Novel ERCC2 variant in trichothiodystrophy infant: the first case report in China.

BACKGROUND: Trichothiodystrophy (TTD) is a rare, autosomal recessive, multisystem disorder most commonly caused by variants in ERCC2. CASE PRESENTATION: Here, we describe the first Chinese patient with a novel variant in ERCC2. A male infant, who was born to a healthy non-consanguineous couple, exhibited brittle hair, hair loss ichthyosis, eczema, retinal pigmentation and hypospadias. He carried a novel heterozygous ERCC2 variant. The maternal variant (c.2191-18_2213del) is a previous described genomic deletion that affects the splicing of intron 22. The paternal variant (c.1666-1G > A), that occurs in the splice site of intron 17 and likely alters ERCC2 gene function through aberrant splicing, has not been reported previously. CONCLUSIONS: Our case reported a novel pathogenic variant in ERCC2, which expanded the known genetic variants associated with TTD.

Impact of weather factors on hand, foot and mouth disease, and its role in short-term incidence trend forecast in Huainan City, Anhui Province.

Hand, foot, and mouth disease (HFMD) is one of the most common communicable diseases in China, and current climate change had been recognized as a significant contributor. Nevertheless, no reliable models have been put forward to predict the dynamics of HFMD cases based on short-term weather variations. The present study aimed to examine the association between weather factors and HFMD, and to explore the accuracy of seasonal auto-regressive integrated moving average (SARIMA) model with local weather conditions in forecasting HFMD. Weather and HFMD data from 2009 to 2014 in Huainan, China, were used. Poisson regression model combined with a distributed lag non-linear model (DLNM) was applied to examine the relationship between weather factors and HFMD. The forecasting model for HFMD was performed by using the SARIMA model. The results showed that temperature rise was significantly associated with an elevated risk of HFMD. Yet, no correlations between relative humidity, barometric pressure and rainfall, and HFMD were observed. SARIMA models with temperature variable fitted HFMD data better than the model without it (sR 2 increased, while the BIC decreased), and the SARIMA (0, 1, 1)(0, 1, 0)52 offered the best fit for HFMD data. In addition, compared with females and nursery children, males and scattered children may be more suitable for using SARIMA model to predict the number of HFMD cases and it has high precision. In conclusion, high temperature could increase the risk of contracting HFMD. SARIMA model with temperature variable can effectively improve its forecast accuracy, which can provide valuable information for the policy makers and public health to construct a best-fitting model and optimize HFMD prevention.

Role of bone marrow-derived progenitor cells in the maintenance and regeneration of dental mesenchymal tissues.

While dental mesenchymal stem cells are well-studied, the origin of these cells is still unclear. Bone marrow-derived cells (BMDCs) have the potential to engraft into several tissues after injury, but whether they can become dental tissue-specific progenitor cells under normal conditions and the relationship of these cells to the tissue-resident cells are unknown. Thus, we transplanted green fluorescent protein (GFP)-labeled BMDCs into irradiated wild-type mice. We found that the engraftment of BMDCs participated in the regeneration and differentiated into periodontal specific cells after injury. Under normal conditions, there were more BMDCs engrafting into the dental mesenchymal tissue than other organs, in which the expression of stromal cell-derived factor-1 (SDF-1) was significantly higher than in other organs, and the engraftment of cells increased with time. A small fraction of GFP+ cells maintained the mesenchymal stem cell phenotype positive for CD105, CD106, and CD90, which were significantly less than the tissue-resident stem cells; meanwhile, GFP+/CD45+ cells were rare. Isolation and characterization of the dental pulp cells showed that the number of GFP+/Ki67+ cells were greater than the GFP-/Ki67+ cells. In addition, some GFP+ cells differentiated into the dental-specific cells and expressed dental-specific proteins, and can be found in the odontoblast layer after implantation of the apical bud. In conclusion, these data suggest that bone marrow progenitor cells communicate with dental tissues and become tissue-specific mesenchymal progenitor cells to maintain tissue homeostasis.

Effects of Sleeve Gastrectomy on Fecal Gut Microbiota and Short-Chain Fatty Acid Content in a Rat Model of Polycystic Ovary Syndrome.

