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© 2020 the Author(s)., Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon–haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon’s ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon–haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner’s ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca. Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host., This study was partially supported by grants from the Italian Ministry of University and Research under the RITMARE Flagship Project (2012–2016) and the INMARE Project (Contract H2020-BG-2014-2634486), funded by the European Union’s Horizon 2020 Research Program. P.N.G. and O.V.G. acknowledge support from the Centre for Environmental Biotechnology Project, partly funded by the European Regional Development Fund via the Welsh Assembly Government. D.Y.S. was supported by SYAM-Gravitation Program of the Dutch Ministry of Education and Science Grant 24002002 and Russian Foundation for Basic Research Grant 19-04-00401). S.V.T. acknowledges Kurchatov Centre for Genome Research, and his work was supported by Ministry of Science and Higher Education of Russian Federation Grant 075-15-2019-1659.

This protocol describes a rapid protein extraction method for the archaeon Candidatus Vulcanisaeta moutnovskia, which can be also implemented for other archaea. The utilization of two different methods for protein extraction constitute the main step of the protocol. Method I involves the extraction with a multi-chaotropic lysis buffer containing a non-denaturing zwitterionic detergent, most efficient for extracting cytosolic proteins. Method II involves a denaturing anionic detergent allowing total disruption of the membranes and capable of extracting both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins. The big advantage of the methods is to use general laboratory chemicals to make powerful extraction buffers, resulting in high quality and quantity of proteins. The methods probably are usable for any other archaea or microbial cells, and takes about 14-22 h. Following extraction and further protein digestion, 1D-nano Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MSMS) analysis with Triple TOF 5600 and Orbitrap technologies were used for protein identification and further quantification.

To analyze the presence of total IgA and anti-gliadin antibodies (AGA) in BM from CD mothers who follow a gluten-free diet (GFD) and from mothers on a normal gluten-containing diet (ND). 218 samples of mature milk were obtained at different months of lactation (1–6) from 83 mothers (2 or more samples per mother) from Italy (Naples), The Netherlands (Leiden) and Spain (Madrid, Valencia and Reus): 42 CD mothers on GFD for more than 2 years and 41 non-CD mothers on a ND. Whey samples were analyzed for AGA-IgA by an indirect homemade ELISA and for total IgA (g/L) by a commercial ELISA kit. AGA-IgA was detected in BM, both in mothers on a GFD and mothers on a ND. AGA-IgA levels in both groups of mothers, CD and non-CD, show the same trend towards decreasing slightly along the months of lactation (p = 0.91). A different trend is observed for total IgA levels, decreasing markedly in CD mothers from the first month of lactation onwards but remaining stable in non-CD mothers (p = 0.048). A statistically significant association was found between the means of total IgA and AGA-IgA (p

In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV (GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor signaling pathway, the autophagy pathway, and the activation of the innate immune response, among others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the future design of novel vaccine strategies., This research was funded by the European Research Council, grant number GA639249 (ERC Starting Grant).

Dissimilatory sulfate reduction (DSR)-an important reaction in the biogeochemical sulfur cycle-has been dated to the Palaeoarchaean using geological evidence, but its evolutionary history is poorly understood. Several lineages of bacteria carry out DSR, but in archaea only Archaeoglobus, which acquired DSR genes from bacteria, has been proven to catalyse this reaction. We investigated substantial rates of sulfate reduction in acidic hyperthermal terrestrial springs of the Kamchatka Peninsula and attributed DSR in this environment to Crenarchaeota in the Vulcanisaeta genus. Community profiling, coupled with radioisotope and growth experiments and proteomics, confirmed DSR by 'Candidatus Vulcanisaeta moutnovskia', which has all of the required genes. Other cultivated Thermoproteaceae were briefly reported to use sulfate for respiration but we were unable to detect DSR in these isolates. Phylogenetic studies suggest that DSR is rare in archaea and that it originated in Vulcanisaeta, independent of Archaeoglobus, by separate acquisition of qmoABC genes phylogenetically related to bacterial hdrA genes.

