PD-1/CD80+ small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression.

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.

Exosomes Derived from Human Palatal Mesenchymal Cells Mediate Intercellular Communication During Palatal Fusion by Promoting Oral Epithelial Cell Migration.

Purpose: Exosomes are important "messengers" in cell-cell interactions, but their potential effects on palatal fusion are still unknown. This study aimed to explore the role and mechanism of exosomes derived from palatal mesenchymal cells in epithelial-mesenchymal communication during palatogenesis. Methods: The expression of exosome marker CD63 and CD81 in palatal cells during palatogenesis was detected by immunofluorescence staining. After being purified from the supernatant of human embryonic palatal mesenchymal (HEPM) cells, exosomes (HEPM-EXO) were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. HEPM-EXO were co-cultured with human immortalized oral epithelial cells (HIOEC). The effects of HEPM-EXO on the cell proliferation, migration, apoptosis and epithelial-mesenchymal transition (EMT) of HIOEC were evaluated. The proteins encapsulated in HEPM-EXO were analyzed by proteomic analysis. Results: The extensive expression of CD63 and CD81 in palatal epithelial and mesenchymal cells were continuously detected during E12.5~E14.5, suggesting that exosomes were involved in the process of palatal fusion. The expression of CD63 was also observed in the acellular basement membrane between the palatal epithelium and the mesenchyme in vivo, and HEPM-EXO could be internalized by HIOEC in vitro, suggesting that exosomes are potent to diffuse through the cellular tissue boundary to mediate palatal cell-cell communication. Exposure of HEPM-EXO to HIOEC substantially inhibited the proliferation and stimulated the migration of HIOEC, but had no significant effect on cell apoptosis and EMT. Proteomic analysis revealed the basic characteristics of the proteins in HEPM-EXO and that exosomal THBS1 may potentially regulate the cell behaviors of HIOEC, which needs further verification. Gene ontology (GO) analysis uncovered that the proteins highly expressed in HEPM-EXO are closely related to wound healing, implying a promising therapeutic opportunity of HEPM-EXO in tissue injury treatment with future studies. Conclusion: HEPM-EXO mediated cell-cell communication by regulating cell proliferation and migration of oral epithelial cells during palatogenesis.

The Role of DSPP in Dentine Formation and Hereditary Dentine Defects.

The dentine sialophosphoprotein (DSPP) gene is the only identified causative gene for dentinogenesis imperfecta type 2 (DGI-II), dentinogenesis imperfecta type 3 (DGI-III) and dentine dysplasia type 2 (DD-II). These three disorders may have similar molecular mechanisms involved in bridging the DSPP mutations and the resulting abnormal dentine mineralisation. The DSPP encoding proteins DSP (dentine sialoprotein) and DPP (dentine phosphoprotein) are positive regulators of dentine formation and perform a function during dentinogenesis. The present review focused on the recent findings and viewpoints regarding the relationship between DSPP and dentinogenesis as well as mineralisation from multiple perspectives, involving studies relating to spatial structure and tissue localisation of DSPP, DSP and DPP, the biochemical characteristics and biological function of these molecules, and the causative role of the proteins in phenotypes of the knockout mouse model and in hereditary dentine defects.

Expert consensus on difficulty assessment of endodontic therapy.

Endodontic diseases are a kind of chronic infectious oral disease. Common endodontic treatment concepts are based on the removal of inflamed or necrotic pulp tissue and the replacement by gutta-percha. However, it is very essential for endodontic treatment to debride the root canal system and prevent the root canal system from bacterial reinfection after root canal therapy (RCT). Recent research, encompassing bacterial etiology and advanced imaging techniques, contributes to our understanding of the root canal system's anatomy intricacies and the technique sensitivity of RCT. Success in RCT hinges on factors like patients, infection severity, root canal anatomy, and treatment techniques. Therefore, improving disease management is a key issue to combat endodontic diseases and cure periapical lesions. The clinical difficulty assessment system of RCT is established based on patient conditions, tooth conditions, root canal configuration, and root canal needing retreatment, and emphasizes pre-treatment risk assessment for optimal outcomes. The findings suggest that the presence of risk factors may correlate with the challenge of achieving the high standard required for RCT. These insights contribute not only to improve education but also aid practitioners in treatment planning and referral decision-making within the field of endodontics.