Purpose: Sleeve gastrectomy (SG) is a surgical intervention for polycystic ovary syndrome (PCOS), especially for patients with obesity. Here, we explored the effects of SG on the gut microbiota of rats with PCOS and investigated the association between the intestinal flora and efficacy of SG in PCOS. Methods: Dehydroepiandrosterone (DHEA) injection was administered alone and in combination with a high-fat diet to induce PCOS in rats. SG was performed in rats with PCOS, and the effects of SG on the fecal and gut microbiota and the short-chain fatty acid (SCFA) content were observed. Furthermore, the association among gut microbiota, SCFA content and hyperandrogenism or other hallmarks of PCOS was evaluated. Results: The abundance of Firmicutes reduced and that of Bacteroidetes increased in response to SG in the DHEA-induced PCOS rat model. At the genus level, the abundances of Bacteroides and Blautia increased and those of Ruminococcus, Clostridium, and Alistipes reduced distinctly in the PCOS-SG groups. Moreover, the levels of fecal SCFAs, especially butyric acid, reduced after SG. SG significantly ameliorated PCOS-related symptoms such as hyperandrogenism, disrupted ovary function, and impaired glucose tolerance. Bacteroides and Blautia exhibited a negative correlation and Ruminococcus, Clostridium, and Alistipes exhibited a positive correlation with the levels of fecal SCFAs, luteinizing hormone, testosterone, and inflammatory factors. Conclusions: The amelioration of PCOS-related reproductive and metabolic disorders following SG was associated with the regulation of microbial taxa and SCFA content. Our findings provide a novel perspective on the microbial mechanisms in PCOS after SG.

Effect of Sleeve Gastrectomy on Kisspeptin Expression in the Hypothalamus of Rats with Polycystic Ovary Syndrome.

OBJECTIVES: The purpose of this study was to determine changes in the expression levels of kisspeptin-1 (Kiss1) in the hypothalamus during the development of polycystic ovary syndrome (PCOS) and after treatment with sleeve gastrectomy (SG). METHODS: This study used chronic dehydroepiandrosterone (DHEA) alone and DHEA plus a high-fat diet (HFD) to generate a PCOS rat model. Subsequently, SG was performed in the animals with PCOS and the effects on glucose tolerance, insulin sensitivity, sex hormones, estrous cyclicity, adiponectin, and Kiss1 expression in the hypothalamus were investigated. RESULTS: Impaired glucose tolerance, decreased insulin sensitivity, reduced adiponectin levels, disrupted estrous cyclicity, and elevated sex hormone levels associated with PCOS models were restored to normal following SG. In addition, SG was able to restore the increase in the expression of Kiss1 mRNA and Kiss1-positive neurons in the arcuate nucleus of rats with PCOS. Interestingly, although SG did not result in a significant loss of body weight in rats administered DHEA under a chow diet, it resulted in comparable metabolic improvements and Kiss1 expression in rats that had been administered DHEA along with an HFD. CONCLUSIONS: The recovery of normal levels of Kiss1 expression in the hypothalamus after SG in this study suggests that Kiss1 might play an important role in the development of PCOS and its improvement by SG.

Identification of a novel missense mutation of MSX1 gene in Chinese family with autosomal-dominant oligodontia.

OBJECTIVES: Oligodontia is defined as the congenital absence of 6 or more permanent teeth excluding the third molar. The occurrence of non-syndromic still remains poorly understood, but in recent years some cases have been reported where mutations or polymorphisms of PAX9 and MSX1 had been associated with non-syndromic oligodontia. The objective of the present work was to study the phenotype and genotype of three generations of a Han Chinese family affected by non-syndromic autosomal-dominant oligodontia. DESIGN: We examined all individuals of the oligodontia family by clinical and radiographic examinations. Based on clinical manifestations, candidate genes MSX1 and PAX9 were picked up to analyse and screen mutations. RESULTS: Dental evaluation showed that the most commonly missing teeth are the mandibular second premolars, followed by the maxillary second premolars and maxillary lateral incisors, and subsequently the maxillary first premolars. The probability of missing a particular type of tooth is not always bilaterally symmetrical, and differences exist between maxilla and mandible. PCR-SSCP analysis and DNA sequencing revealed a novel missense mutation c.662C>A in a highly conserved homeobox sequence of MSX1 and a known polymorphisms c.347C>G. CONCLUSION: Our finding suggests the missense transversion (c.662C>A) and the polymorphisms (c.347C>G) may be responsible for oligodontia phenotype in this Chinese family.

Determination of genes involved in the early process of molar root development initiation in rat by modified subtractive hybridization.

Rat molars and incisors have different root patterning mechanisms: the former form a multirooted pattern while the latter form a single-rooted analogue. But the genetic signals that control root patterning are poorly understood. In this study, to identify the special molecular signals which may lead to the molar root development, we firstly observed that at embryonic day 19 the molar and the incisor began differentially developing: the molar formed double-layered cells of the root sheath while the incisor formed a cervical loop. By using the subtractive hybridization method, we successfully constructed a subtraction cDNA library of the rat molar and incisor tissues. Differentially expressed gene clones were evaluated by dot-blot and sequencing. Sel1l, Nfic, Edar, GATA6, and some novel genes were found differentially expressed, which were strongly related the tooth root patterning. It is anticipated that further study of genes identified will provide insights into their specific roles in the tooth root patterning.

ADAM28 participates in the regulation of tooth development.

Disintegrin and metalloprotease (ADAM) proteins are a family of membrane-anchored glycoproteins with diverse functions in fertilisation, development, neurogenesis and protein ectodomain shedding. ADAM28 is a newly discovered member of the ADAM family in humans and murine with autocatalytic activity. Recently, the authors screened ADAM28 genes from patients with congenital hypoplasia of tooth root, and studied the relationship between ADAM28 and tooth development. A polyclonal antibody (pAb) against ADAM28 was preparared, and the expression and localisation of ADAM28 were detected in tooth germ and dental mesenchymal cells. The results indicated that the prokaryotic expression vector pGEX-4T-ADAM28 was constructed successfully. Glutathione S-transferase-ADAM28 fusion protein was generated after inducement by isopropylthio-beta-d-galactoside and isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fusion protein was used as an antigen for production of antibody. Western blot and enzyme-linked immunosorbent assay analyses verified that the antibody had a high specificity and titre. Immunohistochemistry and reverse transcriptase-polymerase chain reaction showed that ADAM28 was expressed at each stage of tooth germ development at different levels. Moreover, it was expressed in human dental follicle cells, human dental papilla cells, human dental pulp stem cells, human periodontal ligament cells and human dental cervical loop epithelial cells at transcription level. In conclusion, it is reasonable to suggest that ADAM28 may participate in tooth development and the regulation of odontogenic mesenchymal cells through progressive reciprocal inductive interactions between the epithelium and the mesenchyme.

[Changes of dentin, dental pulp and periodontium tissue in tail-suspended rats].

OBJECTIVE: To investigate the metabolic changes of calcium and phosphorus in dentin, dental pulp and periodontium in tail-suspended rats, and the functions of TGF-beta 1, c-fos, collagen-I and collagen IV in dentin, dental pulp and periodontium. METHOD: Relative percentage contents of Ca, P in dentin, dental pulp and periodontium were measured with scanning electron microscope and energy spectrum analytical system in 3 groups of rats. The expression of TGF-beta 1, c-fos, collagen-I and collagen IV were also observed. RESULT: In the suspension group, the relative percentage content of Ca declined significantly, while P increased slightly. There were no significant differences of Ca, P in alveolar bone. The expressions of TGF-beta 1, c-fos and collagen-I declined, but the expression of collagen-IV in pulp vessel increased. There were no significant changes of expressions of TGF-beta 1, c-fos, collagen-I and collagen-IV in the vicinity of PDL. After adopting artificial countermeasures, the above expressions restored partly. CONCLUSION: Weightlessness might cause abnormal mineralization in dentin, and 1.5 G artificial countermeasures could eliminate the above changes of mineral metabolism. The poor mineralization of dentin might be associated with the reduced secretion of TGF-beta 1, c-fos and collagen-I in tail-suspended rats.

Scaffold-free cell pellet transplantations can be applied to periodontal regeneration.

Cell transplantation has emerged as a novel therapeutic strategy for periodontitis, and the adoption of cell pellet offers advantages by secreting abundant extracellular matrix (ECM) and eliminating the adverse effect of cell carriers. This study aimed to fabricate scaffold-free periodontal ligament stem cell (PDLSC) pellets (MUCPs) and to evaluate their regeneration potential. We constructed monolayer cell pellets (MCPs) by fabricating and culturing multilayered cell sheets (MUCS) and constructed MUCPs from the MUCS. Immunochemistry, scanning electron microscope, real-time PCR, and Western blot analysis showed higher levels of COL-I, COL-III, fibronectin, and laminin in the MUCPs. Furthermore, the massive increase in ECM secretion improved cell adhesion, migration, and proliferation. Finally, upon transplantation into the omentum sac and periodontal defects, all the transplants formed regular aligned cementum/PDL-like complex, but the mineral deposit and fiber alignment were more obvious in the MUCPs than in the MCPs. Altogether, our results suggest that MUCPs may be a promising alternative to periodontal repair for future clinical application.

Oral and craniofacial manifestations and two novel missense mutations of the NTRK1 gene identified in the patient with congenital insensitivity to pain with anhidrosis.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare inherited disorder of the peripheral nervous system resulting from mutations in neurotrophic tyrosine kinase receptor 1 gene (NTRK1), which encodes the high-affinity nerve growth factor receptor TRKA. Here, we investigated the oral and craniofacial manifestations of a Chinese patient affected by autosomal-recessive CIPA and identified compound heterozygosity in the NTRK1 gene. The affected boy has multisystemic disorder with lack of reaction to pain stimuli accompanied by self-mutilation behavior, the inability to sweat leading to defective thermoregulation, and mental retardation. Oral and craniofacial manifestations included a large number of missing teeth, nasal malformation, submucous cleft palate, severe soft tissue injuries, dental caries and malocclusion. Histopathological evaluation of the skin sample revealed severe peripheral nerve fiber loss as well as mild loss and absent innervation of sweat glands. Ultrastructural and morphometric studies of a shed tooth revealed dental abnormalities, including hypomineralization, dentin hypoplasia, cementogenesis defects and a dysplastic periodontal ligament. Genetic analysis revealed a compound heterozygosity--c.1561T>C and c.2057G>A in the NTRK1 gene. This report extends the spectrum of NTRK1 mutations observed in patients diagnosed with CIPA and provides additional insight for clinical and molecular diagnosis.

Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root.

Tissue engineering strategies to reconstruct tooth roots are an effective therapy for the treatment of tooth loss. However, strategies to successfully regenerate tooth roots have not been developed and optimized. In the present study, rat dental follicle stem cells (DFCs) were characterized, followed by a thorough investigation of tooth roots regeneration for a combination of DFCs seeding cells, treated dentin matrix (TDM) scaffolds, and an inductive alveolar fossa microenvironment. Eighteen clones derived from single DFCs were harvested; however, only three clones were amplified successfully more than five passages and 90-95 days in culture. Following 270 days or 30 passages, the heterogeneous DFCs showed suitable characteristics for seeding cells to regenerate tooth roots. However, various features, such as variable proliferation rates, differentiation characteristics, apoptosis rates, and total lifespan were observed in DFCs and the three clones. Importantly, upon transplantation of DFCs combined with TDM for four weeks, root-like tissues stained positive for markers of dental pulp and periodontal tissues were regenerated in the alveolar fossa, but not in the skull and omental pockets. These results indicate that tooth roots were successfully regenerated and suggest that the combination of DFCs with TDM in the alveolar fossa is a feasible strategy for tooth roots regeneration. This strategy could be a promising approach for the treatment of clinical tooth loss and provides a perspective with potential applications to regeneration of other tissues and organs.

Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells.

