Jue Xu’s research while affiliated with Sichuan University and other places

Aging is a complex multifactorial process that greatly affects animal health. Multi-omics analysis is widely applied in evolutionary biology and biomedical research. However, whether multi-omics can provide sufficient information to reveal comprehensive changes in aged non-human primates remains unclear. Here, we explored changes in host–microbe interactions with aging in Chinese rhesus macaques ( Macaca mulatta lasiota , CRs) using multi-omics analysis. Results showed marked changes in the oral and gut microbiomes between young and aged CRs, including significantly reduced probiotic abundance and increased pathogenic bacterial abundance in aged CRs. Notably, the abundance of Lactobacillus , which can metabolize tryptophan to produce aryl hydrocarbon receptor (AhR) ligands, was decreased in aged CRs. Consistently, metabolomics detected a decrease in the plasma levels of AhR ligands. In addition, free fatty acid, acyl carnitine, heparin, 2-(4-hydroxyphenyl) propionic acid, and docosahexaenoic acid ethyl ester levels were increased in aged CRs, which may contribute to abnormal fatty acid metabolism and cardiovascular disease. Transcriptome analysis identified changes in the expression of genes associated with tryptophan metabolism and inflammation. In conclusion, many potential links among different omics were found, suggesting that aged CRs face multiple metabolic problems, immunological disorders, and oral and gut diseases. We determined that tryptophan metabolism is critical for the physiological health of aged CRs. Our findings demonstrate the value of multi-omics analyses in revealing host–microbe interactions in non-human primates and suggest that similar approaches could be applied in evolutionary and ecological research of other species.

A novel biosensing method is reported for the fluorescence determination of an oral cancer protein biomarker using primer exchange reaction (PER)-synthesized DNAzyme. Specifically, the target protein is captured by antibody-functionalized magnetic beads and recruits antibody-nucleic acid probes through immunoreactions to form sandwich-like complexes. After magnetic separation, the nucleic acid part of the antibody-nucleic acid probe participates in the PER as a catalytic hairpin, mediating the extension of primers. The extended primer forms the complete sequence of RNA-cleaving DNAzyme to effectively catalyze the cleavage of molecular beacons and produce a significantly amplified fluorescence signal. Taking interleukin 6 as a model biomarker, the method allows quantitative determination of the target in a wide linear range from 5 fg/mL to 500 pg/mL with a detection limit of 2.3 fg/mL and shows desirable specificity and usability in biological samples. More importantly, the complete DNAzyme is synthesized by the target-powered PER, which not only ensures the detection specificity, but also enables the decrease of background signals, thus greatly improving the sensitivity. Therefore, the method may provide a valuable tool for the determination of oral cancer protein biomarkers and may expand applications of DNAzyme in biosensing.

As a kind of promising non-invasive biomarker, exosomes naturally occurring in saliva have recently attracted considerable attention in view of their potential use in the diagnosis of oral diseases. Herein, we propose a new electrochemical method for the sensitive and precise detection of salivary exosomes. A red blood cell membrane (RBCM) engineered with CD63 aptamer is the core element of the method and is used to camouflage a gold electrode, thus giving the electrode superior antifouling and targeting ability. Target exosomes presented in saliva are recognized and captured by the highly specific interaction between the exosomal CD63 and the aptamers engineered in RBCM. Then, silver nanoparticles modified with CD63 aptamers are recruited onto the electrode surface to generate significant electrochemical signals, which enables the sensitive detection of target exosomes. By using human oral squamous cell carcinoma CAL27 cell-derived exosomes as a model, the method allows target salivary exosome detection in a wide linear range from 5 × 102 to 1 × 106 particles per mL and a low detection limit of 2.07 × 102 particles per mL. Moreover, the method displays good reproducibility and is feasible for detecting target exosomes with high precision in saliva samples. Overall, the method may provide a useful tool for salivary exosome detection and may have great potential for practical use in the clinical diagnosis of oral diseases.

