Periodontitis is positively linked to cardiovascular disease (CVD), diabetes, cancer, and increased mortality. Empirically derived clusters of IgG antibodies against 19 selected periodontal microorganisms have been associated with hyperglycemia. We further investigated associations between these serum IgG antibody clusters and all-cause and CVD mortality in a representative US population. Participants free of CVD and cancer and aged >= 40 y at baseline (N = 6,491) from the Third National Health and Nutrition Examination Survey (1988 to 1994) were followed up until December 31, 2011. Antibodies were categorized into 4 clusters: red-green, orange-red, yellow-orange, and orange-blue. Over a 23-y follow-up, 2,702 deaths occurred, including 810 CVD-related deaths. In fully adjusted Cox proportional hazard models, the red-green cluster was positively associated with all-cause mortality (tertile 3 vs. tertile 1: hazard ratio [HR] = 1.43, 95% CI = 1.08 to 1.90, P = 0.015). The yellow-orange cluster was inversely associated with all-cause mortality (tertile 3 vs. tertile 1: HR = 0.78, 95% CI = 0.63 to 0.97, P = 0.028) and CVD mortality (tertile 2 vs. tertile 1: HR = 0.57, 95% CI = 0.42 to 0.77, P = 0.005). The orange-blue cluster (composed of antibodies against Eubacterium nodatum and Actinomyces naeslundii) was inversely associated with all-cause mortality (tertile 3 vs. tertile 1: HR = 0.65, 95% CI = 0.55 to 0.78, P < 0.0001) and CVD mortality (tertile 3 vs. tertile 1: HR = 0.65, 95% CI = 0.47 to 0.88, P = 0.007). These antibodies could predict prognosis or be potential intervention targets to prevent systemic effects of periodontal disease if further studies establish a causal relationship.
Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (ASvicR) associated with an adjacent RNase III-encoding (rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the ASvicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing ASvicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from ASvicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5 '-UTR regions of the vicR gene. Furthermore, ASvicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III-encoding genes. There was an increase in ASvicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of ASvicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of ASvicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.
Vertical malocclusion is a developmental condition, resulting from complex interactions among multiple etiological factors during the growth period. As a tricky dentofacial deformity clinically, long-face (LF) morphology is characterized by excessive vertical facial growth with severe disarrangement of jaws and teeth. Since the improvement of LF patients on facial profile and occlusion is often difficult and lacks long-term stability, it becomes important to unravel the etiology of LF pattern formation for early prevention and treatment. In the current studies, we identified a transgenic mouse model that exhibited a dysplastic coronoid process and LF morphology. Although the mutant mice exhibited jaw structures and occlusion comparable to controls at birth, they all acquired typical LF morphology with anterior open bite during postnatal growth, resembling clinical features of the selected skeletal class III patients. Since the coronoid process provides an insertion site for the temporalis attachment, we examined the initial development and differentiation of the temporalis and found identical results in both control and mutant mice before E17.5 when the temporal muscle makes attachment to the coronoid process. However, thereafter, we observed altered orientation and reduced size of the cross-sectional area of the temporalis in mutant mice, which persisted to the weaning stage. Biomechanical analysis and simulation modeling further support the idea that altered morphology of the coronoid process may impair the efficiency of the vertical temporalis contraction and appears to correlate with LF formation. Consistently, we present evidence that a dysplastic mandibular coronoid process was also seen in some human patients with skeletal III LF morphology. Taken together, the results presented in this study establish an association of the craniofacial bony structures with vertical patterning, which will have implications in earlier prediction for clinical precaution and intervention.
Reprogramming diseased cells with mutated genes into induced pluripotent stem cells (iPSCs) can allow studies of disease mechanism and correct the mutation. Oculofaciocardiodental (OFCD) syndrome is a developmental disorder caused by heterozygous mutations in the X-linked BCL-6 corepressor (BCOR) gene. In this present study, we aimed to reprogram stem cells from a tooth apical papilla (SCAP) of a patient with OFCD, termed SCAP-O, into iPSCs. The SCAP-O carry a copy of the BCOR gene having 1 nucleotide deletion in 1 of the alleles, therefore harboring a mixture of cells expressing either normal (SCAP-OBCOR-WT) or mutated (SCAP-OBCOR-mut) BCOR transcripts. We subcloned SCAP-O and separated SCAP-OBCOR-WT and SCAP-OBCOR-mut as verified by sequencing. The selected subclone SCAP-OBCOR-mut expressed only the mutated BCOR transcripts and remained in such condition after multiple passages. We reprogrammed SCAP-O and subclone SCAP-OBCOR-mut into transgene-free iPSCs using an excisable lentiviral vector system (hSTEMCCA-loxP) carrying 4 reprogramming factors in a single cassette, followed by removal of transgenes via Cre-mediated excision. We found that after reprogramming SCAP-O or subclone SCAP-OBCOR-mut into iPSCs, some of the iPSC clones expressed either solely the normal BCOR-WT or BCOR-mut transcripts, while other clones expressed both BCOR-WT and BCOR-mut transcripts. This is our first step toward establishing OFCD study models by generating isogenic control BCOR-WT iPSCs versus BCOR-mut iPSCs.
Dentin is an important structural component of the tooth. Odontoblast differentiation is an essential biological process that guarantees normal dentin formation, which is precisely regulated by various proteins. Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase, and it plays a pivotal role in the differentiation of different cell types, such as osteoblasts and myoblasts. However, whether Mdm2 plays a role in odontoblast differentiation remains unknown. Here, we investigated the spatiotemporal expression of Mdm2 by immunostaining and found that Mdm2 was highly expressed in the odontoblasts and slightly in the dental papilla cells of mouse incisors and molars. Gene knockdown and overexpression experiments verified that Mdm2 promoted the odontoblast-like differentiation of mouse dental papilla cells (mDPCs). Intranuclear colocalization and physical interaction between Mdm2 and distal-less 3 (Dlx3), a transcription factor important for odontoblast differentiation, was found during the odontoblast-like differentiation of mDPCs by double immunofluorescence and immunoprecipitation. Mdm2 was proved to monoubiquitinate Dlx3, which enhanced the expression of Dlx3 target gene Dspp. In addition, p53, the canonical substrate of Mdm2, was validated to be also ubiquitinated but degraded by Mdm2 during the odontoblast-like differentiation of mDPCs. Gene knockdown experiments confirmed that p53 inhibited the odontoblast-like differentiation of mDPCs. p53 and Mdm2 double knockdown partially rescued the reduced odontoblast-like differentiation by knockdown of Mdm2 alone. Taken together, our study revealed that Mdm2 promoted the odontoblast-like differentiation of mDPCs by ubiquitinating both Dlx3 and p53. On one hand, the monoubiquitination of Dlx3 by Mdm2 led to upregulation of Dspp, which is a marker of the odontoblast differentiation. On the other hand, ubiquitination of p53 by Mdm2 resulted in its degradation, which eliminated the inhibitory effect of p53 on the odontoblast-like differentiation of mDPCs.
Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate-dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate-dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in Ildr1(-/-) mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function.
Tooth morphogenesis involves dynamic changes in shape and size as it proceeds through the bud, cap, and bell stages. This process requires exact regulation of cell proliferation and differentiation. Smad7, a general antagonist against transforming growth factor-beta (TGF-beta) signaling, is necessary for maintaining homeostasis and proper functionality in many organs. While TGF-beta signaling is widely involved in tooth morphogenesis, the precise role of Smad7 in tooth development remains unknown. In this study, we showed that Smad7 is expressed in the developing mouse molars with a high level in the dental epithelium but a moderate to weak level in the dental mesenchyme. Smad7 deficiency led to a profound decrease in tooth size primarily due to a severely compromised cell proliferation capability in the dental epithelium. Consistent with the tooth shrinkage phenotype, RNA sequencing (RNA-seq) analysis revealed that Smad7 ablation downregulated genes referred to epithelial cell proliferation and cell cycle G1/S phase transition, whereas the upregulated genes were involved in responding to TGF-beta signaling and cell cycle arrest. Among these genes, the expression of Cdkn1a (encoding p21), a negative cell proliferation regulator, was remarkably elevated in parallel with the diminution of Ccnd1 encoding the crucial cell cycle regulator cyclin D1 in the dental epithelium. Meanwhile, the expression level of p-Smad2/3 was ectopically elevated in the developing tooth germ of Smad7 null mice, indicating the hyperactivation of the canonical TGF-beta signaling. These effects were reversed by addition of TGF-beta signaling inhibitor in cell cultures of Smad7(-/-) molar tooth germs, with rescued expression of cyclin D1 and cell proliferation rate. In sum, our studies demonstrate that Smad7 functions primarily as a positive regulator of cell proliferation via inhibition of the canonical TGF-beta signaling during dental epithelium development and highlight a crucial role for Smad7 in regulating tooth size.
Fibroblast growth factor receptor 2 (FGFR2) in craniofacial bones mediates osteoprogenitor proliferation, differentiation, and apoptosis. The distortion of proper craniofacial bone growth may cause class II and class III skeletal malocclusion and result in compromised function and aesthetics. Here, we investigated the association between variations in FGFR2 and skeletal malocclusions. First, 895 subjects were included in a 2-stage case-control study with independent populations (stage 1: n = 138 class I, 111 class II, and 81 class III; stage 2: n = 279 class I, 187 class II, and 99 class III). Eight candidate single-nucleotide polymorphisms (SNPs) in FGFR2 were screened and validated. Five SNPs (rs2162540, rs2981578, rs1078806, rs11200014, and rs10736303) were found to be associated with skeletal malocclusions (all P < 0.05). That is, rs2162540 was significantly associated with skeletal class II malocclusion, while others were associated with skeletal class III malocclusion. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed that the common genotypes of rs2981578 and rs10736303 contained the binding sites of RUNX2 and SMAD4. Compared with the common genotypes, the minor genotypes at these 2 SNPs decreased the binding affinity and enhancer effect of RUNX2 and SMAD4, as well the levels of FGFR2 expression. In addition, FGFR2 expression contributed positively to osteogenic differentiation in vitro. Thus, we identified FGFR2 as a skeletal malocclusion risk gene, and FGFR2 polymorphisms regulated its transcriptional expression and then osteogenic differentiation.
The oral cavity contains a distinct habitat that supports diverse bacterial flora. Recent observations have provided additional evidence that sRNAs are key regulators of bacterial physiology and pathogenesis. These sRNAs have been divided into 5 functional groups: cis-encoded RNAs, trans-encoded RNAs, RNA regulators of protein activity, bacterial CRISPR (clustered regularly interspaced short palindromic repeat) RNAs, and a novel category of miRNA-size small RNAs (msRNAs). In this review, we discuss a critical group of key commensal and opportunistic oral pathogens. In general, supragingival bacterial sRNAs function synergistically to fine-tune the regulation of cellular processes and stress responses in adaptation to environmental changes. Particularly in the cariogenic bacteria Streptococcus mutans, both the antisense vicR RNA and msRNA1657 can impede the metabolism of bacterial exopolysaccharides, prevent biofilm formation, and suppress its cariogenicity. In Enterococcus faecalis, selected sRNAs control the expression of proteins involved in diverse cellular processes and stress responses. In subgingival plaques, sRNAs from periodontal pathogens can function as novel bacterial signaling molecules that mediate bacterial-human interactions in periodontal homeostasis. In Porphyromonas gingivalis, the expression profiles of putative sRNA101 and sRNA42 were found to respond to hemin availability after hemin starvation. Regarding Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), a major periodontal pathogen associated with aggressive periodontitis, the predicted sRNAs interact with several virulence genes, including those encoding leukotoxin and cytolethal distending toxin. Furthermore, in clinical isolates, these associated RNAs could be explored not only as potential biomarkers for oral disease monitoring but also as alternative types of regulators for drug design. Thus, this emerging subspecialty of bacterial regulatory RNAs could reshape our understanding of bacterial gene regulation from their key roles of endogenous regulatory RNAs to their activities in pathologic processes.
Doctors and patients attempt to accelerate orthodontic tooth movement with a minimally invasive surgery approach. The purpose of this systematic review was to evaluate the evidence of accelerated tooth movement in minimally invasive surgery and the adverse effects from it. A systematic search of the literature was performed in the electronic databases of PubMed, CENTRAL (Cochrane Central Register of Controlled Trials), Embase, Scopus, Web of Science, Science Direct, and Medline and was complemented by a manual search until February 2019. The inclusion criteria were prospective clinical studies of patients treated with a fixed appliance, and the intervention was accelerated orthodontic treatment with minimally invasive surgery. Nineteen articles (538 participants) were included in the review: 9 studies assessed the rate of upper canine movement; 5 considered the treatment time; 1 evaluated the en masse retraction time; and 4 studied adverse effects. We performed a meta-analysis for the rate of canine movement and treatment time and described the results for the adverse effects in a systematic review. The results of the subgroup analysis according to micro-osteoperforation and piezocision were included in the study. No accelerated tooth movement was found in the micro-osteoperforation group. After flapless corticotomy procedures, increased tooth movement rates were identified by weighted mean differences of 0.63 (95%CI = 0.22, 1.03, P = 0.003) and 0.64 (95% CI, -25 to 1.53; P = 0.16) for 1 and 2 mo, respectively. The mean treatment time was 68.42 d (95% CI, -113.19 to -23.65; P = 0.003) less that than for minimally invasive surgery. Moreover, no significant adverse effect was found. Because of the high heterogeneity of the meta-analysis, the results must be validated by additional large-sample multicenter clinical trials. There is not sufficient evidence to support that the single use of micro-osteoperforation could accelerate tooth movement, and there is only low-quality evidence to prove that flapless corticotomy could accelerate tooth movement.
