Comparisons of different extraction methods and solvents for saliva samples.

INTRODUCTION: Changes in the categories and concentrations of salivary metabolites may be closely related to oral, intestinal or systemic diseases. To study salivary metabolites, the first analytical step is to extract them from saliva samples as much as possible, while reducing interferences to a minimum. Frequently used extraction methods are protein precipitation (PPT), liquid-liquid extraction (LLE) and solid-phase extraction (SPE), with various organic solvents. The types and quantities of metabolites extracted with different methods may vary greatly, but few studies have systematically evaluated them. OBJECTIVES: This study aimed to select the most suitable methods and solvents for the extraction of saliva according to different analytical targets. METHODS: An untargeted metabolomics approach based on liquid chromatography-mass spectrometry was applied to obtain the raw data. The numbers of metabolites, repeatability of the data and intensities of mass spectrometry signals were used as evaluation criteria. RESULTS: PPT resulted in the highest coverage. Among the PPT solvents, acetonitrile displayed the best repeatability and the highest coverage, while acetone resulted in the best signal intensities for the extracted compounds. LLE with the mixture of chloroform and methanol was the most suitable for the extraction of small hydrophobic compounds. CONCLUSION: PPT with acetonitrile or acetone was recommended for untargeted analysis, while LLE with the mixture of chloroform and methanol was recommended for small hydrophobic compounds.

The Nrf2 Pathway Alleviates Overloading Force-Induced TMJ Degeneration by Downregulating Oxidative Stress Reactions.

Objective: Oxidative stress is involved in the mechanisms associated with temporomandibular joint (TMJ) diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial oxidative stress marker, but the specific mechanisms of its regulation in the early stages of mandibular condylar cartilage (MCC) degeneration remain unclear. This study aimed to explore the regulatory role of Nrf2 and its related oxidative stress signaling pathway in the early stage of MCC degeneration. Materials and Methods: Overloading force-induced MCC degeneration was performed in wild-type and Nrf2 knockout mice, as well as in mice after treatment with the Nrf2 activator cardamonin. Changes in MCC degeneration and the expression of oxidative stress markers in the corresponding situations were observed. Results: Nrf2 and NADPH oxidase 2 (NOX2) expression were elevated during early MCC degeneration induced by an overloading force. MCC degeneration was aggravated when Nrf2 was knocked out, accompanied by increased NOX2 and superoxide dismutase 2 (SOD2) expression. The MCC degeneration process was alleviated after cardamonin treatment, with activation of the Nrf2 pathway and decreased NOX2 and SOD2 expression. Conclusion: Early MCC degeneration is accompanied by mild oxidative stress progression. Activated Nrf2 and related pathways could alleviate the degeneration of MCC.

Prognostic potential of serum mesencephalic astrocyte-derived neurotrophic factor in acute intracerebral hemorrhage: a prospective observational study.