Expert consensus on irrigation and intracanal medication in root canal therapy.

Chemical cleaning and disinfection are crucial steps for eliminating infection in root canal treatment. However, irrigant selection or irrigation procedures are far from clear. The vapor lock effect in the apical region has yet to be solved, impeding irrigation efficacy and resulting in residual infections and compromised treatment outcomes. Additionally, ambiguous clinical indications for root canal medication and non-standardized dressing protocols must be clarified. Inappropriate intracanal medication may present side effects and jeopardize the therapeutic outcomes. Indeed, clinicians have been aware of these concerns for years. Based on the current evidence of studies, this article reviews the properties of various irrigants and intracanal medicaments and elucidates their effectiveness and interactions. The evolution of different kinetic irrigation methods, their effects, limitations, the paradigm shift, current indications, and effective operational procedures regarding intracanal medication are also discussed. This expert consensus aims to establish the clinical operation guidelines for root canal irrigation and a position statement on intracanal medication, thus facilitating a better understanding of infection control, standardizing clinical practice, and ultimately improving the success of endodontic therapy.

Human Dental Pulp Stem Cells Are Subjected to Metabolic Reprogramming and Repressed Proliferation and Migration by the Sympathetic Nervous System via α1B-Adrenergic Receptor.

INTRODUCTION: Human dental pulp stem cells (hDPSCs) reside in specialized microenvironments in the dental pulp, termed "niches," which are composed of diverse cellular components including nerves. Sensory nerves can positively regulate the expansion and differentiation of pulp cells, while the biological effects of the sympathetic nervous system (SNS) on hDPSCs remain elusive. This study is devoted to investigating the effects and underlying mechanisms of the SNS on the proliferation and migration of hDPSCs. METHODS: The distribution of sympathetic nerve fibers in human dental pulp was examined by immunofluorescence staining of tyrosine hydroxylase. The concentration of norepinephrine in healthy and carious human dental pulp tissues was detected using enzyme-linked immunosorbent assay. RNA-sequencing was applied to identify the dominant sympathetic neurotransmitter receptor in hDPSCs. Seahorse metabolic assay, adenosine triphosphate assay, lactate assay, and mitochondrial DNA copy number were performed to determine the level of glycometabolism. Transwell assay, wound healing assay, 5-ethynyl-2'-deoxyuridine staining assay, cell cycle assay, and Cell Counting Kit-8 assay were conducted to analyze the migratory and proliferative capacities of hDPSCs. RESULTS: Sprouting of sympathetic nerve fibers and an increased concentration of norepinephrine were observed in inflammatory pulp tissues. Sympathetic nerve fibers were mainly distributed along blood vessels, and aldehyde dehydrogenase 1-positive hDPSCs resided in close proximity to neurovascular bundles. ADRA1B was identified as the major sympathetic neurotransmitter receptor expressed in hDPSCs, and its expression was enhanced in inflammatory pulp tissues. In addition, the SNS inhibited the proliferation and migration of hDPSCs through metabolic reprogramming via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways. CONCLUSIONS: This study demonstrates that the SNS can shift the metabolism of hDPSCs from oxidative phosphorylation to anaerobic glycolysis via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways, thereby inhibiting the proliferative and migratory abilities of hDPSCs. This metabolic shift may facilitate the maintenance of the quiescent state of hDPSCs.

Clinical and radiological outcomes of dynamic navigation in endodontic microsurgery: a prospective study.

OBJECTIVES: This study was aimed at evaluating the clinical and radiological outcomes of novel dynamic navigation (DN)-aided endodontic microsurgery (EMS), with an analysis of potential prognostic factors. MATERIALS AND METHODS: Forty-six teeth from 32 patients who received DN-aided EMS were included. Clinical and radiographic assessments were performed at least 1 year postoperatively. Two calibrated endodontists assessed radiological outcomes according to two-dimensional (2D) periapical radiography (PA) and three-dimensional (3D) cone-beam computed tomography (CBCT) imaging using Rud's and Molven's criteria and modified PENN 3D criteria, respectively. Fisher's exact test was used for statistical analysis of the predisposing factors. RESULTS: Of the 32 patients with 46 treated teeth, 28 with 40 teeth were available for follow-up. Of the 28 patients, four (five teeth) refused to undergo CBCT and only underwent clinical and PA examinations, and the remaining 24 (35 teeth) underwent clinical, PA, and CBCT examinations. Combined clinical and radiographic data revealed a 95% (38/40) success rate in 2D healing evaluations and a 94.3% (33/35) success rate in 3D healing evaluations. No significant effect was found in sex, age, tooth type, arch type, preoperative lesion volume, preoperative maximum lesion size, presence/absence of crown and post, and the root canal filling state on the outcome of DN-aided EMS. CONCLUSIONS: DN-aided EMS has a favorable prognosis and could be considered an effective and reliable treatment strategy. Further investigations with larger sample sizes are required to confirm these results. CLINICAL RELEVANCE: DN-aided EMS could be considered an effective and reliable treatment strategy.

Genome-wide meta-analyses identify five new risk loci for nonsyndromic orofacial clefts in the Chinese Han population.

BACKGROUND: Nonsyndromic orofacial clefts (NSOFCs) are the most common craniofacial birth malformations in humans and are generally classified as nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Genome-wide association studies (GWASs) of NSOFCs have demonstrated multiple risk loci and candidate genes; however, published risk factors are able to explain only a small fraction of the observed NSOFCs heritability. METHODS: Here, we performed GWASs of 1615 NSCPO cases and 2340 controls, and then conducted genome-wide meta-analyses of NSOFCs, totaling 6812 NSCL/P cases, 2614 NSCPO cases, and 19,165 controls from the Chinese Han population. RESULTS: We identify 47 risk loci with genome-wide pmeta -value <5.0 × 10-8 , 5 risk loci (1p32.1, 3p14.1, 3p14.3, 3p21.31, and 13q22.1) of which are new. All of the 47 susceptibility loci conjointly account for 44.12% of the NSOFCs' heritability in the Chinese Han population. CONCLUSION: Our results improve the comprehending of genetic susceptibility to NSOFCs and provide new views into the genetic etiology of craniofacial anomalies.

Analysis of the accuracy of a dynamic navigation system in endodontic microsurgery: A prospective case series study.

OBJECTIVES: To evaluate the accuracy of a dynamic navigation system (DNS) for guided osteotomy and root-end resection during endodontic microsurgery (EMS) and assess its prognosis. METHODS: Nine patients who met inclusion criteria underwent DNS-guided EMS. Osteotomy and root-end resection were performed with assistance of DNS (DHC-ENDO1, DCARER Medical Technology, Suzhou, China). The preoperative virtually planned path and postoperative cone-beam computed tomography images were superimposed using DNS software. Accuracy was assessed based on deviations in the platform, apex, and angle of the osteotomy, as well as in the length and angle of the root-end resection. Follow-up evaluations were performed after at least a year postoperatively. RESULTS: Among the nine patients (11 teeth with 12 roots), the mean platform, apex, and angular deviation of the osteotomy were 1.05 mm, 1.2 mm, and 6.24°, respectively. The mean length and angle deviation of the root-end resection were 0.46 mm and 4.9°, respectively. Significant differences were observed according to tooth position. The platform and apex deviated significantly less in the posterior than in the anterior teeth (p < .05). No significant differences were observed according to arch type, side, and depth of the surgical path (p > .05). Eight patients were evaluated after at least a year postoperatively; clinical and radiographic evaluation revealed a 90% success rate (9/10 teeth). CONCLUSIONS: This study demonstrated high accuracy of DNS in EMS. Furthermore, DNS-guided EMS had a success rate similar to that of freehand EMS over a short-term follow-up. Further study with a larger sample size is necessary. CLINICAL SIGNIFICANCE: The present novel DNS technology is a viable method for guided osteotomy and root-end resection in EMS. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR2100042312.

The effects of dimethyl fumarate on cytoplasmic LPS-induced noncanonical pyroptosis in periodontal ligament fibroblasts and dental pulp cells.

AIM: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs. METHODOLOGY: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT. RESULTS: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs. CONCLUSIONS: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis.

Mutation analysis in patients with nonsyndromic tooth agenesis using exome sequencing.