OBJECTIVE: This study focused on the characterization of stem cells from human exfoliated deciduous teeth (SHED) in comparison with dental pulp stem cells (DPSCs) to certify SHED as a key element in tissue engineering. METHODS: In the present study, SHED and DPSCs were assayed for their cell surface antigens and proliferation by measuring the cell cycles, growth rates, Ki67-positive efficiencies, and colony-forming units (CFUs). The evaluation of multi-differentiation was performed using alizarin red and oil red O and real-time PCR in vitro. The mineralization capability of the cells was examined in vivo by implanting with ceramic bovine bone (CBB) into subcutaneous of immunocompromised mice for 8weeks. A three-dimensional pellet cultivation system is proposed for SHED and DPSCs to recreate the biological microenvironment that is similar to that of a regenerative milieu. RESULTS: SHED showed a higher proliferation rate and differentiation capability in comparison with DPSCs in vitro, and the results of the in vivo transplantation suggest that SHED have a higher capability of mineralization than the DPSCs. The mRNA expression levels of inflammatory cytokines, including matrix metalloproteinase-1 (MMP1), tissue inhibitors of metalloproteinase-1 (TIMP1), matrix metalloproteinase-2 (MMP2), tissue inhibitors of metalloproteinase-2 (TIMP2) and interleukin-6 (IL-6) were higher in SHED than that in DPSCs. In addition, the expression levels of Col I and proliferating cell nuclear antigen (PCNA) in SHED sheets were significantly higher than those in DPSCs sheets. CONCLUSIONS: This study systematically demonstrated the differences in the growth and differentiation characteristics between SHED and DPSCs. Consequently, SHED may represent a suitable, accessible and potential alternative source for regenerative medicine and therapeutic applications.

Targeting nicotinic acetylcholine receptor to treat smoking-related periodontitis.

Tobacco smoking is considered to be one of the major risk factors for periodontitis. Nicotine, the major component in tobacco smoke, has been considered playing an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. Recently studies found that nAChRs could be expressed on oral gingival and periodontal tissues. We hypothesize that nicotine may act on periodontal tissues directly and specifically through nAChRs to affect periodontitis activity, and that nicotine-induced periodontitis could be prevented by tissue-selective nAChR inhibitors targeting periodontal nAChRs. Thus, periodontal nAChRs may provide to be novel molecular targets to treat smoking-related periodontitis, effectively blocking of periodontal nAChRs may offer an optimistic outlook for the therapy of smoking- related periodontitis.

Changes of the unique odontogenic properties of rat apical bud cells under the developing apical complex microenvironment.

AIM: To characterize the odontogenic capability of apical bud and phenotypical change of apical bud cells (ABCs) in different microenvironment. METHODOLOGY: Incisor apical bud tissues from neonatal SD rat were dissected and transplanted into the renal capsules to determine their odontogenic capability. Meanwhile ABCs were cultured and purified by repeated differential trypsinization. Then ABCs were cultured with conditioned medium from developing apical complex cells (DAC-CM). Immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and scanning electron microscope (SEM) were performed to compare the biological change ofABC treated with or without DAC-CM. RESULTS: First we confirmed the ability of apical bud to form crown-like structure ectopically. Equally important, by using the developing apical complex (DAC) conditioned medium, we found the microenvironment created by root could abrogate the "crown" features of ABCs and promote their proliferation and differentiation. CONCLUSION: ABCs possess odontogenic capability to form crown-like tissues and this property can be affected by root-produced microenvironment.

Essential role of ADAM28 in regulating the proliferation and differentiation of human dental papilla mesenchymal cells (hDPMCs).

Dental papilla mesenchymal cells (DPMCs) have been supposed to possess the relatively independent and critical role for tooth development and morphogenesis. Here, we characterized the role of ADAM28, a member of a disintegrin and metalloproteinase (ADAM) family, in the regulative mechanisms of odontogenic capability of hDPMCs. Immunofluorescence staining showed the ubiquitous expression of ADAM28 in multiple human dental mesenchymal and epithelial cells. After confirming the effect of eukaryotic expression plasmid containing ADAM28 coding region and ADAM28 antisense oligodeoxynucleotide (AS-ODN), we respectively transfected them into hDPMCs and observed the biological markers for proliferation and differentiation. Overexpression of ADAM28 favored the proliferation and lineage-specific differentiation of hDPMCs, while blockage of ADAM28 exerted the opposite effects and induced apoptosis. These results identified an unrecognized hypothesis that ADAM28 may function as positive regulator of growth and differentiation of hDPMCs and act as an important molecule mediating reciprocal epithelial-mesenchymal signaling during tooth organ development.

