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Journal of Molecular Histology Apr 2024Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes... (Review)
Review
Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.
Topics: Animals; Rats; Ameloblasts; Amelogenesis; Molar; Muscle Proteins; Nerve Tissue Proteins; Odontogenesis; Proteins
PubMed: 38407765
DOI: 10.1007/s10735-024-10182-9 -
Biomaterials Advances May 2024Obtaining rapid mineralisation is a challenge in current bone graft materials, which has been attributed to the difficulty of guiding the biological processes towards...
Obtaining rapid mineralisation is a challenge in current bone graft materials, which has been attributed to the difficulty of guiding the biological processes towards osteogenesis. Amelogenin, a key protein in enamel formation, inspired the design of two intrinsically disordered peptides (P2 and P6) that enhance in vivo bone formation, but the process is not fully understood. In this study, we have elucidated the mechanism by which these peptides induce improved mineralisation. Our molecular dynamics analysis demonstrated that in an aqueous environment, P2 and P6 fold to interact with the surrounding Ca, PO and OH ions, which can lead to apatite nucleation. Although P2 has a less stable backbone, it folds to a stable structure that allows for the nucleation of larger calcium phosphate aggregates than P6. These results were validated experimentally in a concentrated simulated body fluid solution, where the peptide solutions accelerated the mineralisation process compared to the control and yielded mineral structures mimicking the amorphous calcium phosphate crystals that can be found in lamella bone. A pH drop for the peptide groups suggests depletion of calcium and phosphate, a prerequisite for intrinsic osteoinduction, while S/TEM and SEM suggested that the peptide regulated the mineral nucleation into lamella flakes. Evidently, the peptides accelerate and guide mineral formation, elucidating the mechanism for how these peptides can improve the efficacy of P2 or P6 containing devices for bone regeneration. The work also demonstrates how experimental mineralisation study coupled with molecular dynamics is a valid method for understanding and predicting in vivo performance prior to animal trials.
Topics: Animals; Osteogenesis; Bone Regeneration; Apatites; Peptides; Bone and Bones
PubMed: 38401402
DOI: 10.1016/j.bioadv.2024.213801 -
Forensic Science International Mar 2024With the increasing importance of X-chromosome (Chr-X) genotyping in kinship identification, the exploitation of X chromosome genetic marker multiplex kits is...
With the increasing importance of X-chromosome (Chr-X) genotyping in kinship identification, the exploitation of X chromosome genetic marker multiplex kits is increasing. The Human X-InDels amplification kit is a novel developed system which contained 38 X-chromosomal Insertion/deletion markers (X-InDels) and Amelogenin. Herein, we investigated the genetic diversity of the 38 X-InDels in the Tibetan ethnic minority (n = 792) from seven regions and evaluated the application potential of this novel panel. The rs16368 was the least variable locus, whereas the most polymorphic locus was the rs59605609 in Tibetan population. We confirmed three linkage groups with the haplotype diversities ranged from 0.5032 to 0.5976. The overall combined power of discrimination (PD) in males and females were 0.999999999582066 and 0.999999999999993, respectively. And the overall combined mean exclusion chance (MEC) values were not lower than 0.999125526990159. In addition, we explored the genetic relationships among the Tibetans in seven different regions via series of population comparison analyses, finding that the genetic relationship between the Ngari Tibetan and Chamdo Tibetan was the farthest, which was consistent with geographical distribution.
Topics: Male; Female; Humans; Gene Frequency; Genetics, Population; Tibet; Ethnicity; Forensic Genetics; Minority Groups; X Chromosome; Genetic Structures; China; East Asian People
PubMed: 38377671
DOI: 10.1016/j.forsciint.2024.111961 -
Heliyon Jan 2024Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix...
