Dimethyl-pepep: a DNA probe in two-photon excitation cellular imaging

Dimethyl-pepep (D-pepep), a newly developed and very efficient two-photon absorber, has been tested here for two-photon excitation (TPE) cellular imaging. The spectral characteristics of the dye following one-photon excitation (OPE) and TPE (excitation and emission spectra, fluorescence lifetime, molecular brightness, saturation intensity) are reported. In vitro interaction studies with biomolecules show that dimethyl-pepep has a large affinity for DNA. A comparison with a widely used DNA stainer, 4-6-diamidino-2-phenylindole (DAPI) bound to DNA shows that the D-pepep brightness is one order of magnitude higher than that of DAPI, making this dye suitable for microscopy and imaging applications. TPE images taken from double-stained yeast Saccharomyces cerevisiae cells have revealed that D-pepep localizes mainly in the nucleus, similarly to DAPI, and in mitochondria, although to a minor extent. Preliminary tests have shown that the dye cellular toxicity is negligible.     

"Modeled microgravity" affects cell response to ionizing radiation and increases genomic damage

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.     

A novel <Emphasis Type="Italic">Dictyostelium</Emphasis> RasGEF required for chemotaxis and development

Background  

Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.     

Redox proteomics: identification and functional role of glutathionylated proteins

Although radical oxygen and nitrogen species are harmful molecules that destroy cell functions, many operate as mediators of important cell signaling pathways when not in excess. Oxidants can modify protein function through the covalent, reversible addition of glutathione to cysteine. This review addresses different proteomic methods of identifying glutathionylation targets and emphasizes ways of defining their pattern of modification in response to oxidative stimuli in cells. Finally, the literature on nonproteomic studies that investigate the functional changes induced by glutathionylation are reviewed and future studies are commented on.     

Vitamin D Supplementation and Breast Cancer Prevention: A Systematic Review and Meta-Analysis of Randomized Clinical Trials

In recent years, the scientific evidence linking vitamin D status or supplementation to breast cancer has grown notably. To investigate the role of vitamin D supplementation on breast cancer incidence, we conducted a systematic review and meta-analysis of randomized controlled trials comparing vitamin D with placebo or no treatment. We used OVID to search MEDLINE (R), EMBASE and CENTRAL until April 2012. We screened the reference lists of included studies and used the “Related Article” feature in PubMed to identify additional articles. No language restrictions were applied. Two reviewers independently extracted data on methodological quality, participants, intervention, comparison and outcomes. Risk Ratios and 95% Confident Intervals for breast cancer were pooled using a random-effects model. Heterogeneity was assessed using the I² test. In sensitivity analysis, we assessed the impact of vitamin D dosage and mode of administration on treatment effects. Only two randomized controlled trials fulfilled the pre-set inclusion criteria. The pooled analysis included 5372 postmenopausal women. Overall, Risk Ratios and 95% Confident Intervals were 1.11 and 0.74–1.68. We found no evidence of heterogeneity. Neither vitamin D dosage nor mode of administration significantly affected breast cancer risk. However, treatment efficacy was somewhat greater when vitamin D was administered at the highest dosage and in combination with calcium (Risk Ratio 0.58, 95% Confident Interval 0.23–1.47 and Risk Ratio 0.93, 95% Confident Interval 0.54–1.60, respectively). In conclusions, vitamin D use seems not to be associated with a reduced risk of breast cancer development in postmenopausal women. However, the available evidence is still limited and inadequate to draw firm conclusions. Study protocol code: FARM8L2B5L.     

Phylogenetic characterization of bioemulsifier-producing bacteria

Bacteria able to produce biological emulsifiers were isolated from different environments using different isolation media with the aim of discovering the widest diversity. The phylogenetic diversity of the isolates was evaluated by 16S rRNA gene analysis. Among 190 isolated strains, 127 released extracellular emulsifiers able to stabilize oil-water emulsions when grown on low-cost substrates. Among these, the 35 isolates that showed the highest emulsifier production on different substrates were found to belong to 16 different bacterial genera. Overall, this is the first systematic study of the diversity of bioemulsifier-producing bacteria and of their ability to produce bioemulsifiers on low-cost substrates.     

Cullin3 - BTB Interface: A Novel Target for Stapled Peptides

Cullin3 (Cul3), a key factor of protein ubiquitination, is able to interact with dozens of different proteins containing a BTB (Bric-a-brac, Tramtrack and Broad Complex) domain. We here targeted the Cul3–BTB interface by using the intriguing approach of stabilizing the α-helical conformation of Cul3-based peptides through the “stapling” with a hydrocarbon cross-linker. In particular, by combining theoretical and experimental techniques, we designed and characterized stapled Cul3-based peptides embedding the helix 2 of the protein (residues 49–68). Intriguingly, CD and NMR experiments demonstrate that these stapled peptides were able to adopt the helical structure that the fragment assumes in the parent protein. We also show that some of these peptides were able to bind to the BTB of the tetrameric KCTD11, a substrate adaptor involved in HDAC1 degradation, with high affinity (~ 300–600 nM). Cul3-derived staple peptides are also able to bind the BTB of the pentameric KCTD5. Interestingly, the affinity of these peptides is of the same order of magnitude of that reported for the interaction of full-length Cul3 with some BTB containing proteins. Moreover, present data indicate that stapling endows these peptides with an increased serum stability. Altogether, these findings indicate that the designed stapled peptides can efficiently mimic protein-protein interactions and are potentially able to modulate fundamental biological processes involving Cul3.     

Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.