Development and evaluation of reparative tricalcium silicate-ZrO2 -Biosilicate composites.

Biosilicate is a bioactive glass-ceramic used in medical and dental applications. This study evaluated novel reparative materials composed of pure tricalcium silicate (TCS), 30% zirconium oxide (ZrO2 ) and 10 or 20% biosilicate, in comparison with Biodentine. Setting time was evaluated based on ISO 6876 standard, radiopacity by radiographic analysis, solubility by mass loss, and pH by using a pH meter. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and NR assays. Alkaline phosphatase (ALP) activity and alizarin red were used to evaluate cell bioactivity. Antimicrobial activity was assessed on Enterococcus faecalis by the direct contact test. The data were submitted to analysis of variance (ANOVA)/Tukey; Bonferroni and Kruskal-Wallis, and Dunn tests (α = 0.05). The association of Biosilicate with TCS + ZrO2 had appropriate setting time, radiopacity, and solubility, alkaline pH, and antimicrobial activity. TCS and Biodentine showed higher ALP activity in 14 days than the control (serum-free medium). All cements produced mineralized nodules. In conclusion, Biosilicate + TCS ZrO2 decreased the setting time and increased the radiopacity in comparison to TCS. Biosilicate + TCS ZrO2 presented lower solubility and higher radiopacity than Biodentine. In addition, these experimental cements promoted antimicrobial activity and mineralization nodules formation, suggesting their potential for clinical use.

Cytotoxicity, genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles and farnesol as irrigating solutions.

OBJECTIVE: To evaluate the cytotoxicity, genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles (AgNPs-PVA) and farnesol (FAR). DESIGN: The cytotoxicity (% of cell viability) was evaluated by MTT assay and the genotoxicity (% of DNA in the tail) was evaluated by Comet assay. Root canal disinfection with different irrigating protocols was evaluated ex vivo in human teeth contaminated with Enterococcus faecalis for 21days. Three microbiological samples were collected: initial (after contamination); post-irrigation (after irrigation); and final (after 7days). After each sample, the number of log 10 CFU mL-1 was determined. Statistical analyses was performed using two-way ANOVA and Bonferroni post-hoc tests for MTT assay, Kruskal-Wallis and Dunn post-hoc tests for Cometa and antibacterial assays (α=0.05). RESULTS: The MTT assay showed that AgNPs and FAR were less cytotoxic that sodium hypochlorite (NaOCl) and showed a lower% of DNA in the tail, in comparison with H2O2 (positive control - C+). In the post-irrigation microbiological sample, all the irrigating protocols were more effective than C+ (without irrigation). NaOCl/saline, NaOCl/saline/AgNPs-PVA and NaOCl/saline/FAR led to complete bacterial elimination (p >0.05). In comparison with the initial sample, both the post-irrigation and the final samples showed microbial reduction (p < 0.05). CONCLUSIONS: AgNPs-PVA and FAR showed low cytotoxicity and genotoxicity, and exhibit potential for use as a final endodontic irrigation protocols.

Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum.

Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies.

Characterization of the ubiquitylating components of the human malaria parasite's protein degradation pathway.

Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.