Ameloblastin is a protein in biomineralization of tooth enamel. However recent results indicate that this is probably not its only role in an organism. Enamel matrix formation represents a complex process enabled via specific crosslinking of two proteins - the most abundant amelogenin and the ameloblastin (AMBN). The human AMBN (hAMBN) gene possesses 13 protein coding exons with alternatively spliced transcripts and the longest isoform about 447 amino acid residues. It has been described that AMBN molecules assemble into oligomers via a sequence encoded by exon 5. Enamel is formed by the processing of enamel proteins by two specific proteases - enamelysin (MMP-20) and kallikrein 4 (KLK-4). The scaffold made of AMEL and non-amelogenin proteins is cleaved and removed from the developed tooth enamel. The hAMBN is expressed in two isoforms (ISO I and II), which could lead to their different utilization determined by distinct proteolytic profiles. In this study, we compared proteolytic profiles of both isoforms of hAMBN expressed in after proteolysis by MMP-20, KLK-4, and their 1:2 mixture. Proteolysis products were analysed and cleavage sites were identified by mass spectrometry. The proteolytic profiles of two AMBN isoforms showed different results, although we have to determine that the analysed AMBN was not post-translationally modified as expressed in prokaryotic cells. These results may lead to the suggestion of potentially divergent roles of AMBN isoforms cleavage products in various cell signalling pathways such as calcium buffering or signalling cascades.
PubMed: 38298721
DOI: 10.1016/j.heliyon.2024.e24564 -
Frontiers in Physiology 2023Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor...
Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. In this study, enamel formation in mice was analyzed when transgenic was overexpressed from the amelogenin promoter encoding full-length Ambn. was under- and overexpressed at six increasing concentrations in separate mouse lines. Mice overexpressing displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17 kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.
PubMed: 38274050
DOI: 10.3389/fphys.2023.1233391 -
RSC Advances Jan 2024Due to persistent inflammation and limited osteogenesis, jawbone defects present a considerable challenge in regenerative medicine. Amelogenin, a major protein...
Due to persistent inflammation and limited osteogenesis, jawbone defects present a considerable challenge in regenerative medicine. Amelogenin, a major protein constituent of the developing enamel matrix, demonstrates promising capabilities in inducing regeneration of periodontal supporting tissues and exerting immunomodulatory effects. These properties render it a potential therapeutic agent for enhancing jawbone osteogenesis. Nevertheless, its clinical application is hindered by the limitations of monotherapy and its rapid release characteristics, which compromise its efficacy and delivery efficiency. In this context, calcium alginate hydrogel, recognized for its superior physicochemical properties and biocompatibility, emerges as a candidate for developing a synergistic bioengineered drug delivery system. This study describes the synthesis of an injectable calcium amelogenin/calcium alginate hydrogel using calcium alginate loaded with amelogenin. We comprehensively investigated its physical properties, its role in modulating the immunological environment conducive to bone healing, and its osteogenic efficacy in areas of jawbone defects. Our experimental findings indicate that this synthesized composite hydrogel possesses desirable mechanical properties such as injectability, biocompatibility, and biodegradability. Furthermore, it facilitates jawbone formation by regulating the bone-healing microenvironment and directly inducing osteogenesis. This research provides novel insights into the development of bone-tissue regeneration materials, potentially advancing their clinical application.
PubMed: 38196914
DOI: 10.1039/d3ra05046g -
Journal of Forensic Sciences Mar 2024"Touch DNA" is a form of trace DNA that is presumed to be deposited when an individual touches something and leaves behind DNA-containing skin cells, sweat, or other...
"Touch DNA" is a form of trace DNA that is presumed to be deposited when an individual touches something and leaves behind DNA-containing skin cells, sweat, or other fluids. While touch DNA is often the result of direct contact (i.e., primary transfer), it can also be indirectly transferred between surfaces or individuals (e.g., secondary or tertiary transfer). Even experts cannot distinguish between different types of transfer and do not fully understand which variables affect direct versus indirect transfer or how often each type of transfer occurs. In this study, we utilize an innovative protocol that combines a paired male and female transfer DNA experimental design with an Amelogenin qPCR assay to generate data on primary, secondary, and tertiary DNA transfer. We report frequencies of indirect DNA transfer and also investigate the potential effects of participant age, self-identified ethnicity, and skin conditions on DNA transfer. Out of 22 experimental trials, we detected primary transfer (male + female) in 71% of trials, secondary DNA transfer in 50% of trials, and tertiary DNA transfer in 27% of trials. No significant associations were found between primary DNA transfer and age, self-identified ancestry, or skin conditions, however, all individuals with sloughing skin conditions demonstrated primary DNA transfer and we suggest this variable be explored in larger samples. These results contribute to a better understanding of the conditions under which secondary and tertiary DNA transfer occurs and can be used to propose realistic DNA transfer scenarios in court cases.
Topics: Humans; Male; Female; Research Design; DNA Fingerprinting; Skin; Touch; DNA
PubMed: 38108622
DOI: 10.1111/1556-4029.15444 -
Forensic Science International. Genetics Mar 2024Massively parallel sequencing (MPS) techniques were developed approximately 15 years ago. Meanwhile, several MPS kits for forensic identification, phenotypic...
