Biological Responses of Human Gingival Fibroblasts (HGFs) in an Innovative Co-Culture Model with Streptococcus mitis to Thermosets Coated with a Silver Polysaccharide Antimicrobial System

This study sought to evaluate the in vitro biological response of human gingival fibroblasts (HGFs) co-coltured with Streptococcus mitis to bisphenol A glycidylmethacrylate/triethylene glycol dimethacrylate (BisGMA/TEGDMA) thermosets coated with Chitlac-nAg, a nanocomposite system with antimicrobial properties. To avoid bacterial adhesion to dental devices and to reduce cytotoxicity against eukaryotic cells, we coated BisGMA/TEGDMA methacrylic thermosets with a new material, Chitlac-nAg, formed by stabilizing silver nanoparticles, which have well-known antimicrobial properties, with a polyelectrolyte solution containing Chitlac. Cytotoxicity, cell morphology, cell migration and inflammatory interleukine-6 (IL-6) and prostaglandin E₂ (PGE₂) secretion were evaluated. Our results showed that the cytotoxicity exerted on HGFs by our nanocomposite material was absent in our co-culture model, where fibroblasts are able to adhere and migrate. After 24 h thermosets coated with Chitlac as well as those coated with Chitlac-nAg exerted a minimal cytotoxic effect on HGFs, while after 48 h LDH release rises up 20%. Moreover the presence of S. mitis reduced this release in a greater amount with Chitlac-nAg coated thermosets. The secretion of IL-6 was significant in both Chitlac and Chitlac-nAg coated thermosets, but PGE₂ production was minimal, suggesting that the IL-6 production was not related to an inflammatory response. Co-culture and the addiction of saliva did not influence IL-6 and PGE₂ secretion. Data obtained in the present work suggest that Chitlac n-Ag coated thermosets could significantly improve the success rates of restorative dentistry, since they limit bacterial adhesion and are not toxic to HGFs.     

The universal ancestor lived in a thermophilic or hyperthermophilic environment

Galtier et al. (Science 1999, 283, 220-221) exploit the correlation between the optimal growth temperature in prokaryotes and the G+C content of rRNAs and establish that the last universal common ancestor (LUCA) lived in a mesophilic environment. This result was achieved by estimating the G+C content of the ancestral sequences of the rRNAs of the LUCA through use of a complex Markov model. I have re-analysed their alignments of the rDNAs with maximum parsimony and I have found that their result is not robust and is, in all likelihood, incorrect. In particular, the rRNA ancestral sequences reconstructed with maximum parsimony from these rDNA alignments as well as those reconstructed after eliminating all the sites that turn out to be ambiguous to the parsimony algorithm and to a site-by-site inspection of these alignments, are such as to suggest that the LUCA lived in a thermophilic or hyperthermophilic environment. This finding is also supported by some tRNA ancestral sequences. The main conclusion of this analysis is that if the LUCA was a progenote then the origin of life might have taken place at a high temperature.     

Revised Systematics of Holospora-Like Bacteria and Characterization of “Candidatus Gortzia infectiva”, a Novel Macronuclear Symbiont of Paramecium jenningsi

The genus Holospora (Rickettsiales) includes highly infectious nuclear symbionts of the ciliate Paramecium with unique morphology and life cycle. To date, nine species have been described, but a molecular characterization is lacking for most of them. In this study, we have characterized a novel Holospora-like bacterium (HLB) living in the macronuclei of a Paramecium jenningsi population. This bacterium was morphologically and ultrastructurally investigated in detail, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and developed a specific probe for fluorescence in situ hybridization experiments. A new taxon, “Candidatus Gortzia infectiva”, was established for this HLB according to its unique characteristics and the relatively low DNA sequence similarities shared with other bacteria. The phylogeny of the order Rickettsiales based on 16S rRNA gene sequences has been inferred, adding to the available data the sequence of the novel bacterium and those of two Holospora species (Holospora obtusa and Holospora undulata) characterized for the purpose. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and showed a possible pattern of evolution for some of their features. We suggested to classify inside the family Holosporaceae only HLBs, excluding other more distantly related and phenotypically different Paramecium endosymbionts.     

Anti-angiogenic therapy induces integrin-linked kinase 1 up-regulation in a mouse model of glioblastoma

Background

In order to improve our understanding of the molecular pathways that mediate tumor proliferation and angiogenesis, and to evaluate the biological response to anti-angiogenic therapy, we analyzed the changes in the protein profile of glioblastoma in response to treatment with recombinant human Platelet Factor 4-DLR mutated protein (PF4-DLR), an inhibitor of angiogenesis.

Methodology/Principal Findings

U87-derived experimental glioblastomas were grown in the brain of xenografted nude mice, treated with PF4-DLR, and processed for proteomic analysis. More than fifty proteins were differentially expressed in response to PF4-DLR treatment. Among them, integrin-linked kinase 1 (ILK1) signaling pathway was first down-regulated but then up-regulated after treatment for prolonged period. The activity of PF4-DLR can be increased by simultaneously treating mice orthotopically implanted with glioblastomas, with ILK1-specific siRNA. As ILK1 is related to malignant progression and a poor prognosis in various types of tumors, we measured ILK1 expression in human glioblatomas, astrocytomas and oligodendrogliomas, and found that it varied widely; however, a high level of ILK1 expression was correlated to a poor prognosis.

Conclusions/Significance

Our results suggest that identifying the molecular pathways induced by anti-angiogenic therapies may help the development of combinaatorial treatment strategies that increase the therapeutic efficacy of angiogenesis inhibitors by association with specific agents that disrupt signaling in tumor cells.     

Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates

The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10⁻⁸ per base per generation and a rate of 1.26 × 10⁻⁹ for <20 bp indels. By quantifying how estimates varied as a function of allele frequency, we inferred the probability that a site is involved in non-crossover gene conversion as 5.99 × 10⁻⁶. We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction.     

In Vitro and In Vivo Antitumor Activity of [Pt(O,O′-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O′-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC–siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.     

Interspecific analysis of the glycosidases of the sperm plasma membrane in Drosophila

We have studied the presence of four sperm glycosidases, alpha-mannosidase, alpha-L- fucosidase and two beta-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and beta-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha-L-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and beta-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross-species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of beta-hexosaminases and alpha-L-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface.     

Effects of tunable excitation in carotenoids explained by the vibrational energy relaxation approach

Carotenoids are fundamental building blocks of natural light harvesters with convoluted and ultrafast energy deactivation networks. In order to disentangle such complex relaxation dynamics, several studies focused on transient absorption measurements and their dependence on the pump wavelength. However, such findings are inconclusive and sometimes contradictory. In this study, we compare internal conversion dynamics in \(\beta\)-carotene, pumped at the first, second, and third vibronic progression peak. Instead of employing data fitting algorithms based on global analysis of the transient absorption spectra, we apply a fully quantum mechanical model to treat the high-frequency symmetric carbon–carbon (C=C and C–C) stretching modes explicitly. This model successfully describes observed population dynamics as well as spectral line shapes in their time-dependence and allows us to reach two conclusions: Firstly, the broadening of the induced absorption upon excess excitation is an effect of vibrational cooling in the first excited state (\(S_{1}\)). Secondly, the internal conversion rate between the second excited state (\(S_{2}\)) and \(S_{1}\) crucially depends on the relative curve displacement. The latter point serves as a new perspective on solvent- and excitation wavelength-dependent experiments and lifts contradictions between several studies found in literature.