22 research outputs found

    OTUs most significantly altered between room temperature and −80°C storage for all samples and each individual.

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    <p>OTUs most significantly altered between room temperature and −80°C storage for all samples and each individual.</p

    Abundances of dominant phyla in samples.

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    <p>Abundances (% of total 16S rRNA sequences) of the predominant bacterial phyla in healthy control and IBS patient fecal sample aliquots exposed to room temperature and −80°C for different lengths of time.</p

    Induction of MPER-specific antibody production by long-term immunization.

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    <p>Mice received GAD19 orally every 2 weeks for 14 weeks. (a) Diluted serum (1/100) was analyzed by ELISA at each time point. Arrows represent timing of the gavage. (b) Endpoint titers (or absorbance at 450 nm) of MPER-specific serum IgG, cecal IgA, vaginal IgA, and vaginal IgG. Each symbol represents an individual mouse.</p

    Mucosal Immunogenicity of Genetically Modified <i>Lactobacillus acidophilus</i> Expressing an HIV-1 Epitope within the Surface Layer Protein

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    <div><p>Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified <i>Lactobacillus acidophilus</i> strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the <i>Lactobacillus</i> S-layer protein for development of oral vaccines targeting specific peptides.</p></div

    β-diversity analysis of samples.

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    <p>Principal coordinates analysis (PCoA) of weighted and unweighted UniFrac distances of IBS patient (green and orange triangles) and healthy control (blue squares and red circles) fecal sample aliquots exposed to room temperature and −80°C for different lengths of time (room temperature - 1, 4, 6, 8 and 24 hours; −80°C −1 week and 1, 2, 3, 4, 5 and 6 months). PCoA plots illustrate the subject each sample aliquot originated from (<b>A&E</b>) and the temperature they were stored at (<b>B&F</b>). Average weighted UniFrac distances for all sample aliquots based on storage at room temperature (<b>C&G</b>) or −80°C (<b>D&H</b>) indicate that sample aliquot microbiotas show significantly similarity (*<i>p</i><0.05).</p

    Validation of genetically modified <i>L</i>. <i>acidophilus</i> producing MPER-displaying S-layer proteins.

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    <p>The <i>L</i>. <i>acidophilus slpA</i> gene in NCK1909 was replaced with the modified <i>slpA</i> gene including MPER-encoding sequences by homologous recombination in NCK2208. (a) The gene replacement of <i>slpA</i> with the modified <i>slpA</i> was confirmed by PCR. L, DNA ladder marker. Amplified DNA fragments using primers, AK_62 and AK_65 (lane 1 and 4), AK_62 and AK_57 (lane 2 and 5), or AK_56 and AK_65 (lane 3 and 6). (b) Detection of the MPER epitope in S-layer (SlpA) protein using 2F5 mAb. Total cell proteins and purified S-layer proteins of NCK1909 and NCK2208 were separated by SDS-PAGE. The gels were stained with CBB or blotted onto PVDF membrane followed by western blot analysis using 2F5 (anti-MPER monoclonal human IgG). (c) The exposed MPER epitope was detected by flow cytometry. The <i>L</i>. <i>acidophilus</i> strains labeled with 2F5 and Alexa Fluor 488-conjugated anti-human IgG were analyzed. Relative fluorescence intensity of each strain was shown as histogram plot.</p

    Typing of classes and subclasses of MPER-specific antibodies.

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    <p>Sera from GAD19-immunized mice were analyzed by ELISA. Each value plus SD (standard deviation) was shown.</p

    Reconstructed uptake and catabolic pathways in <i>L. acidophilus</i> NCFM.

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    <p>Proteins are listed by locus tag LBA numbers, transporters are colored by class (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044409#pone-0044409-g003" target="_blank">Figure 3</a>) and glycoside hydrolases are listed with GH family number. The polydextrose fraction transported by the ABC transporter (LBA0502–LBA0505) is uncertain and thus the hydrolytic pathway is marked as unknown. The present data outlines the PTS permease LBA0606 (higher level of induction compared to LBA0502–LBA0505) and associated hydrolytic pathway, as the main route of polydextrose utilization by <i>L. acidophilus</i> NCFM.</p

    Transcriptional Analysis of Prebiotic Uptake and Catabolism by <em>Lactobacillus acidophilus</em> NCFM

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    <div><p>The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in <em>Lactobacillus acidophilus</em> NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β- linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of <em>L. acidophilus</em> NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.</p> </div