Dental pulp can initiate its damage repair after an injury of the pulp–dentin complex by rearrangement of odontoblasts and formation of newly differentiated odontoblast-like cells. Connexin 43 (Cx43) is one of the gap junction proteins that participates in multiple tissue repair processes. However, the role of Cx43 in the repair of the dental pulp remains unclear. This study aimed to determine the function of Cx43 in the odontoblast arrangement patterns and odontoblastic differentiation. Human teeth for in vitro experiments were acquired, and a pulp injury model in Sprague-Dawley rats was used for in vivo analysis. The odontoblast arrangement pattern and the expression of Cx43 and dentin sialophosphoprotein (DSPP) were assessed. To investigate the function of Cx43 in odontoblastic differentiation, we overexpressed or inhibited Cx43. The results indicated that polarized odontoblasts were arranged along the pulp–dentin interface and had high levels of Cx43 expression in the healthy teeth; however, the odontoblast arrangement pattern was slightly changed concomitant to an increase in the Cx43 expression in the carious teeth. Regularly arranged odontoblast-like cells had high levels of the Cx43 expression during the formation of mature dentin, but the odontoblast-like cells were not regularly arranged beneath immature osteodentin in the pulp injury models. Subsequent in vitro experiments demonstrated that Cx43 is upregulated during odontoblastic differentiation of the dental pulp cells, and inhibition or overexpression of Cx43 influence the odontoblastic differentiation. Thus, Cx43 may be involved in the maintenance of odontoblast arrangement patterns, and influence the pulp repair outcomes by the regulation of odontoblastic differentiation.

The first branchial arch (BA1), which is derived from cranial neural crest (CNC) cells, gives rise to various orofacial tissues. Cre mice are widely used for the determination of CNC and exploration of gene functions in orofacial development. However, there is a lack of Cre mice specifically marked BA1’s cells. Pax2 -Cre allele was previously generated and has been widely used in the field of inner ear development. Here, by compounding Pax2 -Cre and R26R- mTmG mice, we found a specific expression pattern of Pax2 ⁺ cells that marked BA1’s mesenchymal cells and the BA1-derivatives. Compared to Pax2 -Cre; R26R -mTmG allele, GFP ⁺ cells were abundantly found both in BA1 and second branchial arch in Wnt1 -Cre; R26R -mTmG mice. As BMP4 signaling is required for orofacial development, we over-activated Bmp4 by using Pax2 -Cre; pMes -BMP4 strain. Interestingly, our results showed bilateral hyperplasia between the upper and lower teeth. We also compare the phenotypes of Wnt1 -Cre; pMes -BMP4 and Pax2 -Cre; pMes -BMP4 strains and found severe deformation of molar buds, palate, and maxilla-mandibular bony structures in Wnt1 -Cre; pMes -BMP4 mice; however, the morphology of these orofacial organs were comparable between controls and Pax2 -Cre; pMes -BMP4 mice except for bilateral hyperplastic tissues. We further explore the properties of the hyperplastic tissue and found it is not derived from Runx2 ⁺ cells but expresses Msx1, and probably caused by abnormal cell proliferation and altered expression pattern of p-Smad1/5/8. In sum, our findings suggest altering BMP4 signaling in BA1-specific cell lineage may lead to unique phenotypes in orofacial regions, further hinting that Pax2 -Cre mice could be a new model for genetic manipulation of BA1-derived organogenesis in the orofacial region.

Haploinsufficiency of Meis homeobox 2 ( MEIS2 ), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 in palate development. However, its functional mechanisms remain unknown. Here, we observed widespread MEIS2 expression in the developing palate in mice. Wnt1 Cre -mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of Wnt1 Cre ; Meis2 f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with a complete absence of the palatal bones, results from integrative analyses of MEIS2 by ChIP-Seq, RNA-Seq, andassay for transposase-accessible chromatin (ATAC)-Seq identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP-Seq analyses revealed genome-wide co-occupancies of MEIS2 and SHOX2, in addition to their co-localization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2 -mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

