del SIDA (RES-RETIC)/ISCII/FEDER, Agencia Canaria de Investigación, Innovación y Sociedad de la Información/Fundación CajaCanarias/FEDER, Fundación Canaria Dr. Manuel Morales, Cabildo Tenerife, Fondo de Investigaciones Sanitarias (FIS)/ Instituto de Salud Carlos III (ISCIII), Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (∼2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase subunit B inhibitor), or by seconeolitsine (a topoisomerase I inhibitor), has revealed the existence of topological domains that specifically respond to such changes. The control of DNA-supercoiling in S. pneumoniae occurs mainly via the regulation of topoisomerase gene transcription: relaxation triggers the up-regulation of gyrase and the down-regulation of topoisomerases I and IV, while hypernegative supercoiling down-regulates the expression of topoisomerase I. Relaxation affects 13% of the genome, with the majority of the genes affected located in 15 domains. Hypernegative supercoiling affects 10% of the genome, with one quarter of the genes affected located in 12 domains. However, all the above domains overlap, suggesting that the chromosome is organized into topological domains with fixed locations. Based on its response to relaxation, the pneumococcal chromosome can be said to be organized into five types of domain: up-regulated, down-regulated, position-conser, Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III-MINECO, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Wine originally emerged as a serendipitous mix of chemistry and biology, where microorganisms played a decisive role. From these ancient fermentations to the current monitored industrial processes, winegrowers and winemakers have been continuously changing their practices according to scientific knowledge and advances. A new enology direction is emerging and aiming to blend the complexity of spontaneous fermentations with industrial safety of monitored fermentations. In this context, wines with distinctive autochthonous peculiarities have a great acceptance among consumers, causing important economic returns. The concept of terroir, far from being a rural term, conceals a wide range of analytical parameters that are the basis of the knowledge-based enology trend. In this sense, the biological aspect of soils has been underestimated for years, when actually it contains a great microbial diversity. This soil-associated microbiota has been described as determinant, not only for the chemistry and nutritional properties of soils, but also for health, yield, and quality of the grapevine. Additionally, recent works describe the soil microbiome as the reservoir of the grapevine associated microbiota, and as a contributor to the final sensory properties of wines. To understand the crucial roles of microorganisms on the entire wine making process, we must understand their ecological niches, population dynamics, and relationships between ‘microbiome- vine health’ and ‘microbiome-wine metabolome.’ These are critical steps for designing precision enology practices. For that purpose, current metagenomic techniques are expanding from laboratories, to the food industry. This review focuses on the current knowledge about vine and wine microbiomes, with emphasis on their biological roles and the technical basis of next-generation sequencing pipelines. An overview of molecular and informatics tools is included and new directions are proposed, highlighting the importance of –omics tech, WineSeq Project, BiomeMakers Inc., Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub