Exploring human porphobilinogen synthase metalloprotein by quantum biochemistry and evolutionary methods.

Previous studies have shown the porphobilinogen synthase (PBGS) zinc-binding mechanism and its conservation among the living cells. However, the precise molecular interaction of zinc with the active center of the enzyme is unknown. In particular, quantum chemistry techniques within the density functional theory (DFT) framework have been the key methodology to describe metalloproteins, when one is looking for a compromise between accuracy and computational feasibility. Considering this, we used DFT-based models within the molecular fractionation with conjugate caps scheme to evaluate the binding energy features of zinc interacting with the human PBGS. Besides, phylogenetic and clustering analyses were successfully employed in extracting useful information from protein sequences to identify groups of conserved residues that build the ions-binding site. Our results also report a conservative assessment of the relevant amino acids, as well as the benchmark analysis of the calculation models used. The most relevant intermolecular interactions in Zn2+-PBGS are due to the amino acids CYS0122, CYS0124, CYS0132, ASP0169, SER0168, ARG0221, HIS0131, ASP0120, GLY0133, VAL0121, ARG0209, and ARG0174. Among these residues, we highlighted ASP0120, GLY0133, HIS0131, SER0168, and ARG0209 by co-occurring in all clusters generated by unsupervised clustering analysis. On the other hand, the triple cysteines at 2.5 Å from zinc (CYS0122, CYS0124, and CYS0132) have the highest energy attraction and are absent in the taxa Viridiplantae, Sar, Rhodophyta, and some Bacteria. Additionally, the performance of the DFT-based models shows that the processing time-dependence is more associated with the choice of the basis set than the exchange-correlation functional.

In vitro effect of children's toothpaste on brushing abrasion of eroded primary enamel.

PURPOSE: Emerging data have suggested that acid erosion has become an increasing clinical problem in pediatric dentistry. This study aimed to investigate the abrasive effects of two commercial toothpastes on primary enamel eroded by orange juice using an in vitro model. METHODS: Thirty enamel slabs were obtained from primary teeth and then randomly assigned to three groups (n = 8) comprising two different toothpastes: G1-control-distilled water; G2-Dentifrice A (containing no fluoride); and G3-Dentifrice B (1.100 ppmF-NaF). Each slab had one half protected to provide a control side, and the other one was subjected to treatments. The slabs were submitted to daily erosive challenges (3×/day, 2 min) in concentrated orange juice (pH 3.38) associated with abrasive challenges using a tooth-brushing machine (150 brush movements for each cycle). During the experimental period, the slabs were kept in 37 °C artificial saliva, and the experiment was carried out for 5 days. The depths of the resulting eroded areas were measured by stylus profilometry. The data were analyzed using ANOVA and the Tukey-Kramer test (α = 5%). RESULTS: Tooth loss (µm, mean ± SD) was 2.46 ± 1.18 for G1, 3.32 ± 2.12 for G2 and 2.14 ± 1.03 for G3. Therefore, the NaF dentifrice (Dentifrice B) produced significantly less mineral loss (p = 0.04) than dentifrice A. CONCLUSIONS: The findings suggest that fluoride toothpaste could protect primary enamel against erosion.

Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil.

The genomic sequences of 20 Leishmania infantum isolates collected in northeastern Brazil were compared with each other and with the available genomic sequences of 29 L. infantum/donovani isolates from Nepal and Turkey. The Brazilian isolates were obtained in the early 1990s or since 2009 from patients with visceral or non-ulcerating cutaneous leishmaniasis, asymptomatic humans, or dogs with visceral leishmaniasis. Two isolates were from the blood and bone marrow of the same visceral leishmaniasis patient. All 20 genomic sequences display 99.95% identity with each other and slightly less identity with a reference L. infantum genome from a Spanish isolate. Despite the high identity, analysis of individual differences among the 32 million base pair genomes showed sufficient variation to allow the isolates to be clustered based on the primary sequence. A major source of variation detected was in chromosome somy, with only four of the 36 chromosomes being predominantly disomic in all 49 isolates examined. In contrast, chromosome 31 was predominantly tetrasomic/pentasomic, consistent with its regions of synteny on two different disomic chromosomes of Trypanosoma brucei. In the Brazilian isolates, evidence for recombination was detected in 27 of the 36 chromosomes. Clustering analyses suggested two populations, in which two of the five older isolates from the 1990s clustered with a majority of recent isolates. Overall the analyses do not suggest individual sequence variants account for differences in clinical outcome or adaptation to different hosts. For the first known time, DNA of isolates from asymptomatic subjects were sequenced. Of interest, these displayed lower diversity than isolates from symptomatic subjects, an observation that deserves further investigation with additional isolates from asymptomatic subjects.

Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

In situ effects of restorative materials on dental biofilm and enamel demineralisation.

OBJECTIVES: Since secondary caries is one of the main reasons for replacing restorations, this study assessed the effects of different restorative materials on the microbiological composition of dental biofilm and on enamel demineralisation around the restoration. METHODS: A randomized, double-blind, split-mouth in situ design was conducted in one phase of 14 days, during which, 20 volunteers wore palatal devices containing five human dental enamel slabs. Each slab was randomly restored with one of the following materials: Filtek-Z-250/Single Bond, control group (composite resin), Permite (amalgam), Fuji II (encapsulated resin-modified glass ionomer), Vitremer (resin-modified glass ionomer) and Ketac Molar (conventional glass ionomer). The volunteers used fluoride dentifrice, 3x/day and a 20% sucrose solution was dripped onto the slabs 8x/day. The biofilm formed on the slabs was analyzed to determine the counts of total streptococci, mutans streptococci and lactobacilli. Enamel demineralisation was determined by cross-sectional microhardness (CSMH) at 20 and 70 microm from the margin of the restoration. Kruskal-Wallis and analysis of variance, followed by least mean squares (LMS) test, were used to evaluate microbiota and CSMH among the groups. The significance level used was 5%. RESULTS: No statistically significant differences were found in the cariogenic microbiota grown on the slabs. At a 20-mum distance, only Fuji II statistically differed from the other groups, showing the lowest demineralisation. At 70 microm, Fuji II significantly inhibited demineralisation when compared to Permite, Filtek-Z-250 and Ketac Molar. CONCLUSIONS: In the context of fluoride dentifrice and under the cariogenic exposure conditions of this study, only the encapsulated resin-modified glass ionomer material provided additional protection against secondary caries.