Exploration of the Genomic Diversity and Core Genome of the Bifidobacterium adolescentis Phylogenetic Group by Means of a Polyphasic Approach

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota.     

Shape-Related Toxicity of Titanium Dioxide Nanofibres

Titanium dioxide (TiO₂) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO₂ nanoparticles have been extensively studied, the toxicological characterization of TiO₂ nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO₂ nanofibres using TiO₂ nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO₂ nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO₂ NP did not decrease cell viability. TiO₂ nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO₂ nanoparticles or crocidolite. Moreover, TiO₂ nanofibres and crocidolite, but not TiO₂ nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO₂ nanofibres, as well as by TiO₂ nanoparticles and crocidolite. This study indicates that TiO₂ nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO₂ nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO₂ nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.     

Mammalian prion amyloid formation in bacteria

Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrP^Sc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrP^Sc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.     

A Surface-Induced Asymmetric Program Promotes Tissue Colonization by Pseudomonas aeruginosa

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