12 p.-5 fig.-1 tab., Anthroponotic visceral leishmaniasis is a life-threatening disease caused by Leishmania donovani (Kinetoplastida: Trypanosomatidae) in East Africa and the Indian subcontinent. Unlike promastigote growth and differentiation in the sand fly gut or in axenic culture, L. donovani promastigote-into-amastigote development has been studied by high-throughput gene expression profiling. In this study, we have identified abundant constitutive proteins in axenically cultured promastigotes by two-dimension electrophoresis and matrix-assisted laser desorption-ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Most proteins involved in the trypanothione-based redox antioxidant system are expressed constitutively throughout axenic L. donovani promastigote growth and differentiation (tryparedoxin, trypanothione peroxidase, generic peroxidoxin, iron-superoxide dismutase, and elongation factor 1β). These findings are in agreement with previous data on other Old World species (i.e., L. major and L. infantum), whereas New World species (i.e., L. amazonensis and L. pifanoi) and Crithidia fasciculata show different expression patterns., We acknowledge Alfredo Toraño and Mercedes Domínguez for kind supply of the L. donovani strain used in this study.The CIB-CSIC Laboratory of Molecular Parasitology thanks the Ramón Areces Foundation (2016 call) for a contract. The CNB-CSIC Proteomics Facility belongs to ProteoRed (PRB2-ISCIII) and has been funded with grant PT13/0001.

8 pages, 5 figures, 3 tables, To better understand spring viremia of carp virus (SVCV) pathogenesis in zebrafish proteomic analysis was used to examine the plasma protein profile in SVCV-infected zebrafish. A total of 3062 proteins were identified. Of those 137, 63 and 31 proteins were enriched in blood samples harvested at 1, 2 and 5 days post SVCV infection, respectively. These altered host proteins were classified based on their biological function: 23 proteins under the response to stimulus term were identified. Interestingly, at the top of the up-regulated proteins during SVCV infection were the proteins of the vitellogenin family (Vtg) and the grass carp reovirus-induced gene (Gig) proteins. Real-time RT-PCR evaluation of samples from internal organs verified that SVCV infection induced vtg and gig2 gene expression already at day 1 post-infection. Western blot analysis revealed the presence of Vtg protein only in blood of SVCV-infected fish. This is the first proteomic study to reveal the involvement of Vtg proteins in adult fish response to viral challenge. It also highlights the role of Gig proteins as important factors in antiviral response in fish. This work provides valuable relevant insight into virus-host interaction and the identification of molecular markers of fish response to virus, This work has been funded by projects AGL2014-51773-C3 and BIO2018-782851-C3 from Spanish Ministerio de Economía y Competitividad. Dr. Ortega-Villaizán is the leader of ERC Grant GA639249. Melissa Belló-Perez is supported by a Generalitat Valenciana ACIF/2016/207 Fellowship. The Proteomics facility (CNB-CSIC) was supported by Grant PT13/0001

Composition of the gut microbiota changes during ageing, but questions remain about whether age is also associated with deficits in microbiome function and whether these changes occur sharply or progressively. The ability to define these deficits in populations of different ages may help determine a chronological age threshold at which deficits occur and subsequently identify innovative dietary strategies for active and healthy ageing. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics in three well‐defined age groups consisting of 30 healthy volunteers, namely, ten infants, ten adults and ten elderly individuals. Samples from each volunteer at intervals of up to 6 months (n = 83 samples) were used for validation. Ageing gradually increases the diversity of gut bacteria that actively synthesize proteins, that is by 1.4‐fold from infants to elderly individuals. An analysis of functional deficits consistently identifies a relationship between tryptophan and indole metabolism and ageing (p < 2.8e−8). Indeed, the synthesis of proteins involved in tryptophan and indole production and the faecal concentrations of these metabolites are directly correlated (r2 > .987) and progressively decrease with age (r2 > .948). An age threshold for a 50% decrease is observed ca. 11–31 years old, and a greater than 90% reduction is observed from the ages of 34–54 years. Based on recent investigations linking tryptophan with abundance of indole and other “healthy” longevity molecules and on the results from this small cohort study, dietary interventions aimed at manipulating tryptophan deficits since a relatively “young” age of 34 and, particularly, in the elderly are recommended., S.R. was recipient of a postdoctoral “Sara Borrell” contract from the Instituto de Salud Carlos III. This work was funded by grants SAF2015‐65878‐R and RTI2018‐095166‐B‐I00 from the Ministry of Science, Innovation and Universities with the cofunds of the European Regional Development Fund (ERDF) and the Agencia Estatal de Investigación (AEI), grant PIE14/00045 from the Instituto de Salud Carlos III, grant AC17/00022 from the Instituto de Salud Carlos III and Fundación Agencia Española contra el Cáncer (AECC) within the ERA NET TRANSCAN‐2, grant 2017‐1485 from the AECC and grant Prometeo/2018/A/133 from the Generalitat Valenciana. M.F. acknowledges the support of BIO2017‐85522‐R, from the Ministry of Science, Innovation and Universities, with the cofunds of the ERDF and the Agencia Estatal de Investigación (AEI). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB‐CSIC) that belongs to ProteoRed, PRB3‐ISCIII, supported by grant PT17/0019. The funding bodies did not have a role in the design or conduct of the study, the analysis and interpretation of the results, the writing of the report, or the decision to publish.

