RESUMO
Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of ß-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the CaCa distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.
Assuntos
Proteínas do Esmalte Dentário , Nanotubos de Carbono , Amelogenina/química , Proteínas Amiloidogênicas , Sítios de Ligação , Fosfatos de CálcioRESUMO
Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.
Assuntos
Restos Mortais , Impressões Digitais de DNA , Humanos , Marcadores Genéticos , Impressões Digitais de DNA/métodos , Amelogenina/genética , Alelos , Degradação Necrótica do DNA , Repetições de Microssatélites , Elementos Nucleotídeos Curtos e Dispersos , Reação em Cadeia da Polimerase , MasculinoRESUMO
Massively parallel sequencing allows for integrated genotyping of different types of forensic markers, which reduces DNA consumption, simplifies experimental processes, and provides additional sequence-based genetic information. The STRseqTyper122 kit genotypes 63 autosomal STRs, 16 X-STRs, 42 Y-STRs, and the Amelogenin locus. Amplicon sizes of 117 loci were below 300 bp. In this study, MiSeq FGx sequencing metrics for STRseqTyper122 were presented. The genotyping accuracy of this kit was examined by comparing to certified genotypes of NIST standard reference materials and results from five capillary electrophoresis-based kits. The sensitivity of STRseqTyper122 reached 125 pg, and > 80% of the loci were correctly called with 62.5 pg and 31.25 pg input genomic DNA. Repeatability, species specificity, and tolerance for DNA degradation and PCR inhibitors of this kit were also evaluated. STRseqTyper122 demonstrated reliable performance with routine case-work samples and provided a powerful tool for forensic applications.
Assuntos
Impressões Digitais de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Humanos , Impressões Digitais de DNA/métodos , Amelogenina/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Genótipo , Reação em Cadeia da Polimerase , Especificidade da Espécie , Masculino , Animais , Degradação Necrótica do DNA , Eletroforese Capilar , FemininoRESUMO
OBJECTIVES: In order to evaluate the effect of methacrylated hyaluronic acid (HAMA) hydrogels containing the recombinant human amelogenin (rhAm) in vitro and in vivo. BACKGROUND: The ultimate goal in treating periodontal disease is to control inflammation and achieve regeneration of periodontal tissues. In recent years, methacrylated hyaluronic acid (HAMA) containing recombinant human amyloid protein (rhAm) has been widely used as a new type of biomaterial in tissue engineering and regenerative medicine. However, there is a lack of comprehensive research on the periodontal regeneration effects of this hydrogel. This experiment aims to explore the application of photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel for periodontal tissue regeneration and provide valuable insights into its potential use in this field. MATERIALS AND METHODS: The effects of rhAm-HAMA hydrogel on the proliferation of human periodontal ligament cells (hPDLCs) were assessed using the CCK-8 kit. The osteogenic differentiation of hPDLCs was evaluated through ALP staining and real-time PCR. Calvarial parietal defects were created in 4-week-old Sprague Dawley rats and implanted with deproteinized bovine bone matrix in different treatment groups. The animals were euthanized after 4 and 8 weeks of healing. The bone volume of the defect was observed by micro-CT and histological analysis. RESULTS: Stimulating hPDLCs with rhAm-HAMA hydrogel did not significantly affect their proliferation (p > .05). ALP staining and real-time PCR results demonstrated that the rhAm-HAMA group exhibited a significant upregulation of osteoclastic gene expression (p < .05). Micro-CT results revealed a significant increase in mineralized tissue volume fraction (MTV/TV%), trabecular bone number (Tb.N), and mineralized tissue density (MTD) of the bone defect area in the rhAm-HAMA group compared to the other groups (p < .05). The results of hematoxylin and eosin staining and Masson staining at 8 weeks post-surgery further supported the results of the micro-CT. CONCLUSIONS: The results of this study indicate that rhAm-HAMA hydrogel could effectively promote the osteogenic differentiation of hPDLCs and stabilize bone substitutes in the defects that enhance the bone regeneration in vivo.
Assuntos
Amelogenina , Regeneração Óssea , Diferenciação Celular , Proliferação de Células , Ácido Hialurônico , Hidrogéis , Ligamento Periodontal , Ratos Sprague-Dawley , Ácido Hialurônico/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Amelogenina/farmacologia , Amelogenina/uso terapêutico , Humanos , Ligamento Periodontal/efeitos dos fármacos , Ratos , Proliferação de Células/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Osteogênese/efeitos dos fármacos , Masculino , Microtomografia por Raio-X , Células Cultivadas , Metacrilatos , Materiais Biocompatíveis/farmacologiaRESUMO
AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.