BACKGROUND: Tooth agenesis (TA) is a congenital abnormality that may present as syndromic or nonsyndromic. Considering its complex genetic aetiology, the aim of this study was to uncover the pathogenic mutants in patients with nonsyndromic TA and analyse the characteristics of these mutants. METHODS: Exome sequencing was performed to detect pathogenic variants in 72 patients from 43 unrelated families with nonsyndromic TA. All candidate variants were validated using Sanger sequencing. Bioinformatics and conformational analyses were performed to determine the pathogenic mechanisms of the mutants. RESULTS: The following eight mutations (six novel and two known) in six genes were identified in eight families: WNT10A [c.742C > T (p.R248*)], LRP6 [c.1518G > A (p.W506*), c.2791 + 1G > T], AXIN2 [c.133_134insGCCAGG (p.44_45insGQ)], PAX9 [c.439C > T (p.Q147*), c.453_454insCCAGC (p.L154QfsTer60)], MSX1 [c.603_604del (p.A203GfsTer10)] and PITX2 [c.522C > G (p.Y174*)]. Bioinformatics and conformational analyses showed that the protein structures were severely altered in these mutants, and indicated that these structural abnormalities may cause functional disabilities. CONCLUSIONS: Our study extends the mutation spectrum in patients with nonsyndromic TA and provides valuable data for genetic counselling. The pathogenic mechanisms of TA in patients/families with unknown causative variants need to be explored further.

Circulating small extracellular vesicles from patients with periodontitis contribute to development of insulin resistance.

BACKGROUND: Epidemiological studies have identified the role of periodontitis in the pathogenesis of type 2 diabetes, but the underlying mechanism is poorly understood. It is well-known that small extracellular vesicles are lipid bilayer vesicles derived from cells with a diameter around 30 to 200 nm. The purpose of this study was to investigate whether periodontitis induced or exacerbated insulin resistance via circulating small extracellular vesicles. METHODS: Plasma small extracellular vesicles from control and periodontitis rats were intravenously injected into type 2 diabetic rats. Insulin tolerance tests, glucose tolerance tests, and the activation of the insulin signaling pathway were measured to detect the effect of the plasma small extracellular vesicles on insulin sensitivity. In addition, circulating small extracellular vesicles from patients with periodontitis with or without diabetes were isolated and co-cultured with HepG2 cells. The ability of glucose uptake was assessed using the fluorescence of 2-NBDG via flow cytometry. The activation of insulin signaling pathway was examined via Western blotting. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of enzyme related to glycolysis and gluconeogenesis. RESULTS: Small extracellular vesicles derived from the plasma of periodontitis rats further impaired glucose tolerance and insulin tolerance in diabetic rats and significantly reduced the activation of the insulin signaling pathway in liver tissues, as evidenced by the decreased levels of p-AKT and p-GSK3ß and the reduced hepatic glycogen content. For small extracellular vesicles isolated from human plasma, the concentration of small extracellular vesicles in patients with type 2 diabetes combined with periodontitis was higher than that of the healthy control and periodontitis alone. Moreover, circulating small extracellular vesicles from patients with periodontitis significantly inhibited the glucose uptake capacity and inhibited insulin signaling of HepG2 cells. CONCLUSION: Periodontitis acted as a contributing factor to exacerbate insulin resistance of type 2 diabetic rats. Plasma small extracellular vesicles played a critical role in periodontitis aggravating insulin resistance.

Endodontic Microsurgery of Posterior Teeth with the Assistance of Dynamic Navigation Technology: A Report of Three Cases.

When nonsurgical endodontic treatment fails, surgical treatment is an alternative approach for treating periapical disease. However, endodontic microsurgery (EMS), particularly in anatomically challenging areas, such as the posterior teeth, is a skill-sensitive task that can present a unique set of challenges for the surgeon. In recent years, digital guidance technology has been applied more frequently in dentistry. Dynamic navigation (DN) is a pioneering technology that uses an optical positioning device controlled by a sophisticated computerized interface and dedicated three-dimensional surgical path planning software program. This technique has also recently been introduced in the field of EMS to improve accuracy and avoid related complications. This case report presents a novel approach to DN-assisted EMS and describes its application in posterior teeth. After undergoing DN-assisted EMS, all patients were completely asymptomatic at the follow-up visit. Radiographic examinations performed immediately and 3-9 months after EMS revealed that the root resection was performed accurately without complications. The DN technique has been proven to be a feasible, predictable, and time-saving system for assisting EMS in cases requiring treatment in anatomically challenging areas, such as in the posterior teeth.

Mesenchymal Mycn participates in odontoblastic lineage commitment by regulating Krüppel-like Factor 4 (Klf4) in mice.

BACKGROUND: Commitment of mouse dental papilla cells (mDPCs) to the odontoblast lineage is critical for dentin formation, and this biological process is regulated by a complex transcription factor network. The transcription factor Mycn is a proto-oncogene that plays an important role in tumorigenesis and normal embryonic development. An early study revealed that Mycn is exclusively expressed in dental mesenchymal cells at E15.5, which implies a potential role of Mycn in dentinogenesis. However, the role of Mycn in dentin formation remains elusive. Thus, it is of considerable interest to elucidate the role of Mycn in dentin formation. METHODS: Mycnfl/fl; Osr2IresCre (MycnOsr2) and Mycnfl/fl; K14Cre (MycnK14) transgenic mice were generated, and micro-CT scans were performed to quantitatively analyse the volumetric differences in the molars and incisors of the mutants and their littermates. Mycn was also knocked down in vitro, and alkaline phosphatase (ALP) and alizarin red staining (ARS) were conducted. Cleavage under targets and tagmentation (CUT&Tag) analysis and dual luciferase assays were performed to identify direct downstream targets of Mycn. Immunofluorescence and immunochemistry staining and western blotting (WB) were performed to analyse the expression levels of potential targets. Quantitative PCR, WB, ALP and ARS were performed to test the rescue efficiency. RESULTS: Mesenchymal ablation of Mycn (MycnOsr2) led to defective dentin formation, while epithelial deletion (MycnK14) had no obvious effects on tooth development. ALP and ARS staining revealed that the commitment capacity of mDPCs to the odontoblast lineage was compromised in MycnOsr2 mice. CUT&Tag analysis identified Klf4 as a potential direct target of Mycn, and a dual luciferase reporter assay verified that Mycn could bind to the promotor region of Klf4 and directly activate its transcription. Reciprocally, forced expression of Klf4 partially recovered the odontoblastic differentiation capacity of mDPCs with Mycn knockdown. CONCLUSIONS: Our results elucidated that mesenchymal Mycn modulates the odontoblastic commitment of dental papilla cells by directly regulating Klf4. Our study illustrated the role of Mycn in dentin development and furthers our general comprehension of the transcription factor networks involved in the dentinogenesis process. Thus, these results may provide new insight into dentin hypoplasia and bioengineered dentin regeneration.

PIEZO1 Ion Channels Mediate Mechanotransduction in Odontoblasts.

INTRODUCTION: Odontoblasts, terminally differentiated dentin-forming cells with their processes that penetrate into dentin, have been considered potential sensory cells. Current research suggests that odontoblasts sense external stimuli and transmit pain signals. PIEZO1, as a specific mechanically activated ion channel, may play an important role in mechanical transduction in odontoblasts. In this study, we devoted to investigating the functions and underlying molecular mechanisms of PIEZO1 ion channels in odontoblast mechanotransduction. METHODS: Human dental pulp stem cells were cultured in vitro and induced to differentiate into odontoblast-like cells (OLCs). The expression of PIEZO1 protein in pulp, dental pulp stem cells, and OLCs was detected by immunohistochemistry or immunofluorescence. The mechanical sensitivity of OLCs was detected by a constructed fluid shear stress model and examined by calcium fluorescence intensity. A single-cell mechanical stimulation model was used to detect the PIEZO1 electrophysiological properties of OLCs. Yoda1 (a PIEZO1-specific agonist), GsMTx4 (a PIEZO1 antagonist), and non-calcium ion extracellular solution were utilized to confirm PIEZO1 mechanotransduction in OLCs in both fluid shear stress and single-cell mechanical stimulation assays. The amount of ATP released by OLCs was measured under stimulation with Yoda1 and GsMTx4. Rat trigeminal ganglion neurons were cultured in vitro and detected by whole-cell patch-clamp recording under ATP stimulation. RESULTS: PIEZO1 ion channels were positively expressed in OLCs and odontoblastic bodies and processes but weakly expressed in dental pulp cells. After the treatment of OLCs with shearing stress or Yoda1, the fluorescence intensity of intracellular calcium ions increased rapidly but did not noticeably change after treatment with GsMTx4 or the non-calcium ion extracellular solution. When single-cell mechanical stimuli were applied to OLCs, the evoked inward currents were recorded by patch-clamp electrophysiology. The inward currents increased and current inactivation became slower after Yoda1 treatment, but these currents almost completely disappeared after the addition of GsMTx4. The amount of ATP released by OLCs increased significantly after Yoda1 stimulation, while GsMTx4 reversed the release of ATP. Whole-cell patch-clamp detection showed that ATP evoked slow inward currents and increased the frequency of action potentials of trigeminal ganglion neurons. CONCLUSIONS: Taken together, these findings indicated that odontoblasts evoked a fast inward current via PIEZO1 ion channels after the application of external mechanical stimuli and released ATP to transmit signals to adjacent cells. Thus, PIEZO1 ion channels in odontoblasts mediate mechanotransduction under various pathophysiological conditions in dentin.