We appreciate the interest of Aouad and colleagues1 in our work on the archaeal class Methanonatronarchaeia2,3 and their effort to clarify the phylogenetic position of this unique group of extremely halophilic, methyl-reducing methanogens. In our analysis, Methanonatronarchaeia formed a clade with the class Halobacteria — the non-methanogenic euryarchaeal extreme halophiles. Notably, this phylogenetic placement is 100% bootstrap-supported in maximum-likelihood (ML) phylogenetic trees for both 16S ribosomal (r)RNA and concatenated alignments of ribosomal proteins3. Given the congruence of the two trees, the strong support for the Methanonatronarchaeia–Halobacteria clade, the biological plausibility of this affinity and the fact that these trees conformed with the currently favoured solutions for difficult problems in archaeal phylogeny (such as the monophyly of the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaeota (DPANN) superphylum and the euryarchaeal assemblage including Class I methanogens and Thermococci), we did not perform a more thorough phylogenetic analysis. Such an in-depth analysis was undertaken by Aouad and colleagues1. Their results suggest a different position for Methanonatronarchaeia — much deeper in the archaeal tree, sitting outside the branch that consists of Methanomicrobia (formerly, Class II Methanogens), including Halobacteria (denoted ‘Stenosarchaea’ by Aouad et al.) and the class Archaeoglobi, and at the base of the group (which Aouad et al. denote the ‘superclass Methanotecta’)1. This difference between the results of the two phylogenetic analyses stems primarily from the increasingly stringent removal of fast-evolving sites from the alignment prior to the phylogenetic tree construction that was applied by Aouad and colleagues. After a certain fraction of the fastest sites was removed, the tree topology abruptly transitioned to the deep placement of Methanonatronarchaeia. This procedure is supposed to eliminate the false signal produced by sites with multiple substitutions, and therefore Aouad et al. conclude that the affinity of Methanonatronarchaeia with Halobacteria was an artefact caused by such sites. Aouad et al. also obtained the ‘deeper’ placement of Methanonatronarchaeia with extended sets of conserved protein families and expanded taxon sampling in these cases, even without removing the fast-evolving sites.

31 p.-4 fig.-2 tab.-1 graph. abst., Leishmaniasis is a term that encompasses a compendium of neglected tropical diseases caused by dimorphic and digenetic protozoan parasites from the genus Leishmania (Kinetoplastida: Trypanosomatidae). The clinical manifestations of neotropical cutaneous leishmaniasis (NCL) caused by Leishmania pifanoi and other species of the “Leishmania mexicana complex” mainly correspond to anergic diffuse cutaneous leishmaniasis (ADCL), which is the origin of considerable morbidity. Despite the outstanding advances in the characterization of the trypanosomatid genomes and proteomes, the biology of this species has been scarcely explored. However, the close relation of L. pifanoi to the sequenced species L. mexicana and others included in the “L. mexicana complex” allowed us to perform a two-dimension electrophoresis (2DE) approach to the promastigote proteome at the differential expression level. Protein identifications were performed by matrix-assisted laser desorption-ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). This insight has revealed similarities and differences between L. pifanoi and other species responsible for cutaneous and visceral leishmaniasis. Interestingly, certain proteins that were previously described as immunostimulatory (elongation factor 1β, trypanothione peroxidase, heat shock protein 70, enolase, GDP-forming succinyl-CoA and aldehyde dehydrogenase) are more abundant in the final growth stages of promastigotes (late-logarithmic and/or stationary phase) in the case of L. pifanoi., This work has been funded with project AGL2010-21806-C02-01 (Spanish Ministry of Economy and Competitiveness, formerly Spanish Ministry of Science and Innovation) and contract 050204100014, OTT code 20100338 (Ramón Areces Foundation).

