9 research outputs found

    Profound changes in fecal microbiota composition during melioidosis.

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    <p>Fecal pellets were sampled from eight mice before (t = 0) they were infected intranasally with 150 CFU of <i>B</i>. <i>pseudomallei</i> and 72 hours after (t = 72). Microbial composition was analysed by IS-pro, using the number of nucleotides between the genes for ribosomal subunit 16 and 23 in the DNA (interspacer region) of the bacterium as a unique classification characteristic. (A) Clustering analysis, by unweighted pair group method with arithmetic mean (UPGMA) on cosine distances, shows the similarity of samples; individual mice are indicated by a number. Colors represent the most important bacterial phyla (purple, Actinobacteria; red, Bacteroidetes; blue, Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV); yellow, Proteobacteria). Length of the interspacer regions in basepairs is indicated on the y-axis; lines indicate the presence of PCR products. Color intensity increases with the presence of PCR product. (B) Diversity of microbial communities before and 72 hours after induction of melioidosis, expressed as Shannon index (green: total bacteria; red: Bacteroidetes; Blue: Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV); yellow: Proteobacteria). Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. ** p<0.01 pre- versus post-infection.</p

    Antibiotic pre-treated mice show increased growth and dissemination of <i>B</i>. <i>pseudomallei</i> during experimental melioidosis.

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    <p>(A) Study design. (B) Before infection, the fecal microbiota of control- and antibiotic treated mice was analysed by IS-pro. Colors represent the most important bacterial phyla (purple, Actinobacteria; red, Bacteroidetes; blue, Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV); yellow, Proteobacteria). Length of the interspacer regions in basepairs is indicated on the x-axis; height of the peaks indicates the presence of PCR products. Samples are pooled from eight mice per group; representative of two experiments. Control and antibiotic pre-treated mice were inoculated intranasally with 150 CFU (C-E) or 500 CFU (F-H) <i>B</i>. <i>pseudomallei</i> and sacrificed at the indicated time points. Bacterial loads in lung homogenate (C, F), blood (D, G) and liver homogenate (E, H) are depicted as scatter dot plots with a line at the median. Numbers in the boxes below (D) and (G) indicate the number of positive blood cultures for the total number of mice. White dots represent control mice, grey dots antibiotic treated mice. N = 6–8 mice per group. * p<0.05, ** p<0.01, *** p<0.001 control versus antibiotic treated.</p

    Limited effect of antibiotic induced gut microbiota disruption on survival and organ damage.

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    <p>Survival (A) and clinical observation score (B) of control (white dots) and antibiotic treated mice (grey dots) after intranasal inoculation with 150 CFU <i>B</i>. <i>pseudomallei</i> (n = 20 mice per group, depicted is the mean). No statistically significant differences were detected. Aspartate aminotranspherase (AST, C), alanine aminotranspherase (ALT, D), urea (E) and lactate dehydrogenase (LDH, F) were measured in plasma after inoculation with 500 CFU <i>B</i>. <i>pseudomallei</i> as markers for liver-, renal- and general damage. Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. White bars represent control mice, grey bars antibiotic treated mice. N = 5–6 samples per group. ND = not detectable.</p

    Antibiotic microbiota disruption does not affect neutrophil influx.

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    <p>Lungs were obtained at the indicated time points after intranasal inoculation with 500 CFU <i>B</i>. <i>pseudomallei</i>. Paraffin-embedded lung tissue sections were stained with haematoxylin/eosin to score different parameters for pathology (A-B, 2x magnification, representative images). The combined score, given by a blinded pathologist, was not different between the two groups (C). Sections from the same samples were stained for Ly-6GC as a neutrophil marker (representative microphotographs, 2x magnification) (D-E). The percentage Ly-6GC-positive surface of the total lung surface was calculated using ImageJ (F). The number of cells per mL BALF was counted using a Coulter counter (G). Myeloperoxidase was quantified in lung homogenates as a measure for neutrophil degranulation (H). Bone marrow and blood was obtained from naïve control and antibiotic pre-treated mice and using FACS analysis the percentage of Ly6GC+, CD11b+ cells within the viable CD45+ population was determined (I). Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. White bars represent control mice, grey bars antibiotic treated mice. No statistically significant differences were found.</p

    MiRNA-30c transgenic mice develop severely dilated cardiomyopathy.

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    <p>(a) Kaplan-Meier survival curve for both transgenic lines. We observed no mortality in wildtype littermates. 50% mortality was reached at 37 and 21 weeks for line A and B, respectively. (b) Transgenic mice of both lines develop dilated hearts with enlarged right atria during the end-stage of disease progression. In line A this occurs after an age of 12 weeks, while in line B the first signs of dilation are noted at 6 weeks of age. .(c and d) Cardiac function as determined by echocardiography in line B at 6 weeks of age (N = 3). LVID;d/TL denotes LV internal diameter during diastole, corrected for tibia length. LVID;s/TL denotes LV internal diameter during systole, corrected for tibia length. Differences between LVID;d/TL and LVID;s/TL were analysed by 2-way ANOVA for repeated measures (diagonal line indicates significant interaction effect). (e) Heart weight corrected for tibia length shows no significant difference between wildtype and miRNA-30c TG at 6 weeks of age in line B (N = 3). (f) ANF mRNA expression as evaluated by qPCR (N≥3) shows a significant increase in transgenic mice in line B. Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p

    Differentially regulated pathways at 4 weeks of age.

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    <p>Significantly down- and upregulated pathways based on the microarray expression data of left ventricular tissue in line A. Count represents the number of differentially expressed genes and Genes is the total number of genes in the given pathway.</p

    MiRNA-30c transgenic mice have a mitochondrial phenotype.

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    <p>(a) Western blot of mitochondrial OXPHOS proteins from wildtype and transgenic littermates, before the onset of the phenotype, at 4 weeks of age in line B. (b) Quantification of western blots of mitochondrial OXPHOS proteins of panel A (N = 6). (c) Western blot of mitochondrial OXPHOS proteins in line B at 10 weeks of age, after DCM starts to develop. (d) Overview images of electron microscopy from wildtype (left) and miRNA-30c TG mice (right) of line B. Mitochondria are indicated with a black arrowhead. (e) Quantification of mitochondrial area (N = 3, 18 images per heart) at 4 weeks of age. (f) Quantification of mitochondrial to genomic DNA ratio by qPCR (N = 4) at 4 weeks of age in line B. (g) Mitochondrial citrate synthase activity at 4 weeks of age (N = 4) in line B. Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p

    MiRNA-30c expression in the heart and the generation of αMHC-miRNA-30c transgenic mice (miRNA-30c TG).

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    <p>(a) MiRNA-30c in situ hybridization on adult wildtype hearts shows expression in the nuclei of both cardiomyocytes (black arrowheads) and interstitial cells (grey arrowheads). The cytoplasm is also miRNA-30c positive. (b) Schematic overview of the miRNA-30c overexpression construct used for the generation of transgenic mice. (c) Northern blot for miRNA-30c in wildtype and transgenic littermates in line B at 8 weeks of age. U6 was used as a loading control and shows similar loading. (d) Quantification of miRNA-30c overexpression by qPCR in both transgenic lines at 4 weeks of age (N≥6). (e) MiRNA-30c expression in left en right ventricular tissue of line B at 4 weeks of age (N = 6). Error bars represent s.e.m. and * denotes a p-value ≤0.05.</p
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