Massively parallel sequencing (MPS) techniques were developed approximately 15 years ago. Meanwhile, several MPS kits for forensic identification, phenotypic information, ancestry, and mitochondrial DNA analysis have been developed and their use has been established. Sequencing short tandem repeats (STRs) has certain advantages over the currently used length-based genotyping methods, which are based on PCR amplification followed by capillary electrophoresis (CE). MPS is more discriminative and includes the possibility of testing high numbers of targets (> 100), different types of markers [STRs and single nucleotide polymorphisms (SNPs)], as well as the use of smaller amplicons (< 300 bp). This study evaluated in 24 experimental runs the Precision ID GlobalFiler™ NGS STR panel v2 from ThermoFisher, which targets 31 autosomal STRs, amelogenin, and three Y-markers (one STR, SRY, and Yindel). Single-source samples were used in 18 experimental runs, for systematic evaluation. These included assessing library preparation benchmark conditions, limited DNA input, as well as testing repeatability, number of samples per run, and degraded DNA samples. Full profiles were consistently obtained from as little as 50 pg DNA input. Using the optional recovery PCR method improved outcomes for samples with low DNA input. Full profiles were also obtained from severely degraded DNA samples with degradation indices (DI) of > 60. In addition, six experimental runs were performed testing various two-person mixtures with mixture ratios ranging from 1:20 to 20:1. Major and minor contributors were distinguishable by their read counts (coverage), because less DNA input yielded lower read counts, analogous to the traditional CE technology, where less DNA produces lower peak heights. Mixture ratios of approximately 1:1 were indistinguishable, while a greater imbalance, i.e., higher mixture ratios, made the mixture more distinguishable between major and minor contributors. Based on this information, the highest success rate of correctly deconvoluted four-allelic loci was from mixtures with 1:3 ratios. At higher mixture ratios, the drop-out rate of the minor contributor increased, reducing the number of four-allelic loci.
Topics: Humans; DNA Fingerprinting; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Genotyping Techniques; DNA, Mitochondrial; Microsatellite Repeats; Polymorphism, Single Nucleotide
PubMed: 38065030
DOI: 10.1016/j.fsigen.2023.102995 -
Forensic Science International Jan 2024DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and...
Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry.
UNLABELLED
DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation.
AIM
To evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results.
MATERIAL & METHODS
Biological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively.
RESULTS
After DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CV% ≤ 9.55) within the dynamic range analyzed (0.016-50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms.
CONCLUSION
We demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.
Topics: Male; Humans; Female; DNA Fingerprinting; Reproducibility of Results; Microsatellite Repeats; DNA; Chromosomes, Human, Y
PubMed: 38064775
DOI: 10.1016/j.forsciint.2023.111893 -
Electrophoresis Mar 2024The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study,...
The continual investigation of novel genetic markers has yielded promising solutions for addressing the challenges encountered in forensic DNA analysis. In this study, we have introduced a custom-designed panel capable of simultaneously amplifying 41 novel Multi-insertion/deletion (Multi-InDel) markers and an amelogenin locus using the capillary electrophoresis platform. Through a developmental validation study conducted in accordance with guidelines recommended by the Scientific Working Group on DNA Analysis Methods, we demonstrated that the new Multi-InDel system exhibited the sensitivity to produce reliable genotyping profiles with as little as 62.5 pg of template DNA. Accurate and complete genotyping profiles could be obtained even in the presence of specific concentrations of PCR inhibitors. Furthermore, the maximum amplicon size for this system was limited to under 220 bp in the genotyping profile, resulting in its superior efficiency compared to commercially available short tandem repeat kits for both naturally and artificially degraded samples. In the context of mixed DNA analysis, the Multi-InDel system was proved informative in the identification of two-person DNA mixture, even when the template DNA of the minor contributor was as low as 50 pg. In conclusion, a series of performance evaluation studies have provided compelling evidence that the new Multi-InDel system holds promise as a valuable tool for forensic DNA analysis.
Topics: Humans; Genotype; DNA; DNA Fingerprinting; Microsatellite Repeats; DNA Primers; Forensic Genetics; Multiplex Polymerase Chain Reaction
PubMed: 38037290
DOI: 10.1002/elps.202300192