The Pine Wood Nematode (PWN) Bursaphelenchus xylophilus is a severe forest pathogen in countries where it has been introduced and is considered a worldwide quarantine organism. In this study, protein markers for differentiating populations of this nematode were identified by studying differences among four selected Iberian and one American population. These populations were compared by quantitative proteomics (iTRAQ). From a total of 2860 proteins identified using the public database from the B. xylophilus genome project, 216 were unambiguous and significantly differentially regulated in the studied populations. Comparisons of their pairwise ratio were statistically treated and supported in order to convert them into discrete character states, suggesting that 141 proteins were not informative as population specific markers. Application of the Character Compatibility methodology on the remaining 75 proteins (belonging to families with different biological functions) excludes 27 which are incompatible among them. Considering only the compatible proteins, the method selects a subset of 30 specific unique protein markers which allowed the compared classification of the Iberian isolates. This approach makes it easier search for diagnostic tools and phylogenetic inference within species and populations of a pathogen exhibiting a high level of genetic diversity., This work has been funded by the CSIC (PI:201130E033), and the EU project REPHRAME FP7‐KBBE‐2010‐4.

In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen., This work was partially supported by Spanish National project AGL2015-68248-C2-1-R and National Natural Science Museum. The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.

The ubiquity of strictly anaerobic sulfur-respiring haloarchaea in hypersaline systems with circumneutral pH has shaken a traditional concept of this group as predominantly aerobic heterotrophs. Here, we demonstrated that this functional group of haloarchaea also has its representatives in hypersaline alkaline lakes. Sediments from various hypersaline soda lakes showed high activity of sulfur reduction only partially inhibited by antibiotics. Eight pure cultures of sulfur-reducing natronoarchaea were isolated from such sediments using formate and butyrate as electron donors and sulfur as an electron acceptor. Unlike strict anaerobic haloarchaea, these novel sulfur-reducing natronoarchaea are facultative anaerobes, whose metabolic capabilities were inferred from cultivation experiments and genomic/proteomic reconstruction. While sharing many physiological traits with strict anaerobic haloarchaea, following metabolic distinctions make these new organisms be successful in both anoxic and aerobic habitats: the recruiting of heme-copper quinol oxidases as terminal electron sink in aerobic respiratory chain and the utilization of formate, hydrogen or short-chain fatty acids as electron donors during anaerobic growth with elemental sulfur. Obtained results significantly advance the emerging concept of halo(natrono)archaea as important players in the anaerobic sulfur and carbon cycling in various salt-saturated habitats.

Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against fish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine ex vivo and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor a (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches. This work was supported by the European Research Council (ERC Starting Grant GA639249) The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) belonging to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019 This work was supported by the European Research Council (ERC Starting Grant GA639249). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) of ProteoRed, PRB3- ISCIII, supported by grant PT17/0019

The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography–electrospray ionization tandem mass spectrometry data obtained for “Methanonatronarchaeum thermophilum” AMET1 grown in different physiological conditions, including variation of the growth temperature and substrates. Analysis of these data allows us to refine the current understanding of the key biosynthetic pathways of this triple extremophilic methanogenic euryarchaeon and identify proteins that are likely to be involved in its response to growth condition changes., D.Y.S. was supported by Gravitation-SIAM Program (grant 24002002, Dutch Ministry of Education and Science) and by RFBR (grant 16-04-00035). K.S.M., Y.I.W. and E.V.K. are supported by the intramural program of the U.S. Department of Health and Human Services (to the National Library of Medicine). The proteomic analysis was performed in the Proteomics Facility of The Spanish National Centre for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. This project has received funding from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement No [634486]. This work was further funded by grants BIO2014-54494-R, BIO2017-85522-R, PCIN-2014-107 (within the ERA NET-IB2 program), PCIN-2017-078 (within the ERA-MarineBiotech program) from the Spanish Ministry of Economy, Industry and Competitiveness and grant AC17/00022 from the Instituto de Salud Carlos III (within the ERA TRANSCAN-2 program).