[Expression feature and bioinformatic analysis of molar root patterning gene 1].

OBJECTIVE: To examine the expression of molar root patterning gene 1 (Mrp1) and predict the Mrp1 structure by bioinformatics analysis. METHODS: A pair of Mrp1-specific PCR primers were designed, and RT-PCR method was used to study the mRNA's expression pattern in rat molar root and other organs. Gene positioning and other protein sequence prediction were carried out by chromosome analysis and other bioinformatics analysis. RESULTS: Mrp1 was expressed not only in the molar but also in the developing pancreas, liver, lung and kidney tissues. Mrp1 was located in the 18q12.3 chromosome of the rats and the Mrp1 amino acids sequence had about 37% homology with a known protein Uroplakin IIIb (p35) which was an urothelial differentiation membrane molecular marker. A trans-membrane structure, 5 PKC phosphorylation sites and 4 CKII phosphorylation sites in Mrp1 were found. CONCLUSIONS: Mrp1 has a broad expression in different developing organs, and it may have a important function in the rat tooth root development.

[Effects and molecular mechanism of nicotine on odontoblasts].

OBJECTIVE: To observe the effects of nicotine on the proliferation of odontoblasts and explore the possible mechanism. METHODS: Odontoblasts MDPC-23 were cultured, inoculated and divided into two groups randomly. With no stimuli added for the control group, the experimental group was stimulated by 100 microg/mL nicotine. After 8 hours, 10 micromol/L BrdU was added to label cells at S stage in cell cycle. 24 hours later, odontoblasts were fixed and immunofluorescence staining was performed with specific mouse BrdU antibody. After counterstaining with propidium iodide, BrdU positive cells were arbitrarily scored microscopically by an independent estimation conducted three times, and the corresponding total cell number in the same vision were counted in both groups. BrdU positive cell rates were calculated and compared statistically. At the same time, odontoblasts MDPC-23 were cultured and stimulated by 100 microg/mL nicotine, the dynamic Ca2+ concentration inside the cytoplasm were detected immediately by a confocal laser scanning microscope. RESULTS: The ratio of S stage cells in the experimental group was 36.3% significantly lower than that (48.2%) in the control group. After the addition of 100 microg/mL nicotine, the Ca2+ concentration inside the cytoplasm rose rapidly, sustained at a high level for a short time and then relapsed gradually. CONCLUSION: Nicotine had inhibitory effects on the proliferation of odontoblasts MDPC-23, which might be related to the increased Ca2+ concentration in the cytoplasm.

[Regulation of osteopontin expression in odontoblasts by upstream stimulatory factor 1].

OBJECTIVE: To investigate the regulation effects of upstream stimulatory factor 1 (USF1) on osteopontin expression in odontoblasts. METHODS: Odontoblast MDPC-23 was cultured and stably transfected with PCMV-USF1 or A-USF plasmids. Total RNA was extracted and osteopontin expression examined by semi-quantitative RT-PCR. Gray value of osteopontin was measured and statistic analysis performed. RESULTS: Clones of stable PCMV-USF1 and A-USF plasmids transfection were obtained. Compared with the control, osteopontin was upregulated in PCMV-USF1 transfection group, and downregulated in A-USF transfection group. CONCLUSIONS: Upstream stimulatory factor 1 could regulate the osteopontin expression in odontoblasts, which could be blocked partly by A-USF.

[Expression, subcellular localization and nuclear translocation of transcription factor up stream stimulatory factor-1 in odontoblasts].

OBJECTIVE: To examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli. METHODS: Odontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control. RESULTS: The staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2. CONCLUSIONS: There exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.