Recent studies reveal a great value of interleukin-8 (IL-8), a pro-inflammatory cytokine, as a potent biomarker for early diagnosis of oral cancer. Herein, a new electrochemical method is proposed to detect IL-8 by facilely incorporating DNA-templated quantum dots (QDs). In principle, target IL-8 is first treated with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to yield active thiols and then captured by antibody-functionalized magnetic beads (MBs). Thereafter, via the Michael addition reaction between the active thiol and maleimide group, a maleimide-modified DNA probe is linked to the surface of MBs, which can initiate a process of rolling circle amplification. In this way, long-range DNA strands are generated on the MB surface, subsequently recruiting DNA-templated CdTe/CdS QDs (DNA-QDs) to act as electrochemical reporters. By tracing the responses of DNA-QDs, the method allows IL-8 detection in a linear range from 5 to 5000 fg/mL with a detection limit of 3.36 fg/mL. The selectivity, reproducibility, and applicability in complex serum samples are also demonstrated to be favorable, indicating that the method may have a great potential in the future. More importantly, the use of TCEP treatment in the method not only provides a facile way to incorporate DNA-QDs, avoiding the complicated and time-consuming preparation process of antibody-DNA conjugates or functional nanomaterials; but also makes the method capable of being extended to detect other protein biomarkers in view of widespread presence of disulfides, which may hold a broad potential to facilitate efficient biosensing designs.

Objective: Fibroblast growth factor 8 (FGF8) signaling is essential in regulating craniofacial osteogenesis. This study aims to explore the effect of altered FGF8 signaling in maxillomandibular development during embryogenesis. Materials and methods: Dmp1Cre ;R26RmTmG mice were generated to trace Dmp1+ cell lineage, and Dmp1Cre ;R26RFgf8 mice were generated to explore the effects of augmented FGF8 signaling in Dmp1+ cells on osteogenesis with a focus on maxillomandibular development during embryogenesis, as assessed by whole mount skeletal staining, histology, and immunostaining. Additionally, cell proliferation rate and the expression of osteogenic genes were examined. Results: Osteocytes of maxillomandibular bones were found Dmp1 positive prenatally and Fgf8 over-expression in Dmp1+ cells led to mandibular hypoplasia. While Dmp1Cre allele functions in the osteocytes of the developing mandibular bone at as early as E13.5, and enhanced cell proliferation rate is observed in the bone forming region of the mandible in Dmp1Cre ;R26RFgf8 mice at E14.5, histological examination showed that osteogenesis was initially impacted at E15.5, along with an inhibition of osteogenic differentiation markers. Conclusions: Augmented FGF8 signaling in Dmp1+ cells lead to osteogenic deficiency in the mandibular bones, resulting in mandibular hypoplasia.

During mammalian palatogenesis, cranial neural crest–derived mesenchymal cells undergo osteogenic differentiation and form the hard palate, which is divided into palatine process of the maxilla and the palatine. However, it remains unknown whether these bony structures originate from the same cell lineage and how the hard palate is patterned at the molecular level. Using mice, here we report that deficiency in short stature homeobox 2 (Shox2), a transcriptional regulator whose expression is restricted to the anterior palatal mesenchyme, leads to a defective palatine process of the maxilla, but does not affect the palatine. Shox2 overexpression in palatal mesenchyme resulted in a hyperplastic palatine process of the maxilla and a hypoplastic palatine. RNA-Seq and assay for transposase-accessible chromatin (ATAC)-Seq analyses revealed that SHOX2 controls the expression of pattern-specification and skeletogenic genes associated with accessible chromatin in the anterior palate. This highlighted a lineage-autonomous function of SHOX2 in patterning and osteogenesis of the hard palate. H3K27ac ChIP-Seq and transient transgenic enhancer assays revealed that SHOX2 binds distal-acting cis-regulatory elements in an anterior palate–specific manner. Our results suggest that the palatine process of the maxilla and palatine arise from different cell lineages and differ in ossification mechanisms. SHOX2 evidently controls osteogenesis of a cell lineage and contributes to the palatine process of the maxilla by interacting with distal cis-regulatory elements to regulate skeletogenic gene expression and to pattern the hard palate. Genome-wide SHOX2 occupancy in the developing palate may provide a marker for identifying active anterior palate–specific gene enhancers.