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation., This work was supported by the European Research Council (ERC Starting Grant GA639249). The authors would like to thank to Remedios Torres and Efren Lucas for their technical assistance. The proteomic analyses were performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC)that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/0001. We would like to thank to the Blast2GO team and to Paulo Almeida (STABVida) for their technical support. We are also grateful to two anonymous reviewers for their valuable comments and suggestions

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs. This work was funded by the Plan Nacional Proyectos de Investigacion en Salud of Instituto de Salud Carlos III (ISCIII), EU FEDER Grant (PI14/00806). All proteomic analyses were performed at the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC) (a member of ProteoRed, PRB2-ISCIII, financed via grant PT13/0001). The authors thank Jose Maria Arribas (Espectrometria de Masas y Analisis Elemental, Universidad de Alcala) for NADPH analysis and Dr. Marcos Marva (Departamento de Fisica y Matematicas, Universidad de Alcala) for statistical analysis. Sí

This work was supported by the European Research Council (ERC Starting Grant GA639249). The authors would like to thank Remedios Torres and Efren Lucas for their technical assistance. The proteomic analyses were performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/0001. We would like to thank the Blast2GO team and Paulo Almeida (STABVida) for their technical support. We are also grateful to two anonymous reviewers for their valuable comments and suggestions., Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright–Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far, methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage 'Methanonatronarchaeia' that is most closely related to the class Halobacteria. Similar to the Halobacteria, 'Methanonatronarchaeia' are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that 'Methanonatronarchaeia' employ the 'salt-in' osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that use C 1 -methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO2 as well as membrane-bound heterodisulfide reductase and cytochromes. These features differentiate 'Methanonatronarchaeia' from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.

We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.

Background:It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study.Methods:Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed toViral Haemorrhagic Septicaemia virus(VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling.Results:VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon (ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-induciblemxandpkrgenes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV.Conclusions:A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

Teleost red blood cells (RBCs) are nucleated and therefore can propagate cellular responses to exogenous stimuli. RBCs can mount an immune response against a variety of fish viruses, including the viral septicemia hemorrhagic virus (VHSV), which is one of the most prevalent fish viruses resulting in aquaculture losses. In this work, RBCs from blood and head kidney samples of rainbow trout challenged with VHSV were analyzed via transcriptomic and proteomic analyses. We detected an overrepresentation of differentially expressed genes (DEGs) related to the type I interferon response and signaling in RBCs from the head kidney and related to complement activation in RBCs from blood. Antigen processing and presentation of peptide antigen was overrepresented in RBCs from both tissues. DEGs shared by both tissues showed an opposite expression profile. In summary, this work has demonstrated that teleost RBCs can modulate the immune response during an in vivo viral infection, thus implicating RBCs as cell targets for the development of novel immunomodulants., This work was supported by the European Research Council (ERC Starting Grant GA639249).

15 p.-4 fig.-4 tab., Purpose:Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO‐sensitive and resistant promastigotes is presented herein., Experimental design: Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases., Results: NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes., Conclusions and clinical relevance: The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future., The authors thank the Ramón Areces Foundation for the grant number 050204100014 (OTT code 20100338) and FIOCRUZ for zymodeme determination.

Sánchez-López, Ángela María et al., Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms., This work was supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional, Spain (grant nos. BIO2010–18239 and BIO2013–49125–C2–1–P), by the Government of Navarra (grant no. IIM010491.RI1), by the I-Link0939 project from the Ministerio de Economía y Competitividad, by the Ministry of Education, Youth, and Sports of the Czech Republic (grant no. LO1204 from the National Program of Sustainability), by Palacky University institutional support, by predoctoral fellowships from the Spanish Ministry of Science and Innovation (to A.M.S.-L. and P.G.-G.), and by postdoctoral fellowships from the Public University of Navarra (to M.B. and G.A.).

A quantitative real-time PCR system (Q-PCR) for the detection and quantification of wheat DNA in food for celiacs has been developed and optimised. The DNA was extracted with a new procedure, based on the SDS/Guanidine–HCl/Proteinase K method, but faster, with a higher yield and enough purity to avoid an additional purification step. This is the first report that compares the information provided by a Q-PCR technique with the prolamin levels determined with the most frequently used commercially available ELISA, based on the R5 monoclonal antibody. With the exception of some hydrolysed and highly processed food samples (such as beers, syrups, malt extracts, breakfast cereals…), the rest of the food with prolamin levels above the R5 ELISA quantification limit (1.5 mg/kg) have given positive signals with the Q-PCR system. Moreover, some food samples with prolamin levels below 1.5 mg/kg have also given positive signals with the Q-PCR system, indicating a better sensitivity of the DNA technique. Therefore, the developed Q-PCR system can be used as a non-immunological tool in order to confirm, by the “DNA pathway”, the presence of wheat in food not only for celiacs but also for individuals with wheat allergy.

The gluten content in various glucose syrups was determined by two sandwich ELISA methods and one competitive ELISA. Different extraction solutions were used for ELISA methods. MALDI-TOF mass spectrometry and SDS-PAGE were also carried out as a complementary technique to ELISA methods. The analysis proved that glucose syrups analyzed in this work and used in many food products and gluten-free food products are safe for patients suffering from celiac disease. Four food products containing glucose syrup were analyzed with satisfactory results as well. One sample of chocolate bar has gluten content lower than the current limit for gluten-free foods. Gluten content above this limit was found in this chocolate bar after cocktail-gelatine extraction. This product is not labelled as gluten-free food. This fact shows that the analyses of gluten-free products are not simple and need more work and attention.

20 p.-5 fig.-3 tab., Leishmania amazonensis is one of the major etiological agents of the neglected, stigmatizing disease termed american cutaneous leishmaniasis (ACL). ACL is a zoonosis and rodents are the main reservoirs. Most cases of ACL are reported in Brazil, Bolivia, Colombia and Peru. The biological cycle of the parasite is digenetic because sand fly vectors transmit the motile promastigote stage to the mammalian host dermis during blood meal intakes. The amastigote stage survives within phagocytes of the mammalian host. The purpose of this study is detection and identification of changes in protein abundance by 2DE/MALDI-TOF/TOF at the main growth phases of L. amazonensis promastigotes in axenic culture and the differentiation process that takes place simultaneously. The average number of proteins detected per gel is 202 and the non-redundant cumulative number is 339. Of those, 63 are differentially abundant throughout growth and simultaneous differentiation of L. amazonensis promastigotes. The main finding is that certain proteins involved in resistance to nitrosative and oxidative stress are more abundant at the last stages of growth and differentiation of cultured L. amazonensis promastigotes. These proteins are the arginase, a light variant of the tryparedoxin peroxidase, the iron superoxide dismutase, the regulatory subunit of the protein kinase A and a light HSP70 variant. These data taken together with the decrease of the stress-inducible protein 1 levels are additional evidence supporting the previously described pre-adaptative hypothesis, which consists of preparation in advance towards the amastigote stage., This work was supported by AGL2010-21806-C02-01 (Spanish Ministry of Economy and Competitiveness, formerly Spanish Ministry of Science and Innovation); 050204100014, OTT code 20100338 (Fundación Ramón Areces).

Background:It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study.Methods:Trout RBCs were obtained from peripheral blood, ficoll purified and exposed toViral Haemorrhagic Septicaemia virus(VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profilingResults:VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon (ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-induciblemxandpkrgenes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV.Conclusions:A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.

Objective:Studies performed on the nutritional status of the Spanish population have been very heterogeneous with respect to methodology, sample size, geographical location, socio-economic level and health status of the subjects involved. In order to gain an overall view of the state of knowledge in this area, a meta-analysis was performed on the results of all such studies undertaken in Spain between 1990 and 1999.Results:The dietetic data reviewed showed mean intakes similar to those recommended. The results obtained for the biochemical parameters analysed were within normal limits. However, results for some vitamins were very close to the lower established limits for the reference intervals (recommended intakes or blood levels), meaning that a variable percentage of subjects show values lower than those recommended. The percentage of subjects with intakes below those recommended for niacin, vitamin B12 and C was small. The percentage of inadequate intakes of thiamine and riboflavin was small as well. However, for all other vitamins, especially D and E, the number of people with intakes below recommended was rather high, particularly so in some studies. At blood level, deficiencies of vitamins B12, A and E were infrequent. However, for all other vitamins, prevalence of deficiency varied within a wide range. With respect to vitamins D, B1, B2 and B6, over 50% of the population showed inadequate levels.Conclusions:The methodologies used in the studies included in this review were very varied and the knowledge gained is still incomplete. Despite the average Spanish diet often being regarded as satisfactory, this review and other studies show the situation can be improved.

