The role of selenium in shaping mice brain metabolome and selenoproteome through the gut-brain axis by combining metabolomics, metallomics, gene expression, and amplicon sequencing

Selenium (Se) is a trace element crucial for human health. Recently, the impact of Se supplementation on gut microbiota has been pointed out as well as its influence on the expression of certain selenoproteins and gut metabolites. This study aims to elucidate the link between Se supplementation, brain selenoproteins and brain metabolome as well as the possible connection with the gut-brain axis. To this end, an in vivo study with 40 BALB/c mice was carried out. The study included conventional ( n = 20) and mice model with microbiota depleted by antibiotics ( n = 20) under a regular or Se supplemented diet. Brain selenoproteome was determined by a transcriptomic/gene expression profile, while brain metabolome and gut microbiota profiles were accomplished by untargeted metabolomics and amplicon sequencing, respectively. The total content of Se in brain was also determined. The selenoproteins genes Dio and Gpx isoenzymes, SelenoH, SelenoI, SelenoT, SelenoV, and SelenoW and 31 metabolites were significantly altered in the brain after Se supplementation in conventional mice, while 11 selenoproteins and 26 metabolites were altered in microbiota depleted mice. The main altered brain metabolites were related to glyoxylate and dicarboxylate metabolism, amino acid metabolism, and gut microbiota that have been previously related with the gut-brain axis ( e.g., members of Lachnospiraceae and Ruminococcaceae families ). Moreover, specific associations were determined between brain selenoproteome and metabolome, which correlated with the same bacteria, suggesting an intertwined mechanism. Our results demonstrated the effect of Se on brain metabolome through specific selenoproteins gene expression and gut microbiota.This work was supported by the projects: PG2018-096608-B- C21 and PID2021-123073NB-C21 from the Spanish Ministry of Science and Innovation (MICIN) . Generación del Conocimiento . MCIN/ AEI /10.13039/50110 0 011033/ FEDER “Una manera de hacer Europa”, UHU-1256905 and UHU-202009 from the FEDER Andalusian Operative Program 2014-2020 (Ministry of Economy, Knowledge, Business and Universities, Regional Government of Andalusia, Spain). S.R.A. thanks the Spanish Ministry of Science and Innovation for a PhD scholarship ( BES-2016-076364 ). The authors are grateful to FEDER (European Community) for financial support, Grant UNHU13-1E-1611 . The authors would like to acknowledge the support from The Ramón Areces Foundation (ref. CIVP19A5918 ). Funding for open access charge: Universidad de Huelva / CBUA

The treatment with the probiotic Shewanella putrefaciens Pdp11 of sepecimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.S

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species

BACKGROUND: Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. RESULTS: A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. CONCLUSION: We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, ΔFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant

Absolute mRNA levels and transcriptional regulation of the mouse testis-specific thioredoxins

28 páginas, 3 figuras más 1 suplementaria, 4 tablas más 1 suplementaria.Thioredoxins function as general protein disulphide reductases. Mammalian male germ cells are equipped with a set of three testis-specific thioredoxins (named Sptrx-1, -2, and -3, respectively) that are expressed either in different structures within the sperm cell or at different stages of sperm development. Previous studies based on qualitative northern-blot and in situ hybridization analyses restricted the presence of Sptrx mRNAs to adult testis, but nothing is known about their transcriptional regulation or relative expression levels in this tissue. In this report, we investigate the transcriptional profiles of the mouse Sptrx genes in terms of the germ cell-specific regulation by promoter analysis in GC-2spd(ts) cells. Besides, we perform a comprehensive quantification of the Sptrx mRNA molecules by real-time PCR in whole-animal experiments. By these means, we show that transcription is differentially regulated for each Sptrx gene and identify the 5′-flanking regions anticipated to contain the cis-regulatory elements responsible, at least in part, for the transcriptional silencing and/or activation of the Sptrx genes. In addition, we show remarkable age-associated variations between the Sptrx mRNA expression patterns.This work was supported by the Swedish Medical Research Council (Projects 03P-14096, 03X-14041, and 13X-10370), the Åke Wibergs Stiftelse, the Karolinska Institutet, and the Spanish Ministerio de Ciencia y Tecnología (Grant BMC2002-00179). A. Jiménez was supported by a postdoctoral fellowship (EX2003-0390) from the Spanish Ministerio de Educación, Cultura y Deporte. M.-J. Prieto-Álamo and J. Jurado were recipients of postdoctoral contracts (Programa Ramón y Cajal) from the Spanish Ministerio de Ciencia y Tecnología.Peer reviewe

Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species-2

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species"</p><p>http://www.biomedcentral.com/1471-2199/8/83</p><p>BMC Molecular Biology 2007;8():83-83.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2098773.</p><p></p>ved as vehicle controls. Total RNA was extracted at the indicated times. Data are the means of c-(triangles) or c--2 (circles) molecules/pg of total RNA ± SEM (n = 3 mice). Data at 0 min represent the mean values ± SEM of 5 control mice. No time-related effect was noted in these vehicle controls. Some error bars are not visible because of small standard errors. Statistical significance was evaluated using analysis of variance followed by multiple comparison according to the Student-Newman-Keuls method. Significant differences relative to control animals are indicated by filled-in symbols. For each transcript, the maximal fold increment is given in parentheses

Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species-3

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species"</p><p>http://www.biomedcentral.com/1471-2199/8/83</p><p>BMC Molecular Biology 2007;8():83-83.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2098773.</p><p></p>e addition of AmD. The half-life (t) values calculated from the resulting decay lines are given for comparisons. B) Rates of transcript decay and synthesis in NIH 3T3 cells. Transcript synthesis rates (in molecules/min) were calculated from the basal amounts of transcript molecules (per pg of total RNA) and their estimated half-lives (in minutes). These calculations are based on the assumption that though mRNA degradation is a complex process, it follows first-order kinetics[36]. The steady-state levels and rates of decay and synthesis of transcripts coding for both types of FosB protein (and Δ) and for heme oxygenase 1 (), thioredoxin 1 () and superoxide dismutase 3 () are included for comparison

Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species-8

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species"</p><p>http://www.biomedcentral.com/1471-2199/8/83</p><p>BMC Molecular Biology 2007;8():83-83.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2098773.</p><p></p>dicated by brick filled in boxes. The beginning and end of exons are indicated by the corresponding nucleotide positions (NCBI/GeneBank accession numbers: and for c-and , respectively). B) Names, sequences and 5'-position of upper (U) and lower (L) primers. C) Agarose (1.5%) gel electrophoresis analysis of RT-PCR products generated by different primer pairs (identified at the top of each line). Total RNA from NIH 3T3 cells was used as template. Forty cycles of PCR were performed as detailed [16]. Genomic DNA was further amplified by the E1U-E2L primer pair to exclude the possibility that our PCR conditions were not optimal for the amplification of the longest theoretical fragment, i.e. the 933 nt amplicon that should be observed if intron 1 remained unspliced in the c-transcript population [see Additional file ]. The molecular weight marker was a 100-nt ladder from Roche

Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species-7

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species"</p><p>http://www.biomedcentral.com/1471-2199/8/83</p><p>BMC Molecular Biology 2007;8():83-83.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2098773.</p><p></p> the mCRD are underlined. The encircled triplets encode the aa residues that contact to DNA. Triplets encoding two out of the five-leucine residues of the LZ motif are indicated in italics. The stop codon in intron 3 is boxed and shaded in grey. B) Western blot analysis of c-Fos proteins purified by immunoaffinity chromatography. A crude extract from NIH 3T3 cells stimulated with serum for 30 min was loaded onto the immunoaffinity column. The column eluate (E-30), as well as the input crude extract (CE-30) and the crude extract from serum-starved (CE-0) cells were subjected to inmunoblot analysis as described under "Methods". The positions of molecular size standards are indicated on the left in kDa. GAPDH was used for loading control

Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species-0

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing of c-pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species"</p><p>http://www.biomedcentral.com/1471-2199/8/83</p><p>BMC Molecular Biology 2007;8():83-83.</p><p>Published online 21 Sep 2007</p><p>PMCID:PMC2098773.</p><p></p>dicated by brick filled in boxes. The beginning and end of exons are indicated by the corresponding nucleotide positions (NCBI/GeneBank accession numbers: and for c-and , respectively). B) Names, sequences and 5'-position of upper (U) and lower (L) primers. C) Agarose (1.5%) gel electrophoresis analysis of RT-PCR products generated by different primer pairs (identified at the top of each line). Total RNA from NIH 3T3 cells was used as template. Forty cycles of PCR were performed as detailed [16]. Genomic DNA was further amplified by the E1U-E2L primer pair to exclude the possibility that our PCR conditions were not optimal for the amplification of the longest theoretical fragment, i.e. the 933 nt amplicon that should be observed if intron 1 remained unspliced in the c-transcript population [see Additional file ]. The molecular weight marker was a 100-nt ladder from Roche