Folding Stability of Pax9 Intronic G-Quadruplex Correlates with Relative Molar Size in Eutherians.

Eutherian dentition has been the focus of a great deal of studies in the areas of evolution, development, and genomics. The development of molar teeth is regulated by an antero-to-posterior cascade mechanism of activators and inhibitors molecules, where the relative sizes of the second (M2) and third (M3) molars are dependent of the inhibitory influence of the first molar (M1). Higher activator/inhibitor ratios will result in higher M2/M1 or M3/M1. Pax9 has been shown to play a key role in tooth development. We have previously shown that a G-quadruplex in the first intron of Pax9 can modulate the splicing efficiency. Using a sliding window approach with we analyzed the association of the folding energy (Mfe) of the Pax9 first intron with the relative molar sizes in 42 mammalian species, representing 9 orders. The Mfe of two regions located in the first intron of Pax9 were shown to be significantly associated with the M2/M1 and M3/M1 areas and mesiodistal lengths. The first region is located at the intron beginning and can fold into a stable G4 structure, whereas the second is downstream the G4 and 265 bp from intron start. Across species, the first intron of Pax9 varied in G-quadruplex structural stability. The correlations were further increased when the Mfe of the two sequences were added. Our results indicate that this region has a role in the evolution of the mammalian dental pattern by influencing the relative size of the molars.

Type 1 diabetes mellitus effects on dental enamel formation revealed by microscopy and microanalysis.

BACKGROUND: Type 1 diabetes mellitus (T1DM) largely affects children, occurring therefore at the same period of deciduous and permanent teeth development. The aim of this work was to investigate birefringence and morphology of the secretory stage enamel organic extracellular matrix (EOECM), and structural and mechanical features of mature enamel from T1DM rats. METHODS: Adult Wistar rats were maintained alive for a period of 56 days after the induction of experimental T1DM with a single dose of streptozotocin (60 mg/kg). After proper euthanasia of the animals, fixed upper incisors were accurately processed, and secretory stage EOECM and mature enamel were analyzed by transmitted polarizing and bright field light microscopies (TPLM and BFLM), energy-dispersive x-ray (EDX) analysis, scanning electron microscopy (SEM), and microhardness testing. RESULTS: Bright field light microscopies and transmitted polarizing light microscopies showed slight morphological changes in the secretory stage EOECM from diabetic rats, which also did not exhibit statistically significant alterations in birefringence brightness when compared to control animals (P > .05). EDX analysis showed that T1DM induced statistically significant little increases in the amount of calcium and phosphorus in outer mature enamel (P < .01) with preservation of calcium/phosphorus ratio in that structure (P > .05). T1DM also caused important ultrastructural alterations in mature enamel as revealed by SEM and induced a statistically significant reduction of about 13.67% in its microhardness at 80 µm from dentin-enamel junction (P < .01). CONCLUSIONS: This study shows that T1DM may disturb enamel development, leading to alterations in mature enamel ultrastructure and in its mechanical features.

Children with a Higher Activity of Carbonic Anhydrase VI in Saliva Are More Likely to Develop Dental Caries.

OBJECTIVE: This study aimed to analyze the concentration and activity of carbonic anhydrase (CA) VI in the saliva of school children. We investigated the relationship among caries, CA VI concentration/activity, flow rate, pH, and buffering capacity. MATERIALS AND METHODS: Seventy-four school children were divided into a caries-free group and a caries group. Clinical examinations were conducted by one examiner according to World Health Organization criteria + early caries lesions. Salivary flow rate, pH, and buffering capacity were analyzed. Salivary CA VI concentration and activity were evaluated by ELISA and zymography, respectively. The data were analyzed using Student's t test and the Mann-Whitney test, and Pearson and Spearman correlation analyses were also done. In multivariate modeling, associations between variables were expressed as odds ratios. RESULTS: The results showed that salivary flow rate, salivary pH, and BC were significantly higher in the saliva of caries-free children. Also, the salivary CA VI concentration was significantly higher in the saliva of caries-free children. The salivary CA VI activity was higher in children with caries. We found a negative correlation between BC and dental caries. Also, in the caries group we found a positive correlation between the concentration and the activity of CA VI and a negative correlation between BC and CA VI activity. A negative correlation between salivary pH and CA VI concentration was observed in the caries-free group. A high activity of CA and a low salivary flow rate were associated with dental caries. CONCLUSION: These results support the conclusion that dental caries is highly affected by the activity of CA VI in saliva as well as by the salivary flow rate.

G-quadruplex formation enhances splicing efficiency of PAX9 intron 1.

G-quadruplexes are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e., interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex-forming region located near exon 1, which is present in all known sequenced placental mammals. Using circular dichroism (CD) analysis and CD melting, we showed that these sequences are able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary, we used a validated double-reporter splicing assay and qPCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically the splicing efficiency of human PAX9 intron 1. The less stable, rat quadruplex had a less efficient splicing when compared to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether, these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1.

Parathyroid hormone (1-34) modulates odontoblast proliferation and apoptosis via PKA and PKC-dependent pathways.

Parathyroid hormone (PTH) plays a key role in the development and homeostasis of mineralized tissues such as bone and dentine. We have reported that PTH (1-34) administration can increase dentine formation in mice and that this hormone modulates in vitro mineralization of odontoblast-like cells. The purpose of the present study was to investigate whether PTH (1-34) participates in the proliferative and apoptotic signaling of odontoblast-like cells (MDPC23). MDPC23 cells were exposed to 50 ng/ml hPTH (1-34) or vehicle for 1 (P1), 24 (P24), or 48 (P48) hours, and the cell proliferation, apoptosis, and cell number were evaluated. To examine whether changes in the proliferative and apoptotic signaling in response to PTH involve protein kinases A (PKA) and/or C (PKC), MDPC23 cells were exposed to PTH with or without PKC or PKA signaling pathway inhibitors. Overall, the results showed that the PKA pathway acts in response to PTH exposure maintaining levels of cell proliferation, while the PKC pathway is mainly involved for longer exposure to PTH (24 or 48 h), leading to the reduction of cell proliferation and increase of apoptosis. The exposure to PTH reduced the cell number in relation to the control group in a time-dependent manner. In conclusion, PTH modulates odontoblast-like cell proliferative and apoptotic response in a time-dependent manner. Both PKC and PKA pathways participate in PTH-induced modulation in an antagonist mode.

Ionizing radiation effects on the secretory-stage ameloblasts and enamel organic extracellular matrix.

This study assessed the effects of high doses of ionizing radiation on eruption rate, odontogenic region morphology, secretory-stage ameloblasts, and enamel organic extracellular matrix (EOECM) of rat maxillary incisors. For the study, 30 male rats were divided into three experimental groups: control (non-irradiated), irradiated by 15 Gy, and irradiated by 25 Gy. Irradiated groups received a single dose of 15 or 25 Gy of X-rays in the head and neck region. The maxillary incisor eruption rate was measured. Sections of 5-µm thickness of the maxillary incisor odontogenic regions were evaluated using bright field light microscopy. Ultrathin sections of secretory ameloblasts and their EOECM were analyzed by transmission electron microscopy (TEM). Irradiated groups showed significantly diminished eruption rate values at the 4th and at the 6th day after irradiation. Reduced optical retardation values were observed in the irradiated groups. The odontogenic region of maxillary incisors from irradiated rats exhibited altered and poorly organized preameloblasts. TEM showed degeneration areas in the secretory-stage EOECM and several autophagosomes in the secretory ameloblasts from irradiated animals. In conclusion, high radiation doses delay eruption and induce disturbances in secretory ameloblasts and EOECM of rat maxillary incisors. These findings may be associated with structural defects of mature enamel.

Porphyromonas gingivalis LPS stimulation downregulates DNMT1, DNMT3a, and JMJD3 gene expression levels in human HaCaT keratinocytes.

OBJECTIVE: The role of epigenetic regulation in inflammatory diseases such as periodontitis is poorly known. The aim of this study was to assess whether Porphyromonas gingivalis lipopolysaccharide (LPS) can modulate gene expression levels of the some enzymes that promote epigenetic events in cultures of the human keratinocytes and gingival fibroblasts. In addition, the same enzymes were evaluated in gingival samples from healthy and periodontitis-affected individuals. MATERIALS AND METHODS: Primary gingival fibroblast and keratinocyte (HaCaT) cultures were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24 h. After this period, cell viability was assessed by MTT test and total RNA extracted to evaluate gene expression levels of the following enzymes by qRT-PCR: DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), histone demethylases Jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX). To evaluate gene expression in healthy and periodontitis-affected individuals, total RNA was extracted from biopsies of gingival tissue from healthy and periodontitis sites, and gene expression of DNMT1, DNAMT3a, JMJD3, and UTX was evaluated by qRT-PCR. RESULTS: No significant differences were found in the gene expression analysis between healthy and periodontitis-affected gingival samples. The results showed that LPS downregulated DNMT1 (p < 0.05), DNMT3a (p < 0.05), and JMJD3 (p < 0.01) gene expression in HaCaT cells, but no modulation was observed in gingival fibroblasts. CONCLUSION: P. gingivalis LPS exposure to human HaCaT keratinocytes downregulates gene expression of the enzymes that promote epigenetic events. CLINICAL RELEVANCE: The advance knowledge about epigenetic modifications caused by periodontopathogens may to possibly led to the development of new periodontal therapies.

Addition of zinc methacrylate in dental polymers: MMP-2 inhibition and ultimate tensile strength evaluation.

This study evaluated the effect of zinc methacrylate (ZM) on the inhibition of matrix metalloproteinase 2 (MMP-2) and the ultimate tensile strength (UTS) of an experimental polymer. Enzymes secreted from mouse gingival tissues were analyzed by gelatin zymography in buffers containing 5 mM CaCl(2) (Tris-CaCl(2)) in 50 mM Tris-HCl buffer with various concentrations of ZM (0.5, 1, 2, 4, 8, and 16 mM). The matrix metalloproteinases present in the conditioned media were characterized by immunoprecipitation. The polymer UTS evaluation was performed in eight groups with various concentrations of ZM (0, 0.5, 1, 2.5, 5, 10, 20, and 30 wt.%), in a mechanical testing machine. MMP-2 (62 kDa) was detected in the zymographic assays and inhibited by ZM in all tested concentrations. UTS data were submitted to one-way ANOVA and Tukey's test (α = 0.05), and no significant differences were observed among groups, except in the polymer containing 30% ZM, presenting a significantly lower value when compared with the control group (p < 0.05). The results suggest that ZM inhibits MMP-2 expression in all concentrations tested, while small concentrations did not affect the ultimate tensile strength of the polymer. Zinc methacrylate is a metalloproteinase inhibitor that can be copolymerized with other methacrylate monomers. Yet, the addition of ZM did not affect the resin bond strength. Thus, in vivo tests should be performed to evaluate the performance of this material.

Structure and Chemical Composition of ca. 10-Million-Year-Old (Late Miocene of Western Amazon) and Present-Day Teeth of Related Species.

Molecular information has been gathered from fossilized dental enamel, the best-preserved tissue of vertebrates. However, the association of morphological features with the possible mineral and organic information of this tissue is still poorly understood in the context of the emerging area of paleoproteomics. This study aims to compare the morphological features and chemical composition of dental enamel of extinct and extant terrestrial vertebrates of Crocodylia: Purussaurus sp. (extinct) and Melanosuchus niger (extant), and Rodentia: Neoepiblema sp. (extinct) and Hydrochoerus hydrochaeris (extant). To obtain structural and chemical data, superficial and internal enamel were analyzed by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (SEM-EDS). Organic, mineral, and water content were obtained using polarizing microscopy and microradiography on ground sections of four teeth, resulting in a higher organic volume than previously expected (up to 49%). It is observed that both modern and fossil tooth enamel exhibit the same major constituents: 36.7% Ca, 17.2% P, and 41% O, characteristic of hydroxyapatite. Additionally, 27 other elements were measured from superficial enamel by inductively coupled mass spectrometry (ICP-MS). Zinc was the most abundant microelement detected, followed by Pb, Fe, Mg, and Al. Morphological features observed include enamel rods in the rodent teeth, while incremental lines and semiprismatic enamel were observed in the alligator species. The fossil enamel was in an excellent state for microscopic analyses. Results show that all major dental enamel's physical, chemical, and morphological features are present both in extant and extinct fossil tooth enamel (>8.5 Ma) in both taxa.

Fluoride effect on the secretory-stage enamel organic extracellular matrix of mice.

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.

TLR2 and TLR4 gene promoter methylation status during chronic periodontitis.

AIM: The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. MATERIAL AND METHODS: Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. RESULTS: The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). CONCLUSION: The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.

Inhibition of the activity of matrix metalloproteinase 2 by triethylene glycol dimethacrylate.

The aim of this study was to evaluate the effect of different concentrations of triethylene glycol dimethacrylate (TEGDMA) on the inhibition of matrix metalloproteinase 2 (MMP-2). Mouse gingival explants were cultured overnight in DMEM and the expression of secreted enzymes was analyzed by gelatin zymography in buffers containing 5 mM CaCl(2) (Tris-CaCl(2)) in 50 mM Tris-HCl buffer with the addition of TEGDMA at different concentrations (0.62%, 1.25%, 2.5%, or 5.0% (v/v)). The gelatinolytic proteinase present in the conditioned media was characterized as matrix metalloproteinase by means of specific chemical inhibition. The matrix metalloproteinases present in the conditioned media were characterized as MMP-2 by immunoprecipitation. The eletrophoretic bands were scanned and the transmittance values were analyzed. Data was plotted and submitted to linear regression to investigate MMP-2 inhibition as a function of TEGDMA concentration. Three major bands were detected in the zymographic assays. These bands were characterized as MMP-2. Zymogene (72 kDa), intermediate (66 kDa) and active forms of MMP-2 (62 kDa) were inhibited by TEGDMA in a dose-dependent way. These findings suggest that TEGDMA could inhibit MMP-2 expression even at small concentrations.

Current use of effect size or confidence interval analyses in clinical and biomedical research.

The isolated use of the statistical hypothesis testing for two group comparison has limitations, and its combination with effect size or confidence interval analysis as complementary statistical tests is recommended. In the present work, we estimate the use of these complementary statistical tests (i.e. effect size or confidence interval) in recently published in research articles in clinical and biomedical areas. Methods: The ProQuest database was used to search published studies in academic journals between 2019 and 2020. The analysis was carried out using terms that represent five areas of clinical and biomedical research: "brain", "liver", "heart", "dental", and "covid-19". A total of 119,558 published articles were retrieved. Results: The relative use of complementary statistical tests in clinical and biomedical publications was low. The highest frequency usage of complementary statistical tests was among articles that also used statistical hypothesis testing for two-sample comparison. Publications with the term "covid-19" showed the lowest usage rate of complementary statistical tests when all article were analyzed but presented the highest rate among articles that used hypothesis testing. Conclusion: The low use of effect size or confidence interval in two-sample comparison suggests that coordinate measures should be taken in order to increase the use of this analysis in clinical and biomedical research. Their use should be emphasized in statistical disciplines for college and graduate students, become a routine procedure in research laboratories, and recommended by reviewers and editors of scientific journals. Supplementary Information: The online version contains supplementary material available at 10.1007/s11192-021-04150-3.

Ancient enamel peptides recovered from the South American Pleistocene species Notiomastodon platensis and Myocastor cf. coypus.

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SIGNIFICANCE: This study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

DNA G-quadruplex stability, position and chromatin accessibility are associated with CpG island methylation.

CpG islands (CGI) are genomic regions associated with gene promoters and involved in gene expression regulation. Despite their high CpG content and unlike bulk genomic DNA methylation pattern, these regions are usually hypomethylated. So far, the mechanisms controlling the CGI methylation patterning remain unclear. G-quadruplex (G4) structures can inhibit DNA methyltransferases 1 enzymatic activity, leading to CGI hypomethylation. Our aim was to analyse the association of G4 forming sequences (G4FS) and CGI methylation as well as to determine the intrinsic and extrinsic characteristics of G4FS that may modulate this phenomenon. Using methylation data from human embryonic stem cells (hESCs) and three hESC-derived populations, we showed that hypomethylated CpGs located inside CGI (CGI/CpG) tend to be associated with highly stable G4FS (Minimum free energy ≤ -30 kcal·mol-1 ). The association of highly stable G4FS and hypomethylation tend to be stronger when these structures are located at shorter distances (~ < 150 bp) from GCI/CpGs, when G4FS and CpGs are located within open chromatin and G4FS are inside CGI. Moreover, this association is not strongly influenced by the CpG content of CGI. Conversely, highly methylated CGI/CpG tend to be associated with low stability G4FS. Although CpGs inside CGIs without a G4FS tend to be more methylated, high stability G4FS within CGI neighbourhood were associated with decreased methylation. In summary, our data indicate that G4FS may act as protective cis elements against CGI methylation, and this effect seems to be influenced by the G4FS folding potential, its presence within CGI, CpG distance from G4FS and chromatin accessibility.

Interactions between superoxide dismutase and paraoxonase polymorphic variants in nonsyndromic cleft lip with or without cleft palate in the Brazilian population.

During development, oxidative stress is hypothesized to mediate embryotoxicity, which may be intensified by exposition to environmental factors and by genetic variations in the enzymes involved in protecting cells from these damaging effects, including superoxide dismutase (SOD) and paraoxonase (PON). The aim of this study was to evaluate the influence of single-nucleotide polymorphisms (SNP) in genes associated with the neutralization of oxidative stress (SOD and PON family members) in the risk of nonsyndromic oral cleft in the Brazilian population. Initially, we tested for association between 28 SNP in SOD1, SOD2, SOD3, PON1, PON2, and PON3 among 325 nonsyndromic cleft lip with or without cleft palate (NSCL±P) case-parent trios. Multiple logistic regression analyses were used to explore gene, GxG and GxE, involving factors that induce oxidative stress accumulation during pregnancy. Signals still significant after both Bonferroni correction and in permutation test were subsequently confirmed in an ancestry-structured case-control analysis with 722 NSCL±P and 866 controls from the same population. In the trio sample, transmission disequilibrium test (TDT) (allele and haplotype) and GxE analysis showed no significant associations, but multiple pairwise GxG interactions involving 10 SNP in PON1, PON2, and PON3 were detected and further examined in the case-control sample. The PON1 rs2237583 and PON2 rs17166879 yielded significant evidence of SNP-SNP interactions after adjustment for multiple tests (both Bonferroni correction and 10,000 permutation test). The C allele and the CT genotype of PON1 rs2237583 were associated with significant protective effects against NSCL±P, while rs3917490 showed a significant association only in the sample composed of patients displaying high African ancestry. Our results reveal associations between rs2237583 and rs3917490 in PON1 and GxG interactions containing rs2237583 and rs17166879 with the susceptibility of NSCL±P in the Brazilian population. Furthermore, this study underlines the recent tendency of taking into account potential GxG interactions to clarify the underlying mechanisms associated with the etiology of this common malformation. Environ. Mol. Mutagen. 60: 185-196, 2019. © 2018 Wiley Periodicals, Inc.

Digital enhancement of dental enamel microstructure images from intact teeth.

Dental enamel is formed by rod-like structures, the enamel prisms. Groups of prisms are packed together in successive horizontal layers of alternating directions, known as Hunter-Schreger bands (HSBs). HSBs are the major microstructural characteristic of mammalian enamel. The pattern of HSBs can vary among mammalian species and this variability may provide relevant information regarding the species life history and taxon identification. In human HSBs can be used as a biometric-based parameter for personal identification in automated systems. The analysis of HSBs has been hampered by technical difficulties. The low contrast between light and dark bands and variations in light intensity may hinder the observation of HSBs in digital images. This article describes a simple and efficient computational procedure that greatly enhances the contrast and minimizes the differences in the intensity of illumination in HSBs images. Its use can significantly increase the quality and the number of HSBs that can be recorded in intact teeth.

Optimizing the analysis of dental enamel microstructure in intact teeth.

In most mammalian species enamel prisms are regularly arranged in layers of alternating directions forming an angle of approximately 90°. These successive layers of prisms are known as Hunter-Schreger bands (HSBs). The analysis of HSBs may provide valuable information regarding the species life history, taxon and personal identification, with evident applicability in physical anthropology and forensics. Obtaining good quality digital images of HSBs in intact specimens is not always a feasible task. The major problems are the low contrast of images; the reflection of incident light, which may create areas of intense shine in digital images; and the abrupt decrease in the degree of illumination that occurs after light crosses the vertical cracks, frequently present in enamel. We show here that the area of intense shine can be minimized by a polarizing filter coupled to the camera objective, and the filling of enamel cracks with corn oil can reduce refraction of light in enamel cracks. These procedures can significantly increase the quality and the area of HSBs that can be recorded in intact teeth.

Bisphosphonates: Pharmacokinetics, bioavailability, mechanisms of action, clinical applications in children, and effects on tooth development.

Bisphosphonates (BPs) avidly bind to calcium crystals and inhibit osteoclastic bone resorption, making them useful for treatment of skeletal disorders such as osteoporosis, Paget's disease, osteogenesis imperfecta and metastatic bone diseases. BPs therapeutically act by causing toxic effects on osteoclasts or interfering with specific intracellular pathways in those cells. BPs that possess nitrogen in their composition are called nitrogen-containing BPs (NBPs) and include alendronate, pamidronate, risedronate, ibandronate, and zoledronate. Simple BPs or non-NBPs do not have nitrogen in their composition, include etiodronate and clodronate, and were the first to be tested in animals and clinically used. Because BPs may be administered to pregnant women or children during deciduous and permanent teeth development, it is expected that they might disturb tooth eruption and development. A review of current literature on pharmacokinetics, bioavailability, mechanisms of action, and clinical applications of BPs in children, and their effects on tooth eruption and development is presented.

High-throughput DNA analysis shows the importance of methylation in the control of immune inflammatory gene transcription in chronic periodontitis.

BACKGROUND: Chronic periodontitis represents a complex disease that is hard to control and is not completely understood. Evidence from past studies suggests that there is a key role for DNA methylation in the pathogenesis of periodontitis. However, all reports have applied technologies that investigate genes in a low throughput. In order to advance in the knowledge of the disease, we analyzed DNA methylation variations associated with gene transcription using a high-throughput assay. Infinium® HumanMethylation450 (Illumina) was performed on gingival samples from 12 periodontitis cases and 11 age-matched healthy individuals. Methylation data of 1,284 immune-related genes and 1,038 cell cycle-related genes from Gene Ontology (GO) and 575 genes from a dataset of stably expressed genes (genes with consistent expression in different physiological states and tissues) were extracted from a microarray dataset and analyzed using bioinformatics tools. DNA methylation variations ranging from -2,000 to +2,000 bp from the transcription start site (TSS) were analyzed, and the results were tested against a differential expression microarray dataset between healthy and periodontitis gingival tissues. Differences were evaluated using tests from the R Statistical Project. RESULTS: The comparison of probes between periodontitis and normal gingival tissues showed that the mean methylation scores and the frequency of methylated probes were significantly lower in genes related to the immune process. In the immune group, these parameters were negatively correlated with gene expression (Mann-Whitney test, p < 2.2e - 16). CONCLUSIONS: Our results show that variations in DNA methylation between healthy and periodontitis cases are higher in genes related to the immune-inflammatory process. Thus, DNA methylation must be modulating chromatin regions and, consequently, modulating the mRNA transcription of immune-inflammatory genes related with periodontitis, impacting the prognosis of disease.