The only treatment for coeliac disease is to follow a strict, life-long gluten-free diet. It is therefore essential to use a highly sensitive, specific technique for gluten analysis in foods. Nowadays, the usual method for determining gluten content in gluten-free foods, internationally accepted by the Codex Alimentarius Commission, is the R5 antibody-based sandwich ELISA, combined with the cocktail-extraction solution. This technique requires at least two epitopes in the protein, but in hydrolysed foods, proteins are fragmented during food processing and converted into peptides in which only one toxic epitope may appear. Consequently, it was necessary to develop a new competitive immunoassay that, together with a reliable, compatible extraction solution, would provide a complete gluten analysis in any kind of food. We analysed commercial foods and home-made maize breads spiked with a known amount of gliadins using the sandwich R5 ELISA and the new competitive R5 ELISA that has been developed. These foods had previously been extracted with 60% ethanol/water, the cocktail solution or the new extracting solution called UPEX (universal prolamin and glutelin extractant solution). The complementary SDS-PAGE and western blot techniques were also used to confirm the gluten content. The limits of detection and quantification of the competitive R5 ELISA were 0.36 and 1.22 ng/ml of gliadins, respectively. The intra- and inter-assay precisions based on two samples were, respectively, 7.3% and 5.4% for the first sample and 9.9% and 6.3% for the second. This new assay was a better technique than the sandwich R5 ELISA for detecting gliadins quantitatively in hydrolysed foods. Regarding the extraction procedure, we did not find any significant interference from components of the UPEX solution at the concentration used. In addition, the UPEX solution extraction was compatible with the R5 western blot and mass spectrometry techniques. The competitive R5 ELISA we developed, combined with the UPEX solution described here, is a very useful tool for detecting and quantifying gluten in any kind of food samples, including heat-treated and/or hydrolysed ones.

We have investigated the extent of contamination with wheat, barley, rye or a mixture of these cereals in a large number of grains and commercial oats. We have also attempted to identify the type of cereal contaminant.Sandwich R5 ELISA (using either gliadins or hordeins as standards), western blot, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and quantitative real-time PCR (Q-PCR) techniques have been used to analyze a total of 134 oats, comprising grains and commercial oat products collected from Europe, the United States and Canada.Twenty-five of the 134 pure, uncontaminated oat varieties were found to have undetectable levels of gluten, whereas most of the 109 grains and commercial oat products were mainly contaminated with mixtures of wheat, barley and rye, barley being the predominant contaminant. The percentages of these cereals in the oat samples have been calculated by specific wheat, barley and rye Q-PCR systems. The oat samples were grouped according to the avenin spectra determined by the mass spectrometric technique. The data confirmed that contaminated oat foods, based on the same variety, could have different levels of wheat, barley and rye contamination.This study has verified that contamination with wheat gliadins or barley hordeins in oat samples can be measured by the Sandwich R5 ELISA, using either gliadins or hordeins as standards, and also the importance of using confirmatory techniques (such as western blot, Q-PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to confirm that most oats are contaminated with mixtures of wheat, barley and rye.

The threat of osteoporosis in later life means that the bone mass women achieve during their youth is important. Eighty seven women aged 18-35 y from the Madrid region were studied to determine the relationship between their calcium, phosphorus and milk product intakes and bone mineral density (BMD). Intakes of these items were moniroed using a three day food intake record. BMD was measured by double photonic densitometry of the lumbar region, hip and right forearm. Mean calcium intake (802.1+/-258.7 mg/day) was less than that recommended for 45% of women. A linear, positive correlation was seen between calcium intake and BMD at the hip (r=0.23) and greater trochanter (r=0.24) (p0.05). Women whose calcium intake was1000 mg/day had greater hip BMDs than those whose intake was below this level (0.97+/-0.11 g/cm2 compared to 0.90+/-0.10 g/cm2). Similar results were seen for the femur head and greater trochanter in subjects whose Ca/P ratio was0.74 (50th percentile). In addition, an intake of more than two rations of milk per day was optimum for achieving adequate bone mass in different areas of the hip. These results show that greater calcium consumption and a Ca/P ratio of0.74 are associated with better BMD values in young women, and that milk is the lactic product best associated with good bone health.

Objective: The aim of this study was to determine the desire to lose weight in a group of university students, and to analyse the influence of this desire on declared food habits. The degree of knowledge on what constitutes a balanced diet was also determined. Design: Cross-sectional population survey. Subjects: The study subjects were 234 university students (48 males and 186 females) aged 22.3±1.5 y. The weight and height of each were recorded. All subjects were questioned on their desire to lose weight, habitual food intake frequencies, and about what they considered to constitute a balanced diet. Results: Approximately half the population (47.9%), both male (47.8%) and female (47.9%), expressed a desire to lose weight, even though the percentage of overweight students was low (11.1% overall, although much greater among male students [39.6%] than female students [3.8]). The body mass index (BMI) of those who wanted to lose weight was greater than that of those who did not. Nonetheless, the majority of subjects (especially females) showed BMIs within the normal range (18–25 kg/m2). Among those who wished to lose weight, the consumption of sweet foods was lower — although these subjects believed they ought to consume even less. It was also thought necessary that egg consumption be reduced, although no differences were recorded in declared consumption between those who wished to lose weight and those who did not. Conclusions: The results show that there is great concern over body weight, although no great differences were seen in the food habits and nutrition knowledge of those who wished to lose weight and those who did not.

