Campylobacter fetus subspecies: Comparative genomics and prediction of potential virulence targets

The genus Campylobacter contains pathogens causing a wide range of diseases, targeting both humans and animals. Among them, the Campylobacter fetus subspecies fetus and venerealis deserve special attention, as they are the etiological agents of human bacterial gastroenteritis and bovine genital campylobacteriosis, respectively. We compare the whole genomes of both subspecies to get insights into genomic architecture, phylogenetic relationships, genome conservation and core virulence factors. Pan-genomic approach was applied to identify the core- and pan-genome for both C. fetus subspecies and members of the genus. The C. fetus subspecies conserved (76%) proteome were then analyzed for their subcellular localization and protein functions in biological processes. Furthermore, with pathogenomic strategies, unique candidate regions in the genomes and several potential core-virulence factors were identified. The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes in an island specific for C. fetus subsp. venerealis. The genomic variations and potential core and unique virulence factors characterized in this study would lead to better insight into the species virulence and to more efficient use of the candidates for antibiotic, drug and vaccine development.     

Relationships between pasture legumes, rhizobacteria and nodule bacteria in heavy metal polluted mine waste of SW Sardinia

Local populations of the pasture legumes Astragalus hamosus, Lotus edulis, Lotus ornithopodioides, Medicago ciliaris and Scorpiurus muricatus from heavy metal polluted and unpolluted sites in Sardinia were compared for tolerance to Zn, Cd and Pb in hydroponics. Tolerance of plants to heavy metals varied significantly depending on the species, origin of the population and metal. The species L. edulis, L. ornithopodioides and M. ciliaris possessed higher metal tolerance and were used in a pot experiment with Zn, Cd and Pb polluted mine waste. Seeds were inoculated with the metal tolerant plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2 or/and with the corresponding symbiotic nodule bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate deaminase. Co-inoculation with the bacteria had synergistic and additive effects on nodule number, root growth and uptake of elements (N, P, Ca, Mg, Na, Mn, Zn and Pb) in shoots of L. edulis and L. ornithopodioides. Shoot biomass and uptake of K, Fe and Cd was increased by a combined inoculation of L. edulis. The ratio between shoot and root contents of Pb in L. ornithopodioides was above 1, suggesting a characteristic trait of hyperaccumulating species. The results suggest that the development of metal tolerant and efficient plant-bacteria systems might be useful for phytostabilization and revegetation of mine wastes.     

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.     

Direct observation of the interconversion of normal and toxic forms of α-synuclein

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.     

Transient expression and activity of human DNA polymerase iota in loach embryos

Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Experimental simulation and SPR validation

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b₅ (b₅) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α‐fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro‐PMP provides enough prey proteins for LC‐MS/MS identification and SPR validation, so this approach can be used for discovery of new protein–protein interactions in the framework of Human Proteome Project.     

Reevaluation of several taxa of Chinese lagomorphs (Mammalia: Lagomorpha) described on the basis of pelage phenotype variation

Melanism is a common phenomenon in the animal kingdom. While the occurrence of melanism in lagomorphs has been less studied, this phenomenon has led to systematic confusion among different forms of pikas (Ochotona) and hares (Lepus). Within Ochotona, the Pianma black pika (O. nigritia) was established primarily based on its pelage phenotype variation compared with the sympatric Forrest's pika (O. forresti). The Gaoligong pika (O. gaoligongensis) was also described based on pelage color differences with O. forresti. However, the specific status of these two taxa has been questioned for several years. We conducted a detailed examination of the cranial characters used for the initial diagnosis of each of these forms (O. forresti, O. gaoligongensis and O. nigritia). Insignificant differences were displayed among these species, thus contradicting the classification of O. gaoligongensis and O. nigritia as separate species. Within Lepus, the Manchurian black hare (Lepus melainus) was also initially described based on the melanistic pelage possessed by this form. We conducted a detailed examination of the cranial characteristics of L. melainus and the sympatric Manchurian hare (L. mandshuricus) and confirmed the synonomy of these forms; L. melainus represents melanistic individuals of L. mandshuricus. These taxonomic mistakes probably resulted from underestimating the prevalence of melanism and the potential for adaptative variation of pelage coloration in nature. A more detailed study on the genetic background leading to the occurrence of melanistic lagomorphs is needed.     

