46 research outputs found

    Generation, annotation, analysis and database integration of 16,500 white spruce EST clusters

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    BACKGROUND: The sequencing and analysis of ESTs is for now the only practical approach for large-scale gene discovery and annotation in conifers because their very large genomes are unlikely to be sequenced in the near future. Our objective was to produce extensive collections of ESTs and cDNA clones to support manufacture of cDNA microarrays and gene discovery in white spruce (Picea glauca [Moench] Voss). RESULTS: We produced 16 cDNA libraries from different tissues and a variety of treatments, and partially sequenced 50,000 cDNA clones. High quality 3' and 5' reads were assembled into 16,578 consensus sequences, 45% of which represented full length inserts. Consensus sequences derived from 5' and 3' reads of the same cDNA clone were linked to define 14,471 transcripts. A large proportion (84%) of the spruce sequences matched a pine sequence, but only 68% of the spruce transcripts had homologs in Arabidopsis or rice. Nearly all the sequences that matched the Populus trichocarpa genome (the only sequenced tree genome) also matched rice or Arabidopsis genomes. We used several sequence similarity search approaches for assignment of putative functions, including blast searches against general and specialized databases (transcription factors, cell wall related proteins), Gene Ontology term assignation and Hidden Markov Model searches against PFAM protein families and domains. In total, 70% of the spruce transcripts displayed matches to proteins of known or unknown function in the Uniref100 database (blastx e-value < 1e-10). We identified multigenic families that appeared larger in spruce than in the Arabidopsis or rice genomes. Detailed analysis of translationally controlled tumour proteins and S-adenosylmethionine synthetase families confirmed a twofold size difference. Sequences and annotations were organized in a dedicated database, SpruceDB. Several search tools were developed to mine the data either based on their occurrence in the cDNA libraries or on functional annotations. CONCLUSION: This report illustrates specific approaches for large-scale gene discovery and annotation in an organism that is very distantly related to any of the fully sequenced genomes. The ArboreaSet sequences and cDNA clones represent a valuable resource for investigations ranging from plant comparative genomics to applied conifer genetics

    Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

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    Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases

    A first update on mapping the human genetic architecture of COVID-19

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    Effect of Carbon Dioxide on the Thermodynamic State of Water in Collagen

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    Thermodynamic activities of polar sites of collagen in the presence of CO2 were observed by inverse gas chromatographic techniques using water as a probe. The interactions between collagen and the water probe were evaluated by determining the specific retention volume (Vg°) and partition coefficient (Kp) at 25°C, 3°C, and 35°C. Thermodynamic parameters were determined from these data. CO2 exhibited a significant effect on the water binding of collagen as shown by increased Vg° and Kp values as compared to N2‐ and He‐treated collagen. The thermodynamic parameters of partial molar Gibb\u27s free energy (ΔT°), partial molar enthalpy (ΔT°) and partial molar entropy (ΔT°) indicated CO2 significantly increased the average energy of water binding by collagen
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