Objective: To determine the vitamin status of different groups within the Spanish population. Design: Pooled analysis of cross-sectional population nutritional regional studies and meta-analysis of smaller studies in population subgroups performed in Spain between 1990 and 1999. Subjects: In total, 10 208 participants in eight random population studies aged 25–60 y and participants in 76 studies included in the meta-analysis. Interventions: The data from different nutritional studies performed on sample populations representative of eight regions of Spain were integrated into a single database. A meta-analysis was also performed on the results of nutritional studies on the Spanish population involving subjects of other ages and status (all published between 1990 and 1999). Results: The mean intakes observed after the integration of the eight regional studies, and in the meta-analysis study, were similar to those recommended, although a considerable percentage of people had insufficient intakes. Conclusions: Further studies are required, but the vitamin status of the Spanish population clearly shows room for improvement, especially with respect to vitamins B2, B6, A, E, D and folates. Bearing in mind that adequate vitamin intakes are associated with protection from a range of diseases, steps should be taken to correct the imbalances recorded. The support of Roche Vitamins SA and Roche Consumer Health Spain was of great help in the undertaking of this research.

In this article the effects of saturated fatty acid (SFA) consumption on energy and nutrient intake, and on a range of serum lipid parameters (indicators of cardiovascular risk) are reported in a group of 110 preschool children. Subjects were divided into 2 groups: high consumption (HC), in which ≧13% (20th percentile) of total energy came from SFAs, and low consumption (LC), in which SFAs provided

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.

Dissimilatory sulfate reduction (DSR)—an important reaction in the biogeochemical sulfur cycle—has been dated to the Palaeoarchaean using geological evidence, but its evolutionary history is poorly understood. Several lineages of bacteria carry out DSR, but in archaea only Archaeoglobus, which acquired DSR genes from bacteria, has been proven to catalyse this reaction. We investigated substantial rates of sulfate reduction in acidic hyperthermal terrestrial springs of the Kamchatka Peninsula and attributed DSR in this environment to Crenarchaeota in the Vulcanisaeta genus. Community profiling, coupled with radioisotope and growth experiments and proteomics, confirmed DSR by ‘Candidatus Vulcanisaeta moutnovskia’, which has all of the required genes. Other cultivated Thermoproteaceae were briefly reported to use sulfate for respiration but we were unable to detect DSR in these isolates. Phylogenetic studies suggest that DSR is rare in archaea and that it originated in Vulcanisaeta, independent of Archaeoglobus, by separate acquisition of qmoABC genes phylogenetically related to bacterial hdrA genes., This work was supported by the Russian Science Foundation (grant number 17-74-30025) and in part by the grant from the Russian Ministry of Science and Higher Education (to N.A.C., A.V.L., E.N.F., M.L.M., A.Y.M., N.V.P. and E.A.B.-O.). Sequencing of PCR amplicons was performed using the scientific equipment of the core research facility ‘Bioengineering’ by T. Kolganova. The proteomics analysis was performed at the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC), which belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001 (to S.C., M.C.M. and M.F.). P.N.G. acknowledges funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC) within the ERA NET-IB2 programme, grant number ERA-IB-14-030 and the European Union Horizon 2020 Research and Innovation programme (Blue Growth: Unlocking the Potential of Seas and Oceans) under grant agreement number 634486, as well as support from the Centre for Environmental Biotechnology project, part funded by the European Regional Development Fund (ERDF) through the Welsh Government, and support from the Centre of Environmental Biotechnology. D.Y.S. was supported by the SIAM/Gravitation Program (Dutch Ministry of Education, Culture and Science; grant 24002002) and RFBR grant 19-04-00401. F.L.S. and S.N. acknowledge support from the Wiener Wissenschafts, Forschungs- und Technologiefonds (Austria) through the grant VRG15-007. F.L.S. gratefully acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme (grant agreement 803768). I.A.C.P. acknowledges support from the Fundação para a Ciência e Tecnologia (Portugal) through grant PTDC/BIA-BQM/29118/2017 and R&D unit MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020).