Validation of fluorescent in situ hybridization combined with flow cytometry for assessing interindividual variation in the composition of human fecal microflora during long-term storage of samples

This work was conducted to assess the accuracy of in situ hybridization to show differences in human microflora composition between volunteers and to optimize the storage of fecal samples to allow delayed analysis of gut microflora composition in humans. Fecal samples from 25 healthy subjects (14 women, 11 men aged 24-51) were collected. The samples were fixed in 4% Paraformaldehyde (PFA) solution at 4 degrees C overnight and stored at -70 degrees C. Twenty samples were analysed to quantify the variation due to interindividual differences in the composition of fecal microflora. The five remaining samples were stored either after PFA fixation or directly frozen at -70 degrees C and were monitored on a 12-month period. The fecal microflora was analysed by in situ hybridization combined with flow cytometry detection. Ribosomal RNA-targeted probes were used to assess the relative proportions of four phylogenetic groups: Clostridium coccoides-Eubacterium rectale (Erec 482), Bacteroides (Bac 303), Faecalibacterium prausnitzii (Fprau 645) and Bifidobacterium (Bif 164). Our results demonstrated that the method used is adapted to detect significant differences in fecal microflora composition in humans. Moreover, samples stored in PFA solution demonstrated a stable composition even after 8 months of storage. Conversely, frozen samples were less stable as the Bifidobacterium and C. coccoides-E. rectale groups showed significant differences after 2 months of storage. In conclusion, the fecal microflora composition can be analysed up to 8 months after 4% PFA fixation and storage at -70 degrees C. It represents an extended time compared with the 2-month period currently recommended. This will give more flexibility for applying this technology in epidemiological studies including a large number of samples.     

Repression of Osteoblast Maturation by ERRα Accounts for Bone Loss Induced by Estrogen Deficiency

ERRα is an orphan member of the nuclear receptor family, the complete inactivation of which confers resistance to bone loss induced by ageing and estrogen withdrawal to female mice in correlation with increased bone formation in vivo. Furthermore ERRα negatively regulates the commitment of mesenchymal cells to the osteoblast lineage ex vivo as well as later steps of osteoblast maturation. We searched to determine whether the activities of ERRα on osteoblast maturation are responsible for one or both types of in vivo induced bone loss. To this end we have generated conditional knock out mice in which the receptor is normally present during early osteoblast differentiation but inactivated upon osteoblast maturation. Bone ageing in these animals was similar to that observed for control animals. In contrast conditional ERRαKO mice were completely resistant to bone loss induced by ovariectomy. We conclude that the late (maturation), but not early (commitment), negative effects of ERRα on the osteoblast lineage contribute to the reduced bone mineral density observed upon estrogen deficiency.     

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Delta cells showed that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.     

Molecular characterization and phylogenetic position of a new mariner-like element in the coastal crab, Pachygrapsus marmoratus

Mariner-like elements (MLEs) are class-II transposable elements that move within the genome of their hosts by means of a DNA-mediated "cut and paste" mechanism. MLEs have been identified in several organisms, from most of the phyla. Nevertheless, only a few of the sequences characterized contain an intact open reading frame. Investigation of the genome of a coastal crab, Pachygrapsus marmoratus, has identified nine Pacmmar elements, two of which have an open reading frame encoding a putatively functional transposase. Nucleic acid analyses and comparison with the previous data showed that the GC contents of MLEs derived from coastal organisms such as P. marmoratus are significantly higher than those of terrestrial MLEs and significantly lower than those of hydrothermal ones. Furthermore, molecular phylogeny analyses have shown that Pacmmar elements constitute a new lineage of the irritans subfamily within the mariner family.     

A Remarkable Case of Micro-Endemism in Laonastes aenigmamus (Diatomyidae,Rodentia) Revealed by Nuclear and Mitochondrial DNA Sequence Data

L. aenigmamus is endemic to the limestone formations of the Khammuan Province (Lao PDR), and is strongly specialized ecologically. From the survey of 137 individuals collected from 38 localities, we studied the phylogeography of this species using one mitochondrial (Cyt b) and two nuclear genes (BFIBR and GHR). Cyt b analyses reveal a strong mtDNA phylogeographical structure: 8 major geographical clades differing by 5–14% sequence divergence were identified, most of them corresponding to distinct karst areas. Nuclear markers display congruent results but with a less genetic structuring. Together, the data strongly suggest an inland insular model for Laonastes population structure. With 8 to 16 evolutionary significant units in a small area (about 200×50 km) this represents an exceptional example of micro-endemism. Our results suggest that L. aenigmamus may represent a complex of species and/or sub-species. The common ancestor of all Laonastes may have been widely distributed within the limestone formations of the Khammuan Province at the end of Miocene/beginning of the Pliocene. Parallel events of karst fragmentation and population isolation would have occurred during the Pleistocene or/and the end of the Pliocene. The limited gene flow detected between populations from different karst blocks restrains the likelihood of survival of Laonastes. This work increases the necessity for a strict protection of this rare animal and its habitat and provides exclusive information, essential to the organization of its protection.     

Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides-Porphyromonas-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus-Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides-E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice (p<0.05) for the C. coccoides-E. rectale and Bacteroides-Porphyromonas-Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.     

Terrestrial small mammal diversity and abundance in central Benin: comparison between habitats,with conservation implications

We performed a terrestrial small mammal species inventory in the Agoua and Wari‐Maro forest reserves (Benin). Four localities were sampled, and in each locality, three habitats were surveyed: dense forest, open forest or woodland savannah and shrub savannah. This is the first comprehensive inventory for small mammals in central Benin. We captured 794 small mammals representing twenty species (six shrew species, fourteen rodent species). Three new species that need to be described were recorded. We observed a mixture of both true forest species and of species adapted to a wider range of habitats ranging from savannah to forest clearings. Species with either Sudanian or Guinea–Congolian affinities were recorded, as well as a new species endemic to Togo and Benin. This rich biodiversity underlines the urgent need for an effective protection of these forests. The Sudanian species Crocidura cf. foxi was more abundant in Wari‐Maro than in Agoua forest, while the Guineo–Congolian species Praomys misonnei and Hylomyscus sp were only captured in Agoua forest. These results are in agreement with the fact that these two forests belong to two distinct chorological zones.