PI3K-dependent phosphorylation of Fbw7 modulates substrate degradation and activity

The Fbw7 tumor suppressor gene encodes the substrate recognition subunit of the SCF ubiquitin ligase, which targets for degradation a range of oncogenic proteins in a phosphorylation-dependent manner. Substrate phosphorylation is thought to be the main mechanism that ensures timely destruction of Fbw7 substrates. We show here that PI3K dependent phosphorylation of Fbw7 stimulates its ability to ubiquitinate and degrade its substrates. Mutation of the phosphorylation site destabilizes Fbw7 and attenuates degradation of cyclin E and Myc leading to the enhanced expression of a subset of Myc target genes. We suggest that PI3K-dependent phosphorylation of Fbw7 controls the balance between turnover of Fbw7 and its substrates to fine-tune their activity.     

Strategies for DNA methylation analysis in developmental studies

Developmental processes in eukaryotes are highly dependent on DNA methylation. 5-methylcytosine (m(5) C) is the most prevalent and best understood DNA modification implicated in maintenance of genomic integrity and function across species. Although m(5) C occurs almost exclusively in symmetrical CpG context in vertebrates, additional asymmetrical distribution in CpHpG and CpHpH sites has been observed in plants and embryonic stem cells. To this end, accurate and reproducible methodology for full analysis of the DNA methylome is highly demanded. Fortunately, a variety of methods enable quantitative DNA methylation mapping at a single-base resolution and in a large scale. Here, we provide a critical overview of methods applied primarily to m(5) C detection with particular emphasis on technical improvements of the classical bisulfite-conversion protocol. We further describe strategies in combination with emerging technologies that allow acquisition of highly reliable data for developmental studies.     

Quality and digestibility of food ingested by various trophic fish groups in the Upper Paraná River floodplain

Determining quality of food ingested and digestibility can be helpful in understanding the mechanisms that determine trophic plasticity, which is the ability of a given species to use a more advantageous food source at a given time. In this study, the chemical composition and digestibility of food ingested by various trophic fish groups at different sites of the Upper River Paraná floodplain are evaluated. Specimens of Pseudoplatystoma corruscans, Prochilodus lineatus, Leporinus friderici, Pterodoras granulosus and Schizodon borellii from the Baía, Ivinheima and Paraná Rivers and from Fechada and Ventura Lagoons were taken as samples (3-16 cm-mesh net). Volume participation analyses of food items were determined and contents from the stomach and the intestine's latter quarter were removed for bromatological analysis (energy, crude protein, ash and dry matter). Internal marker acid-insoluble ash was employed for apparent digestibility coefficients. P. lineatus and P. corruscans had an intake with lower and higher energy and crude protein contents, respectively. P. corruscans had slight variations in food items and composition, whereas P. granulosus had the greatest variation. Whereas P. lineatus had the highest apparent digestibility coefficients in energy, S. borellii showed least variation in diet. P. granulosus and P. lineatus had the higher percentages of dry matter in the stomach in the Paraná River, whereas P. corruscans had similar use in the Baía Rivers and Ventura Lagoon. S. borellii showed low utilization of germanous energy. It may be concluded that the site determined the variation in quality and use of diet by L. friderici, P. granulosus and S. borellii. The generalist species L. friderici demonstrated a good use of different food items ingested at the site; likewise, the generalist species P. corruscans had a similar diet and the same use of food.     

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of ~26 months and a nearly identical maximal life expectancy of ~37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated-weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity-HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure.     

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.     

Effects of long-term storage on the detection of proteins, DNA, and mRNA in tissue microarray slides

Storage of tissue slides has been claimed to induce dramatically reduced antigen detection particularly for immunohistochemistry (IHC). With tissue microarrays, the necessity to serially cut blocks in order to obtain as much material as possible is obvious. The presumed adverse effect of storage might hamper such an approach. The authors designed an experimental setting consisting of four different storage conditions with storage time of tissue slides of up to 1 year. Detection of proteins, DNA, and mRNA was performed using IHC and in situ hybridization techniques. Slight but significant changes in IHC occurred over time. The most important factor is the primary antibody used: four showed no significant changes, whereas limited decreases in 8 antibodies could be detected by image analysis. Whether the antigen was nuclear or cytoplasmic/membranous did not matter. No major differences between different storage conditions could be shown, but storage at 4C was overall the best procedure. Furthermore, gene copy number aberrations, chromosomal translocations, and the presence of mRNA could be detected on slides stored up to 1 year. In conclusion, in tissues optimally formalin fixed and using modern histological techniques, only minute changes in tissue antigenicity are induced by long-term storage.     

Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein-lipid interphase, we used the von Heijne water-membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (> 1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (< 1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies.