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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.

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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.

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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.

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Proteins can evolve by accumulating changes on amino acid sequences. These changes are mainly caused by missense mutations on its DNA coding sequences. Mutations with neutral or positive effects on fitness can be maintained while deleterious mutations tend to be eliminated by natural selection. Amino acid changes are influenced by the biophysical, chemical, and biological properties of amino acids. There is a multiplicity of amino acid properties that can influence the function and expression of proteins. Amino acid properties can be expressed into numerical indexes, which can help to predict functional and structural aspects of proteins and allow statistical inferences of selection pressure on amino acid usage. The accuracy of these analyses may be compromised by the existence of several numerical indexes that measure the same amino acid property, and the lack of objective parameters to determine the most accurate and biologically relevant index. In the present study, the gradient consistency test was used in order to estimate the magnitude of directional selection imparted by amino acid biochemical and biophysical properties on protein evolution.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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OBJECTIVE: To analyze macroscopic, microscopic, and ultrastructural aspects of enamel from head-and-neck cancer patients submitted to radiotherapy. MATERIALS AND METHODS: Twenty sound extracted permanent molars were used and divided into 2 groups. The experimental group consisted of 10 molars from head-and-neck cancer patients submitted to radiotherapy with total doses that ranged from 50 to 70 Gy. Ten molars from patients who did not receive radiotherapy were matched with experimental-group samples by anatomic tooth group and comprised the control group. To perform a macroscopic analysis, standardized photos of different enamel faces were taken with a camera. Teeth were subjected to longitudinal cuts and hand polished to a final thickness of 0.1 mm. Enamel was analyzed under polarized light microscopy, and optical retardation values of birefringence were calculated in cervical, cusp, and occlusal pit areas. Subsequently, the same enamel areas were analyzed by scanning electron microscopy. Data from optical retardation values were statistically analyzed by 2-way ANOVA and Fisher's test (α < 0.05). RESULTS: No macroscopic differences were observed between the irradiated and control groups. Polarized light microscopy analysis revealed that cervical enamel exhibited darker areas characterized by discrete birefringence patterns compared to the control enamel. Optical retardation values were only significantly different in the cervical enamel of the irradiated and control groups (p < 0.0001). Scanning electron microscopy analysis revealed more evident interprismatic spaces in the cervical and outer cusp enamel of irradiated samples. CONCLUSIONS: Head-and-neck radiotherapy reduced optical retardation values of birefringence in cervical enamel, and the interprismatic spaces became more evident.

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The aim of the present work was to investigate birefringence and morphology of the secretory-stage enamel organic extracellular matrix (EOECM), and structural and mechanical properties of mature enamel of upper incisors from adult rats that had been treated with pamidronate disodium (0.5 mg/kg/week for 56 days), using transmitted polarizing and bright-field light microscopies (TPLM and BFLM), energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM) and microhardness testing. BFLM showed no morphological changes of the EOECM in pamidronate and control groups, but TPLM revealed a statistically significant reduction in optical retardation values of birefringence brightness of pamidronate-treated rats when compared with control animals (p0.05). The present study indicates that pamidronate can affect birefringence of the secretory-stage EOECM, which does not seem to be associated with significant changes in morphological and/or mechanical properties of mature enamel.

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Chronic periodontitis (CP) is a chronic inflammatory disease independently associated with higher incidence of oral cavity squamous cell carcinoma (OSCC). However, the molecular mechanism responsible for this increased incidence is unknown. Here we profiled the DNA methylome of CP patients and healthy controls and compared to a large set of OSCC samples from TCGA. We observed a significant overlap between the altered DNA methylation patterns in CP and in OSCC, suggesting an emergence of a pre-neoplastic epigenome in CP. Remarkably, the hypermethylated CpGs in CP were significantly enriched for enhancer elements. This aberrant enhancer methylation is functional and able to disrupt enhancer activity by preventing the binding of chromatin looping factors. This study provides new insights on the molecular mechanisms linking chronic inflammation and tumor predisposition, highlighting the role of epigenetic disruption of transcriptional enhancers.

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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.

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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.

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Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.

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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.

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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.

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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.

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