Assuntos
Amelogênese Imperfeita , Amelogenina , Estudos de Associação Genética , Mutação , Amelogênese Imperfeita/genética , Amelogênese Imperfeita/patologia , Humanos , Amelogenina/genética , Masculino , Feminino , Linhagem , Fenótipo , Criança , Estresse do Retículo Endoplasmático/genética , Genótipo , Sequenciamento do ExomaRESUMO
BACKGROUND: Several methods were introduced for enamel biomimetic remineralization that utilize a biomimetic analogue to interact and absorb bioavailable calcium and phosphate ions and induce crystal nucleation on demineralized enamel. Amelogenin is the most predominant enamel matrix protein that is involved in enamel biomineralization. It plays a major role in developing the enamel's hierarchical microstructure. Therefore, this study was conducted to evaluate the ability of an amelogenin-inspired peptide to promote the remineralization potential of fluoride and a supersaturated calcium phosphate solution in treating artificially induced enamel carious lesions under pH-cycling regimen. METHODS: Fifty enamel slices were prepared with a window (4*4 mm2 ) on the surface. Five samples were set as control healthy enamel and 45 samples were subjected to demineralization for 3 days. Another 5 samples were set as control demineralized enamel and 40 enamel samples were assigned into 8 experimental groups (n=5) (P/I, P/II, P/III, P/AS, NP/I, NP/II, NP/III and NP/AS) according to peptide treatment (peptide P or non-peptide NP) and remineralizing solution used (I; calcium phosphate solution, II; calcium phosphate fluoride solution, III; fluoride solution and AS; artificial saliva). Samples were then subjected to demineralization/remineralization cycles for 9 days. Samples in all experimental groups were evaluated using Raman spectroscopy for mineral content recovery percentage, microhardness and nanoindentation as healthy, demineralized enamel and after pH-cycling. Data were statistically analysed using two-way repeated measures Anova followed by Bonferroni-corrected post hoc test for pairwise multiple comparisons between groups. Statistical significance was set at p= 0.05. Additionally, XRD, FESEM and EDXS were used for crystal orientation, surface morphology and elemental analysis after pH-cycling. RESULTS: Nanocrystals clumped in a directional manner were detected in peptide-treated groups. P/II showed the highest significant mean values in mineral content recovery (63.31%), microhardness (268.81±6.52 VHN), elastic modulus (88.74±2.71 GPa), nanohardness (3.08±0.59 GPa) and the best crystal orientation with I002/I300 (1.87±0.08). CONCLUSION: Despite pH changes, the tested peptide was capable of remineralizing enamel with ordered crystals. Moreover, the supplementary use of calcium phosphate fluoride solution with peptide granted an enhancement in enamel mechanical properties after remineralization.
Assuntos
Cárie Dentária , Fluoretos , Humanos , Fluoretos/farmacologia , Amelogenina/farmacologia , Amelogenina/uso terapêutico , Cariostáticos/farmacologia , Cariostáticos/uso terapêutico , Biomimética , Fosfatos de Cálcio/farmacologia , Fosfatos de Cálcio/uso terapêutico , Minerais , Fosfatos , Remineralização Dentária/métodos , Concentração de Íons de HidrogênioRESUMO
With the increasing number of complex forensic cases in recent years, it's more important to combine the different types of genetic markers such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (InDels), and microhaplotypes (MHs) to provide more genetic information. In this study, we selected totally 201 genetic markers, including 24 autosomes STRs (A-STRs), 24 Y chromosome STRs (Y-STRs), 110 A-SNPs, 24 Y-SNPs, 9 A-InDels, 1 Y-InDel, 8 MHs, and Amelogenin to establish the HID_AM Panel v1.0, a Next-Generation Sequencing (NGS) detection system. According to the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the repeatability, accuracy, sensitivity, suitability for degraded samples, species specificity, and inhibitor resistance of this system were assessed. The typing results on 48 STRs and Amelogenin of this system were completely consistent with those obtained using capillary electrophoresis. This system accurately detected 79 SNPs as parallelly confirmed by a FGx sequencer with the ForenSeq™ DNA Signature Prep Kit. Complete allele typing results could be obtained with a DNA input of no less than 200 pg. The detection success rate of this system was significantly higher than that of the GlobalFiler™ kit when the degradation index of mock degraded sample was greater than 15.87. When the concentration of hematin in the amplification system was ≤40 µmol/L, indigo blue was ≤2 mmol/L, or humic acid was ≤15 ng/µL, amplification was not significantly inhibited. The system barely amplified the DNA extract from duck, mouse, cow, rabbit, and chick. The detection rate of STRs on routine samples of this panel is 99.74%, while all the SNPs, InDels, and MHs were successfully detected. In summary, we setup a NGS individual typing panel including 201 genetic markers with the high accuracy, sensitivity, species specificity, and inhibitors resistance, which is applicable for individual identification of degraded samples.
Assuntos
Impressões Digitais de DNA , Polimorfismo de Nucleotídeo Único , Feminino , Bovinos , Animais , Camundongos , Coelhos , Impressões Digitais de DNA/métodos , Marcadores Genéticos , Amelogenina/genética , Genótipo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Repetições de Microssatélites , DNA , Análise de Sequência de DNA/métodosRESUMO
The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.
Assuntos
Amelogênese , Regulação da Expressão Gênica , Proteína HMGN2 , Proteínas de Homeodomínio , Fator 1 de Ligação ao Facilitador Linfoide , Fatores de Transcrição , Transcrição Gênica , Amelogênese/genética , Amelogenina/genética , Animais , Cromatina/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2RESUMO
Amel, the gene encoding the amelogenin protein involved in enamel formation, is highly alternatively spliced. When exon4 is excised, it can form a mature miRNA (miR-exon4) that has previously been suggested to indirectly regulate expression of the Runt-related transcription factor 2 (Runx2) involved in bone development in ameloblasts and osteoblasts. However, the precise mechanism of this regulation is unclear. In this study, we aimed to identify direct targets of miR-exon4. The transcription factor family nuclear factor I/A (NFI/A) is known to negatively regulate expression of Runx2 and is among the most highly predicted direct targets of miR-exon4 that link to Runx2. Immunostaining detected NFI/A in osteoblasts and ameloblasts in vivo, and reporter assays confirmed direct interaction of the Nfia 3'-UTR and miR-exon4. In addition, silencing of Nfia in MC3T3-E1-M14 osteoblasts resulted in subsequent downregulation of Runx2. In a monoclonal subclone (mi2) of MC3T3-E1 cells wherein mature miR-exon4 was functionally inhibited, we observed significantly downregulated Runx2 expression. We showed that NFI/A was significantly upregulated in mi2 cells at both mRNA and protein levels. Furthermore, quantitative proteomics and pathway analysis of gene expression in mi2 cells suggested that miR-exon4 could directly target Prkch (protein kinase C-eta), possibly leading to RUNX2 regulation through mechanistic target of rapamycin kinase activation. Reporter assays also confirmed the direct interaction of miR-exon4 and the 3'-UTR of Prkch, and Western blot analysis confirmed significantly upregulated mechanistic target of rapamycin kinase phosphorylation in mi2 cells. Taken together, we conclude that Nfia and Prkch expression negatively correlates with miR-exon4-mediated Runx2 regulation in vivo and in vitro, suggesting miR-exon4 directly targets Nfia and Prkch to regulate Runx2.
Assuntos
Amelogenina/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , MicroRNAs , Fatores de Transcrição NFI/metabolismo , Proteína Quinase C/metabolismo , Regiões 3' não Traduzidas , Animais , Diferenciação Celular , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Éxons , Regulação da Expressão Gênica , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição NFI/genética , Osteoblastos/metabolismo , Osteogênese/fisiologia , Sirolimo/metabolismoRESUMO
Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.
Assuntos
Proteínas do Esmalte Dentário , Dente , Animais , Peixes/genética , Amelogenina/genética , Amelogenina/metabolismo , Minerais , Colágeno , Proteínas do Esmalte Dentário/genética , MamíferosRESUMO
BACKGROUND: This study aimed to investigate the differentiation of ameloblastic-like cells and the nature of the secreted eosinophilic materials in adenomatoid odontogenic tumors. METHODS: We studied histological and immunohistochemical characteristics of 20 cases using: cytokeratins 14 and 19, amelogenin, collagen I, laminin, vimentin, and CD34. RESULTS: Rosette cells differentiated into ameloblastic-like cells positioned face-to-face, displaying collagen I-positive material between them. Epithelial cells of the rosettes can differentiate into ameloblastic-like cells. This phenomenon probably occurs due to an induction phenomenon between these cells. The secretion of collagen I is probably a brief event. Amelogenin-positive areas were interspersed by epithelial cells in the lace-like areas, outside the rosettes and distant from the ameloblastic-like cells. CONCLUSIONS: There are at least two types of eosinophilic material in different areas within the tumor, one in the rosette and solid areas and another in lace-like areas. The secreted eosinophilic material in the rosettes and solid areas is probably a product of well-differentiated ameloblastic-like cells. It is positive for collagen I and negative for amelogenin, whereas some eosinophilic materials in the lace-like areas are positive for amelogenin. We hypothesize that the latter eosinophilic material could be a product of odontogenic cuboidal epithelial or intermediate stratum-like epithelial cells.
Assuntos
Ameloblastoma , Proteínas do Esmalte Dentário , Tumores Odontogênicos , Humanos , Amelogenina , Tumores Odontogênicos/patologia , Imuno-Histoquímica , Ameloblastoma/patologia , Células Epiteliais/patologia , Colágeno , Diferenciação CelularRESUMO
Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.
Assuntos
DNA , Peptídeos , Masculino , Feminino , Animais , Humanos , Amelogenina/química , Amelogenina/genética , Amelogenina/metabolismo , Reprodutibilidade dos Testes , Peptídeos/análise , DNA/análise , Isoformas de Proteínas , Esmalte Dentário/química , Esmalte Dentário/metabolismoRESUMO
OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.
Assuntos
Proteínas do Esmalte Dentário , Esmalte Dentário , Camundongos , Animais , Humanos , Amelogenina/genética , Amelogenina/metabolismo , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Amelogênese/genética , Proteínas Supressoras de Tumor , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismoRESUMO
Identifying the sex of human hosts of insect disease vectors, using PCR amplification of the amelogenin gene (AMEL) from the ingested blood meal is an increasingly useful technique for epidemiological studies of vector-borne diseases, as well as within the criminal justice system. Detection of DNA from ingested blood is influenced by the choice of DNA extraction method, genomic target region, type and length of PCR, and rate of degradation in the DNA samples over time. Here, we have tested two types of PCR (i.e. conventional and nested), producing differently-sized PCR products, in time-course assays targeting the human AMEL gene in Anopheles stephensi mosquitoes that were fed on human male and female blood. The fed female mosquitoes were allowed to digest at 28 °C for times ranging from 0 to 120 h. Three AMEL primer pairs were used to amplify three sequences that were 977, 539, and 106 bp for the X chromosome and 790, 355, and 112 bp for Y. We found that time since feeding had a significant negative effect on the success of PCR amplification. The shortest fragments (106 and 112 bp) were amplified for the longest time after blood feeding (up to 60 h), whereas the medium and longest loci were not amplified by conventional PCR even at 0 h. However, the nested PCR protocol, targeting the medium sequence, could detect small amounts of human DNA up to 36 h (1.5 days) after the blood meal. The shortest PCR assay standardized herein successfully detected small amounts of human DNA in female mosquitoes up to 60 h after the blood meal. This assay represents a promising tool for identifying the sex of the human host from the blood meal in field-collected female mosquitoes.
Assuntos
Anopheles , Animais , Humanos , Masculino , Feminino , Anopheles/genética , Amelogenina/genética , Mosquitos Vetores , DNA/análise , Reação em Cadeia da Polimerase/métodos , Comportamento AlimentarRESUMO
As the hardest tissue formed by vertebrates, enamel represents nature's engineering masterpiece with complex organizations of fibrous apatite crystals at the nanometer scale. Supramolecular assemblies of enamel matrix proteins (EMPs) play a key role as the structural scaffolds for regulating mineral morphology during enamel development. However, to achieve maximum tissue hardness, most organic content in enamel is digested and removed at the maturation stage, and thus knowledge of a structural protein template that could guide enamel mineralization is limited at this date. Herein, by examining a gene-modified mouse that lacked enzymatic degradation of EMPs, we demonstrate the presence of protein nanoribbons as the structural scaffolds in developing enamel matrix. Using in vitro mineralization assays we showed that both recombinant and enamel-tissue-based amelogenin nanoribbons are capable of guiding fibrous apatite nanocrystal formation. In accordance with our understanding of the natural process of enamel formation, templated crystal growth was achieved by interaction of amelogenin scaffolds with acidic macromolecules that facilitate the formation of an amorphous calcium phosphate precursor which gradually transforms into oriented apatite fibers along the protein nanoribbons. Furthermore, this study elucidated that matrix metalloproteinase-20 is a critical regulator of the enamel mineralization as only a recombinant analog of a MMP20-cleavage product of amelogenin was capable of guiding apatite mineralization. This study highlights that supramolecular assembly of the scaffold protein, its enzymatic processing, and its ability to interact with acidic carrier proteins are critical steps for proper enamel development.
Assuntos
Amelogenina/química , Esmalte Dentário/metabolismo , Amelogênese , Amelogenina/metabolismo , Animais , Apatitas/química , Apatitas/metabolismo , Esmalte Dentário/química , Proteínas do Esmalte Dentário/química , Proteínas do Esmalte Dentário/metabolismo , Camundongos , Nanofibras/químicaRESUMO
The aim of this study is to investigate a robust and stable calcium-phosphorus system to remineralize human early enamel caries lesions with nanocomplexes of carboxymethyl chitosan/L-serine/amorphous calcium phosphate (CMC-Ser-ACP) to develop an effective method for mimicking the amelogenin (AMEL) mineralization pattern through ACP assembly. A CMC-Ser-ACP nanocomplex solution was first synthesized by a chemical precipitation method, and then 1% sodium hypochlorite (NaClO) was added to induce ACP phase formation. The morphologies of the nanocomplexes were characterized by transmission electron microscopy (TEM), and zeta potential analysis and Fourier transform infrared spectroscopy (FTIR) were performed to detect surface charge and functional group changes. The subtle changes of the demineralized enamel models induced by the remineralization effect were observed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The CMC-Ser-ACP nanocomplex solution could be preserved without any precipitation for 45 days. After the application of NaClO and through the guidance of Ser, ACP nanoparticles transformed into relatively orderly arranged hydroxyapatite (HAP) crystals, generating an aprismatic enamel-like layer closely integrated with the demineralized enamel, which resulted in enhanced mechanical properties for the treatment of early enamel caries lesions. The CMC-Ser-ACP nanocomplex solution is a remineralization system with great solution stability, and when NaClO is added, it can rapidly regenerate an aprismatic enamel-like layer in situ on the demineralized enamel surface. This novel remineralization system has stable chemical properties and can greatly increase the therapeutic effects against early enamel caries.
Assuntos
Calcinose , Quitosana , Cárie Dentária , Humanos , Amelogenina , Cárie Dentária/tratamento farmacológico , SerinaRESUMO
OBJECTIVES: To investigate the variant of an amelogenesis imperfecta (AI) family and to explore the function of the FAM83H (family with sequence similarity 83 member H) in the enamel formation. MATERIALS AND METHODS: We investigated a five-generation Chinese family diagnosed with AI; clinical data was collected, whole-exome sequencing (WES) was conducted to explore the pathogenic gene and variants and Sanger sequencing was used to verify the variants. The three-dimensional protein structures of wild-type and mutant FAM83H were predicted using alpha fold 2. To study the possible regulatory function of Fam83h on amelogenesis, immunolocalization was performed to observe the expression of Fam83h protein in Sprague-Dawley rat postnatal incisors. The mRNA and protein level of amelogenin, enamelin, kallikrein-related peptidase-4 and ameloblastin were also detected after the Fam83h was knocked down by small interfering RNA (siRNA) in HAT-7 cells. RESULTS: A known nonsense variant (c.973 C > T) in exon 5 of FAM83H gene was found in this family, causing a truncated protein (p.R325X). Immunolocalization of Fam83h in Sprague-Dawley rat postnatal incisors showed that Fam83h protein expression was detected in presecretory and secretory stages. When Fam83h expression was reduced by siRNA, the expression of amelogenin, enamelin, kallikrein-related peptidase-4 decreased. However, the expression of ameloblastin increased. CONCLUSIONS: FAM83H gene variant (c.973 C > T) causes AI. FAM83H regulates the secretion of enamel matrix proteins and affects ameloblast differentiation. CLINICAL RELEVANCE: This study provided that FAM83H variants could influence enamel formation and provided new insights into the pathogenesis of AI.
Assuntos
Amelogênese Imperfeita , Proteínas do Esmalte Dentário , Humanos , Ratos , Animais , Amelogênese Imperfeita/genética , Amelogenina/genética , Ratos Sprague-Dawley , População do Leste Asiático , Proteínas do Esmalte Dentário/genética , Proteínas/genética , CalicreínasRESUMO
OBJECTIVES: Dental caries is a widespread multifactorial disease, caused by the demineralization of hard dental tissues. Susceptibility to dental caries is partially genetically conditioned; this study was aimed at finding an association of selected single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in amelogenesis with this disease in children. MATERIALS AND METHODS: In this case-control study, 15 SNPs in ALOX15, AMBN, AMELX, KLK4, TFIP11, and TUFT1 genes were analyzed in 150 children with primary dentition and 611 children with permanent teeth with/without dental caries from the European Longitudinal Study of Pregnancy and Childhood (ELSPAC) cohort. RESULTS: Dental caries in primary dentition was associated with SNPs in AMELX (rs17878486) and KLK4 (rs198968, rs2242670), and dental caries in permanent dentition with SNPs in AMELX (rs17878486) and KLK4 (rs2235091, rs2242670, rs2978642), (p ≤ 0.05). No significant differences between cases and controls were observed in the allele or genotype frequencies of any of the selected SNPs in ALOX15, AMBN, TFIP11, and TUFT1 genes (p > 0.05). Some KLK4 haplotypes were associated with dental caries in permanent dentition (p ≤ 0.05). CONCLUSIONS: Based on this study, we found that although the SNPs in AMELX and KLK4 are localized in intronic regions and their functional significance has not yet been determined, they are associated with susceptibility to dental caries in children. CLINICAL RELEVANCE: AMELX and KLK4 variants could be considered in the risk assessment of dental caries, especially in permanent dentition, in the European Caucasian population.
Assuntos
Amelogênese , Cárie Dentária , Criança , Humanos , Amelogenina/genética , Estudos de Casos e Controles , Amelogênese/genética , Cárie Dentária/genética , Cárie Dentária/epidemiologia , Estudos LongitudinaisRESUMO
OBJECTIVES: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing / regeneration in recession-type defects. MATERIALS AND METHODS: A total of 17 gingival recession-type defects were surgically created in the maxilla of three minipigs. The defects were randomly treated with a coronally advanced flap (CAF) and either rAmelX (test), or a CAF and placebo (control). At three months following reconstructive surgery, the animals were euthanized, and the healing outcomes histologically evaluated. RESULTS: The test group yielded statistically significantly (p = 0.047) greater formation of cementum with inserting collagen fibers compared with the control group (i.e., 4.38 mm ± 0.36 mm vs. 3.48 mm ± 1.13 mm). Bone formation measured 2.15 mm ± 0.8 mm in the test group and 2.24 mm ± 1.23 mm in the control group, respectively, without a statistically significant difference (p = 0.94). CONCLUSIONS: The present data have provided for the first-time evidence for the potential of rAmelX to promote regeneration of periodontal ligament and root cementum in recession-type defects, thus warranting further preclinical and clinical testing. CLINICAL RELEVANCE: The present results set the basis for the potential clinical application of rAmelX in reconstructive periodontal surgery.
Assuntos
Retração Gengival , Humanos , Animais , Suínos , Amelogenina/farmacologia , Porco Miniatura , Retração Gengival/tratamento farmacológico , Retração Gengival/cirurgia , Cicatrização , Cemento Dentário , Resultado do Tratamento , Raiz Dentária/patologia , Tecido ConjuntivoRESUMO
The enamel matrix protein Ameloblastin (Ambn) has critical physiological functions, including regulation of mineral formation, cell differentiation, and cell-matrix adhesion. We investigated localized structural changes in Ambn during its interactions with its targets. We performed biophysical assays and used liposomes as a cell membrane model. The xAB2N and AB2 peptides were rationally designed to encompass regions of Ambn that contained self-assembly and helix-containing membrane-binding motifs. Electron paramagnetic resonance (EPR) on spin-labeled peptides showed localized structural gains in the presence of liposomes, amelogenin (Amel), and Ambn. Vesicle clearance and leakage assays indicated that peptide-membrane interactions were independent from peptide self-association. Tryptophan fluorescence and EPR showed competition between Ambn-Amel and Ambn-membrane interactions. We demonstrate localized structural changes in Ambn upon interaction with different targets via a multitargeting domain, spanning residues 57 to 90 of mouse Ambn. Structural changes of Ambn following its interaction with different targets have relevant implications for the multifunctionality of Ambn in enamel formation.