Mycn deficiency underlies the development of orofacial clefts in mice and humans.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is the most common subphenotype of non-syndromic orofacial clefts arising from genetic and/or environmental perturbations during embryonic development. We previously identified 2p24.2 as a risk locus associated with NSCL/P in the Chinese Han population, and MYCN is a candidate risk gene in this region. To understand the potential function of MYCN in craniofacial development, we generated Wnt1-Cre;Mycnflox/flox mice that exhibited cleft palate, microglossia and micrognathia, resembling the Pierre Robin sequence (PRS) in humans. Further analyses indicated that the cleft palate was secondary to the delayed elevation of palatal shelves caused by micrognathia. The micrognathia resulted from impaired chondrogenic differentiation in Merkel's cartilage, which limited tongue development, leading to microglossia. In terms of mechanism, Mycn deficiency in cranial neural crest cells (CNCCs) downregulated Sox9 expression by inhibiting Wnt5a in a CNCC-derived chondrogenic lineage in Merkel's cartilage. To investigate whether MYCN deficiency contributed to NSCL/P, we performed direct sequencing targeting all exons and exon-intron boundaries of MYCN in 104 multiplex families with Mendelian NSCL/P and identified a novel pathogenic variant in MYCN. Taken together, our data indicate that ablation of Mycn in mouse CNCCs could resemble PRS by suppressing the Wnt5a-Sox9 signaling pathway in Merkel's cartilage and that mutations in MYCN may be novel potential causes of NSCL/P.

Identification of maternal serum biomarkers for prenatal diagnosis of nonsyndromic orofacial clefts.

Nonsyndromic orofacial clefts (NSOFCs) are the most common congenital defects in the oral and maxillofacial regions. It is mainly diagnosed prenatally through fetal ultrasonography. However, the accuracy of ultrasonography for NSOFC is unreliable. Maternal serological screening is a noninvasive method for the diagnosis of fetal malformations. In our study, we sought to identify specific biomarkers in maternal serum for predicting NSOFC prenatally. We quantified the alterations in maternal serum protein profiles between 20 pregnant women with NSOFC fetuses and 20 pregnant women with healthy fetuses by using isobaric tags for relative and absolute quantitation-based mass spectrometry (MS). The serum levels of 75 elevated and 50 decreased proteins in the NSOFC group were detected. Twenty-eight candidate biomarkers were selected for further confirmation by multiple reaction monitoring-MS; of these, 16 proteins were found to be significantly different. More importantly, the levels of three proteins (APOA, HPT, and CRP) were verified by ELISAs to be obviously altered in serum from pregnancies carrying fetuses with NSOFC. Our results indicate that analysis of the maternal serum proteome is a feasible strategy for biomarker discovery of NSOFC, and APOA, HPT, and CRP proteins are potential serum biomarkers for prenatal diagnosis of NSOFC.

Landscape of Cell Communication in Human Dental Pulp.

The cellular atlas of the stroma is not well understood. Here, the cell populations in human dental pulp through single-cell RNA sequencing are profiled. Dental pulp stem cells, pulp cells, T cells, macrophages, endothelial cells, and glial cells are identified in human dental pulp. These cells support each other through sending growth signals. Based on the appearance of ligand-receptor pairs between two cell populations, pulp cells have the greatest communication with other cell types, while T cells have the least communication. In addition, T cells expressing TLR1, TLR2, and TLR4, and endothelial cells expressing TLR4, monitor bacterial invasion. These findings provide the census of normal dental pulp.