Project description:Background. Food can affect the microbial balance in the human intestine, and the ingestion of probiotics may play a role in the current obesity pandemic. The objective of our study was to determine if increased Lactobacillus spp. in the intestinal microflora of mice can promote growth and if changes in the intestinal microflora are associated with modifications in metabolism. Methodology. Female BALBc mice were divided between one control and two experimental groups and inoculated either once or twice with 4×1010 Lactobacillus per animal in PBS or with PBS alone. Fecal samples were collected and tested using qPCR to detect and quantify Lactobacillus spp., Bacteroidetes and Firmicutes. Gene expression by microarray and RT-PCR was studied in liver and adipose tissue. Finally, metabolic parameters in the plasma were tested. Principal Findings. In three independent experiments, we observed an increase in both weight gain and liver weight in mice inoculated with 4×1010 Lactobacillus. Inoculation with Lactobacillus sp. (ostrich) increased the Lactobacillus spp. and Firmicutes DNA copy number in feces. The transcriptional profile of liver tissue from mice inoculated with Lactobacillus sp. (ostrich) was enriched for Gene Ontology terms related to the immune response and metabolic modifications. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumor necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1) and 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) were significantly elevated in liver tissue in experimental group animals. In gonadal adipose tissue, the expression of leptin, peroxisome proliferator-activated receptor gamma (Pparg and Srebp1c was significantly higher in experimental group animals, whereas the expression of adiponectin was significantly lower. Conclusions. Alterations in the intestinal microbiota resulted in increased weight gain. Furthermore, increased Lactobacillus spp. in the intestinal microflora of mice inoculated with Lactobacillus sp. (ostrich) resulted in accelerated weight gain, liver enlargement and metabolic changes in the plasma, liver and adipose tissue.

Project description:Background. Food can affect the microbial balance in the human intestine, and the ingestion of probiotics may play a role in the current obesity pandemic. The objective of our study was to determine if increased Lactobacillus spp. in the intestinal microflora of mice can promote growth and if changes in the intestinal microflora are associated with modifications in metabolism. Methodology. Female BALBc mice were divided between one control and two experimental groups and inoculated either once or twice with 4×1010 Lactobacillus per animal in PBS or with PBS alone. Fecal samples were collected and tested using qPCR to detect and quantify Lactobacillus spp., Bacteroidetes and Firmicutes. Gene expression by microarray and RT-PCR was studied in liver and adipose tissue. Finally, metabolic parameters in the plasma were tested. Principal Findings. In three independent experiments, we observed an increase in both weight gain and liver weight in mice inoculated with 4×1010 Lactobacillus. Inoculation with Lactobacillus sp. (ostrich) increased the Lactobacillus spp. and Firmicutes DNA copy number in feces. The transcriptional profile of liver tissue from mice inoculated with Lactobacillus sp. (ostrich) was enriched for Gene Ontology terms related to the immune response and metabolic modifications. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumor necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1) and 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) were significantly elevated in liver tissue in experimental group animals. In gonadal adipose tissue, the expression of leptin, peroxisome proliferator-activated receptor gamma (Pparg and Srebp1c was significantly higher in experimental group animals, whereas the expression of adiponectin was significantly lower. Conclusions. Alterations in the intestinal microbiota resulted in increased weight gain. Furthermore, increased Lactobacillus spp. in the intestinal microflora of mice inoculated with Lactobacillus sp. (ostrich) resulted in accelerated weight gain, liver enlargement and metabolic changes in the plasma, liver and adipose tissue. For microarray analysis, we used the livers from two LB1 and two control mice that had been euthanized 20 days after inoculation. We used a whole mouse genome oligomicroarray 4x44K kit (44,000 60-mer oligonucleotides) and performed a one-color microarray-based gene expression analysis, as previously described. Labeled RNAs (Low RNA Input Fluorescent Amplification Kit, Perkin Elmer) were deposited on slides and hybridized using an in situ Hybridization Plus Kit (Agilent Technologies) for 17 h. The arrays were scanned using a DNA Microarray Scanner G2505B (Agilent Technologies), and the image analysis and correction of intra-array signals were performed using Feature Extraction Software A.9.1.3 (Agilent Technologies). Microarray data analysis was performed using GeneSpring 10.01 with the default setting for inter-array normalization and inter-replicate corrections. To identify genes that were differentially expressed, we used a Student’s t-test with p<0.05, and an absolute fold change (FC) greater than 2.0 considered significant. An analysis of Gene Ontology (GO) terms was performed to identify any altered biological processes. Additional data analysis was performed using R software, version 2.8.1. RNA extraction and cDNA synthesis were performed as described above. Primers and probes to target the mouse genes for cytochrome P450 2E1 (Cyp2e1), 3-phosphoinositide-dependent protein kinase-1 (Pdpk1) and glyceraldehyde 3-phosphate dehydrogenase (Gapdh, used as a housekeeping gene) were purchased from Applied Biosystems. qPCR reactions were performed as described above. The relative amount of each examined mRNA was normalized to the Gapdh mRNA expression level and is expressed relative to the mean amount of the same mRNA in the control group.

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Miseq Illumina paired-sequencing of 600-bp fragment from one ostrich (paired) female

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