Dietary carbohydrate source influences molecular fingerprints of the rat faecal microbiota

BACKGROUND: A study was designed to elucidate effects of selected carbohydrates on composition and activity of the intestinal microbiota. Five groups of eight rats were fed a western type diet containing cornstarch (reference group), sucrose, potato starch, inulin (a long- chained fructan) or oligofructose (a short-chained fructan). Fructans are, opposite sucrose and starches, not digestible by mammalian gut enzymes, but are known to be fermentable by specific bacteria in the large intestine. RESULTS: Animals fed with diets containing potato starch, or either of the fructans had a significantly (p < 0.05) higher caecal weight and lower caecal pH when compared to the reference group, indicating increased fermentation. Selective cultivation from faeces revealed a higher amount of lactic acid bacteria cultivable on Rogosa agar in these animals. Additionally, the fructan groups had a lower amount of coliform bacteria in faeces. In the inulin and oligofructose groups, higher levels of butyrate and propionate, respectively, were measured. Principal Component Analysis of profiles of the faecal microbiota obtained by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified bacterial 16S rRNA genes as well as of Reverse Transcriptase-PCR amplified bacterial 16S rRNA resulted in different phylogenetic profiles for each of the five animal groups as revealed by Principal Component Analysis (PCA) of band patterns. CONCLUSION: Even though sucrose and cornstarch are both easily digestible and are not expected to reach the large intestine, the DGGE band patterns obtained indicated that these carbohydrates indeed affected the composition of bacteria in the large gut. Also the two fructans resulted in completely different molecular fingerprints of the faecal microbiota, indicating that even though they are chemically similar, different intestinal bacteria ferment them. Comparison of DNA-based and RNA-based profiles suggested that two species within the phylum Bacteroidetes were not abundant in numbers but had a particularly high ribosome content in the animals fed with inulin

Novel de novo BRCA2 mutation in a patient with a family history of breast cancer

<p>Abstract</p> <p>Background</p> <p><it>BRCA2 </it>germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel <it>de novo BRCA2 </it>splice site mutation found in a patient exhibiting a ductal carcinoma at the age of 40.</p> <p>Methods</p> <p>Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the <it>BRCA1 </it>and <it>BRCA2 </it>genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from whole blood. The paternity was determined by single nucleotide polymorphism (SNP) microarray analysis. Parental origin of the <it>de novo </it>mutation was determined by establishing mutation-SNP haplotypes by variant specific PCR, while <it>de novo </it>and mosaic status was investigated by sequencing of DNA from leucocytes and carcinoma tissue.</p> <p>Results</p> <p>A novel <it>BRCA2 </it>variant in the splice donor site of exon 21 (nucleotide 8982+1 G→A/c.8754+1 G→A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon and synthesis of a truncated BRCA2 protein. The aberrant splicing was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. The mutation was not found in any of the patient's parents or in the mother's carcinoma, showing it is a <it>de novo </it>mutation. Variant specific PCR indicates that the mutation arose in the male germ-line.</p> <p>Conclusion</p> <p>We conclude that the novel <it>BRCA2 </it>splice variant is a <it>de novo </it>mutation introduced in the male spermatozoa that can be classified as a disease causing mutation.</p

Effects of apples and specific apple components on the cecal environment of conventional rats: role of apple pectin

<p>Abstract</p> <p>Background</p> <p>Our study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota.</p> <p>Results</p> <p>Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.</p> <p>A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum <it>Bacteroidetes</it>, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum <it>Firmicutes</it>, and specific species belonging to the <it>Clostridium </it>Cluster XIVa.</p> <p>Quantitative real-time PCR confirmed a lower amount of given <it>Bacteroidetes </it>species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of <it>Clostridium coccoides </it>(belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals.</p> <p>Conclusions</p> <p>Our findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and β-glucuronidase producing <it>Clostridiales</it>, and decreases the population of specific species within the <it>Bacteroidetes </it>group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.</p

Kinetic Modelling of [⁶⁸Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections

Background: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. Methods: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. One- and two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal&rsquo;s non-infected left hind limb was used as a control. Results: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. Conclusion: The kinetics of [68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model