Cobalt bis(dicarbollide) versus closo-dodecaborate in boronated chlorin e(6) conjugates: implications for photodynamic and boron-neutron capture therapy

Conjugation of boron nanoparticles with porphyrins is an attractive way to create dual agents for anticancer boron neutron capture therapy (BNCT) and photodynamic therapy (PDT). Properties of chlorin e(6) conjugated with two cobalt bis(dicarbollide) nanoparticles (1) or with a closo-dodecaborate nanoparticle (2) are reported. Fluorescent dianionic conjugates 1 and 2 penetrate in A549 human lung adenocarcinoma cells, stain cytoplasm diffusely and accumulate highly in lysosomes but are not toxic themselves for cells. Average cytoplasmic concentration of boron atoms (B) achieves 270 μM (ca. 2 × 10(8) B/cell) and 27 μM (ca. 2 × 10(7) B/cell) at the 1.5 μM extracellular concentration of 1 and 2, respectively, that makes conjugate 1 especially suitable for BNCT. Conjugate 2 causes photoinduced cell death at micromolar concentrations and can be considered also as a photosensitizer for PDT. Conjugates 1 and 2 have high quantum yields of singlet oxygen generation (0.55 and 0.85 in solution, respectively), identical intracellular localization and similar lipid-like microenvironment but conjugate 1 possesses no photoinduced cytotoxicity. A presence of cobalt complexes in conjugate 1 is supposed to be a reason of the observed antioxidative effect in cellular environment, but an exact mechanism of this intriguing phenomenon is unclear. Due to increased intracellular accumulation and absence of photoinduced cytotoxicity conjugate 1 is promising for fluorescence diagnostics of cancer.     

Differential targeting of androgen and glucocorticoid receptors induces ER stress and apoptosis in prostate cancer cells: A novel therapeutic modality

Androgen (AR) and glucocorticoid (GR) receptor signaling play opposing roles in prostate tumorigenesis: in prostate, AR acts as an oncogene, and GR is a tumor suppressor. Recently, we found that non-steroidal phyto-chemical compound A (CpdA) is AR/GR modulator acting as anti-inflammatory anti-androgen. CpdA inhibits AR and prevents GR transactivation while enhancing GR transrepression. GR and AR are controlled by proteasomal degradation. We found that prolonged exposure of LNCaP, LNCaP-GR, DU145 and PC3 prostate carcinoma (PCa) cells to proteasome inhibitor Bortezomib (BZ) caused AR degradation and GR accumulation. BZ enhanced CpdA ability to inhibit AR and to augment GR transrepression. We also found that CpdA+BZ differentially regulated GR/AR to cooperatively suppress PCa cell growth and survival and to induce endoplasmic reticulum stress (ERS). Importantly, CpdA+BZ differentially regulated GR-responsive genes. CpdA+BZ blocked activation of glucocorticoid-responsive pro-survival genes, including SGK1, but activated BZ-induced ERS-related genes BIP/HSPA5 and CHOP/GADD153. Using ChIP, we showed that SGK1, BIP/HSPA5 and CHOP regulation was due to effects of CpdA and CpdA+BZ on GR loading on their promoters. We also found that AR and GR are abundant in advanced PCa from patients treated by androgen ablation and/or chemotherapy: 56% of carcinomas from treated patients expressed both receptors, and the other 27% expressed either GR or AR. Overall, our data validate the concept of dual AR/GR targeting in prostate cancer (PC) and suggest that BZ combination with dual-target steroid receptor modulator CpdA has high potential for PC therapy.Key words: prostate cancer, proteasome inhibitor, non-steroidal modulator, apoptosis, ER stress     

Exploring massive, genome scale datasets with the GenometriCorr package

We have created a statistically grounded tool for determining the correlation of genomewide data with other datasets or known biological features, intended to guide biological exploration of high-dimensional datasets, rather than providing immediate answers. The software enables several biologically motivated approaches to these data and here we describe the rationale and implementation for each approach. Our models and statistics are implemented in an R package that efficiently calculates the spatial correlation between two sets of genomic intervals (data and/or annotated features), for use as a metric of functional interaction. The software handles any type of pointwise or interval data and instead of running analyses with predefined metrics, it computes the significance and direction of several types of spatial association; this is intended to suggest potentially relevant relationships between the datasets. AVAILABILITY AND IMPLEMENTATION: The package, GenometriCorr, can be freely downloaded at http://genometricorr.sourceforge.net/. Installation guidelines and examples are available from the sourceforge repository. The package is pending submission to Bioconductor.