T cells and dendritic cells (DCs) that are positive for the tissue-resident marker CD103 play a vital role in antitumor immunity. In this study, multiplexed immunohistochemistry was applied to stain CD103 and the T-cell marker CD8 as well as the DC marker CD11c on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues. Then, the density of CD103(+)CD8(+) and CD103(+)CD11c(+) tumor-infiltrating lymphocytes (TILs) in the intratumoral and stromal regions was calculated, and the correlation of CD103(+)CD8(+) TIL and CD103(+)CD11c(+) TIL density with OSCC patient prognosis was analyzed. The results revealed that CD103(+)CD8(+) TILs and CD103(+)CD11c(+) TILs were abundant in the stromal region and that increased stromal CD103(+)CD8(+) TIL and intratumoral CD103(+)CD11c(+) TIL density indicated a favorable prognosis. Moreover, we freshly isolated TILs from OSCC samples and performed flow cytometry to verify that CD103(+)CD8(+) TILs display a tissue-resident memory T-cell (Trm) phenotype, and we discriminated CD103(+)CD11c(+) TILs from tumor-associated macrophages.
Postmenopausal osteoporosis (PMO) is a risk factor for periodontitis, and current therapeutics against PMO prevent the aggravated alveolar bone loss of periodontitis in estrogen-deficient women. Gut microbiota is recognized as a promising therapeutic target for PMO. Berberine extracted from Chinese medicinal plants has shown its effectiveness in the treatment of metabolic diseases such as obesity and diabetes via regulating gut microbiota. Here, we hypothesize that berberine ameliorates periodontal bone loss by improving the intestinal barriers by regulating gut microbiota under an estrogen-deficient condition. Experimental periodontitis was established in ovariectomized (OVX) rats, and the OVX-periodontitis rats were treated with berberine for 7 wk before sacrifice for analyses. Micro-computed tomography and histologic analyses showed that berberine treatment significantly reduced alveolar bone loss and improved bone metabolism of OVX-periodontitis rats as compared with the vehicle-treated OVX-periodontitis rats. In parallel, berberine-treated OVX-periodontitis rats harbored a higher abundance of butyrate-producing gut microbiota with elevated butyrate generation, as demonstrated by 16S rRNA sequencing and high-performance liquid chromatography analysis. Berberine-treated OVX-periodontitis rats consistently showed improved intestinal barrier integrity and decreased intestinal paracellular permeability with a lower level of serum endotoxin. In parallel, IL-17A-related immune responses were attenuated in berberine-treated OVX-periodontitis rats with a lower serum level of proinflammatory cytokines and reduced IL-17A(+) cells in alveolar bone as compared with vehicle-treated OVX-periodontitis rats. Our data indicate that gut microbiota is a potential target for the treatment of estrogen deficiency-aggravated periodontal bone loss, and berberine represents a promising adjuvant therapeutic by modulating gut microbiota.
Precise and efficient genetic manipulations have enabled researchers to understand gene functions in disease and development, providing a platform to search for molecular cures. Over the past decade, the unprecedented advancement of genome editing techniques has revolutionized the biological research fields. Early genome editing strategies involved many naturally occurring nucleases, including meganucleases, zinc finger nucleases, and transcription activator-like effector-based nucleases. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nucleases (CRISPR/Cas) system has greatly enriched genetic manipulation methods in conducting research. Those nucleases generate double-strand breaks in the target gene sequences and then utilize DNA repair mechanisms to permit precise yet versatile genetic manipulations. The oral and craniofacial field harbors a plethora of diseases and developmental defects that require genetic models that can exploit these genome editing techniques. This review provides an overview of the genome editing techniques, particularly the CRISPR/Cas9 technique, for the oral and craniofacial research community. We also discuss the details about the emerging applications of genome editing in oral and craniofacial biology.