APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.     

Neural correlates of experience-induced deficits in learned vocal communication

Songbirds are one of the few vertebrate groups (including humans) that evolved the ability to learn vocalizations. During song learning, social interactions with adult models are crucial and young songbirds raised without direct contacts with adults typically produce abnormal songs showing phonological and syntactical deficits. This raises the question of what functional representation of their vocalizations such deprived animals develop. Here we show that young starlings that we raised without any direct contact with adults not only failed to differentiate starlings' typical song classes in their vocalizations but also failed to develop differential neural responses to these songs. These deficits appear to be linked to a failure to acquire songs' functions and may provide a model for abnormal development of communicative skills, including speech.     

Using SD-AFLP and MSAP to assess CCGG methylation in the banana genome

Two amplified fragment length polymorphism (AFLP)-derived techniques were used to assess methylation at CCGG sites in the banana genome. Assessment of these techniques revealed that, while amplification steps are very reproducible, the ligation step is more subject to variability. Overall, these techniques produced an error rate of 0.2% per analysed band. Statistical approach highlights the fact that sample duplication is necessary to produce reliable results. This study involved 18 primer pairs and found that in the banana genome, roughly 80% of CCGG sites are unmethylated, 5% are methylated at the internal cytosine, and 15% are methylated at the external or both cytosines.     

Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA

Wolbachia endosymbionts are widespread in arthropods and are generally considered reproductive parasites, inducing various phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, Wolbachia infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. DNA purification methods for efficient genomic sequencing of these unculturable bacteria have proven difficult using a variety of techniques. To efficiently capture endosymbiont DNA for studies that examine the biology of symbiosis, we devised a parallel strategy to an earlier array-based method by creating a set of SureSelect? (Agilent) 120-mer target enrichment RNA oligonucleotides (“baits”) for solution hybrid selection. These were designed from Wolbachia complete and partial genome sequences in GenBank and were tiled across each genomic sequence with 60 bp overlap. Baits were filtered for homology against host genomes containing Wolbachia using BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasite Brugia malayi DNA was used as a test case, as the complete sequence of both Wolbachia and its host are known. DNA eluted from capture was size selected and sequencing samples were prepared using the NEBNext® Sample Preparation Kit. One-third of a 50 nt paired-end sequencing lane on the HiSeq? 2000 (Illumina) yielded 53 million reads and the entirety of the Wolbachia genome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly related Wolbachia strain from the crustacean Armadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.     

The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline

Background

Substantial progress in high-throughput metagenomic sequencing methodologies has enabled the characterisation of bacteria from various origins (for example gut and skin). However, the recently-discovered bacterial microbiota present within animal internal tissues has remained unexplored due to technical difficulties associated with these challenging samples.

Results

We have optimized a specific 16S rDNA-targeted metagenomics sequencing (16S metabarcoding) pipeline based on the Illumina MiSeq technology for the analysis of bacterial DNA in human and animal tissues. This was successfully achieved in various mouse tissues despite the high abundance of eukaryotic DNA and PCR inhibitors in these samples. We extensively tested this pipeline on mock communities, negative controls, positive controls and tissues and demonstrated the presence of novel tissue specific bacterial DNA profiles in a variety of organs (including brain, muscle, adipose tissue, liver and heart).

Conclusion

The high throughput and excellent reproducibility of the method ensured exhaustive and precise coverage of the 16S rDNA bacterial variants present in mouse tissues. This optimized 16S metagenomic sequencing pipeline will allow the scientific community to catalogue the bacterial DNA profiles of different tissues and will provide a database to analyse host/bacterial interactions in relation to homeostasis and disease.     

Impact of Selection for Digestive Efficiency on Microbiota Composition in the Chicken

Objectives

Feed efficiency and its digestive component, digestive efficiency, are key factors in the environmental impact and economic output of poultry production. The interaction between the host and intestinal microbiota has a crucial role in the determination of the ability of the bird to digest its food and to the birds’ feed efficiency. We therefore investigated the phenotypic and genetic relationships between birds’ efficiency and the composition of the cecal microbiota in a F2 cross between broiler lines divergently selected for their high or low digestive efficiency.

Methods

Analyses were performed on 144 birds with extreme feed efficiency values at 3 weeks, with feed conversion values of 1.41±0.05 and 2.02±0.04 in the efficient and non-efficient groups, respectively. The total numbers of Lactobacillus, L. salivarius, L. crispatus, C. coccoides, C. leptum and E. coli per gram of cecal content were measured.

Results

The two groups mainly differed in larger counts of Lactobacillus, L. salivarius and E. coli in less efficient birds. The equilibrium between bacterial groups was also affected, efficient birds showing higher C. leptum, C. coccoides and L. salivarius to E. coli ratios. The heritability of the composition of microbiota was also estimated and L. crispatus, C. leptum, and C. coccoides to E. coli ratios were moderately but significantly heritable (0.16 to 0.24). The coefficient of fecal digestive use of dry matter was genetically and positively correlated with L. crispatus, C. leptum, C. coccoides (0.50 to 0.76) and negatively with E. coli (-0.66). Lipid digestibility was negatively correlated with E. coli (-0.64), and AMEn positively correlated with C. coccoides and with the C. coccoides to Lactobacillus ratio (0.48 to 0.64). We also detected 14 Quantitative Trait Loci (QTL) for microbiota on the host genome, mostly on C. leptum and Lactobacillus. The QTL for C. leptum on GGA6 was close to genome-wide significance. This region mainly includes genes involved in anti-inflammatory responses and in the motility of the gastrointestinal tract.     

DNA mismatch repair defects: role in colorectal carcinogenesis

The inactivation of the DNA mismah repair (MMR) system, which is associated with the predisposition to the hereditary non-polyposis colorectal cancer (HNPCC), has also been documented in nearly 20% of the sporadic colorectal cancers. These tumors are characterized by a high frequency of microsatellite instability (MSI(+) phenotype), resulting from the accumulation of small insertions or deletions that frequently arise during replication of these short repeated sequences. A germline mutation of one of the two major MMR genes (hMSH2 or hMLH1) is found in half to two-thirds of the patients with HNPCC, whereas in sporadic cases hypermethylation of the hMLH1 promoter is the major cause of the MMR defect. Germline mutations in hMSH6 are rare and rather confer predisposition to late-onset familial colorectal cancer, and frequent extracolonic tumors. Yet, the genetic background of a number of HNPCC patients remains unexplained, indicating that other genes participate in MMR and play important roles in cancer susceptibility. The tumor-suppressor genes that are potential targets for the MSI-driven mutations because they contain hypermutable repeated sequences are likely to contribute to the etiology and tissue specificity of the MSI-associated carcinogenesis. Because the prognosis and the chemosensitivity of the MSI(+) colorectal tumors differ from those without instability, the determination of the MSI phenotype is expected to improve the clinical management of patients. This review gives an overview of various aspects of the biochemistry and genetics of the DNA mismah repair system, with particular emphasis in its role in colorectal carcinogenesis.     

AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms

Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.     

Structural and biological effects of a beta2- or beta3-amino acid insertion in a peptide.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta2-HAla-NHMe and Ac-beta3-HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta2-HPhe and of Gly9 by HGly (R)-beta2-HAla or (S)-beta3-HAla. [(R)-beta2-HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta3-HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta3-HAla9]SP and [(R)-beta2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta2-HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.     

Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration

BACKGROUND: Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration. RESULTS: Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation. CONCLUSION: We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.