OBJECTIVE: Mesencephalic astrocyte-derived neurotrophic factor (MANF) expressions are dramatically up-regulated in injured brain tissues, thereby conferring neurological protective effects. We intended to determine significance of serum MANF as a prognostic biomarker of intracerebral hemorrhage (ICH). METHODS: In this prospective, observational study done from February 2018 to July 2021, 124 patients with new-onset primary supratentorial ICH were consecutively enrolled. Also, a group of 124 healthy individuals constituted controls. Their serum MANF levels were detected using the Enzyme-Linked Immunosorbent Assay. National Institutes of Health Stroke Scale (NIHSS) and hematoma volume were designated as the two severity indicators. Early neurologic deterioration (END) was referred to as an increase of 4 or greater points in NIHSS scores or death at post-stroke 24 h. Post-stroke 90-day modified Rankin scale (mRS) scores of 3-6 was considered as a poor prognosis. Serum MANF levels were analyzed using multivariate analysis with respect to its association with stroke severity and prognosis. RESULTS: Patients, in comparison to controls, displayed markedly elevated serum MANF levels (median, 24.7 versus 2.7 ng/ml; P < 0.001), and serum MANF levels were independently correlated with NIHSS scores (beta, 3.912; 95% confidence interval (CI), 1.623-6.200; VIF = 2.394; t = 3.385; P = 0.002), hematoma volumes (beta, 1.688; 95% CI, 0.764-2.612; VIF = 2.661; t = 3.617; P = 0.001) and mRS scores (beta, 0.018; 95% CI, 0.013-0.023; VIF = 1.984; t = 2.047; P = 0.043). Serum MANF levels significantly predicted END and poor 90-day prognosis with areas under receiver operating characteristic curve at 0.752 and 0.787 respectively. END and prognostic predictive abilities were similar between serum MANF levels and NIHSS scores plus hematoma volumes (all P > 0.05). Combination of serum MANF levels with NIHSS scores and hematoma volumes had significantly higher prognostic capability than each of them (both P < 0.05). Serum MANF levels above 52.5 ng/ml and 62.0 ng/ml distinguished development of END and poor prognosis respectively with median-high sensitivity and specificity values. Using multivariate analysis, serum MANF levels > 52.5 ng/ml predicted END with odds ratio (OR) value of 2.713 (95% CI, 1.004-7.330; P = 0.042) and > 62.0 ng/ml predicted a poor prognosis with OR value of 3.848 (95% CI, 1.193-12.417; P = 0.024). Using restricted cubic spline, there was a linear correlation between serum MANF levels and poor prognosis or END risk (both P > 0.05). Nomograms were well established to predict END and a poor 90-day prognosis. Under calibration curve, such combination models were comparatively stable (using Hosmer & Lemeshow test, both P > 0.05). CONCLUSION: Increased serum MANF levels after ICH, in independent correlation with disease severity, independently distinguished risks of END and 90-day poor prognosis. Therefore, serum MANF may be a potential prognostic biomarker of ICH.

C-Reactive Protein Knockout Attenuates Temporomandibular Joint Inflammation in Rats.

BACKGROUND: C-reactive protein (CRP), a biomarker of inflammation, is highly expressed in osteoarthritis- (OA-) related diseases, but its exact role remains unknown. In this study, we evaluated the biological effect of CRP on temporomandibular joint (TMJ) inflammation. METHODS: Freund's complete adjuvant (CFA) was used to induce TMJ inflammation in CRP-knockout (CRP-/-) and control rats. Degenerative changes in the TMJ were compared to elucidate the role of CRP in TMJ inflammation. In addition, inflammatory cytokines, macrophage activation, and osteoclast differentiation were evaluated by real-time quantitative polymerase chain reaction, immunohistochemistry, and tartrate-resistant phosphatase staining to explore the potential regulatory mechanism. RESULTS: Compared to the control, CFA induced TMJ inflammation, which increased systemic and local CRP expression. Furthermore, CRP-/- rats exhibited less severe inflammatory symptoms. The downregulation of proinflammatory cytokines (interleukin- (IL-) 1ß and IL-6) and upregulation of the anti-inflammatory cytokine IL-10 were detected in CRP-/- rats, which also exhibited reduced macrophage activation and osteoclast differentiation. CONCLUSION: These results indicated that controlling the highly elevated levels of CRP during inflammation could modify the cytokine profile, macrophage activation, and osteoclast differentiation, thus, providing beneficial effects for TMJ-OA prevention and treatment.

Optimization of Three-Phase Hybrid Stepper Motors for Noise Reduction.

For the optimization of three-phase hybrid stepper motors with complex electromagnetic structures, an optimization method is presented in this paper. The method is a combination of 3D-FEM and the Taguchi optimization method intended to reduce the dependence on FEM results during the optimization calculation. In this paper, the optimization method is used in the optimization of the tooth shape of the three-phase hybrid stepper motor, and the objective is to reduce the noise caused by harmonics in the "torque-angle characteristic" of the motor. It is clear that traditional optimization methods make it very difficult to carry out such an optimization calculation as a large number of finite element calculations have to be used in the optimization process, and the required computation time is extremely long. Using the optimization method presented in the paper, the optimization becomes feasible because the number of finite element calculations is greatly reduced and the computation time is thus greatly reduced. In order to check the effectiveness of the optimization, the waterfall diagram for noise analysis and its application to check torque ripple are also presented in the paper. Both simulation and test results show that the optimized structure can significantly reduce the motor noise caused by torque ripple. Therefore, the optimization method proposed in this paper can be an effective tool for the optimal design of high-performance motors, including stepper motors.

L-arginine protects cementoblasts against hypoxia-induced apoptosis through Sirt1-enhanced autophagy.

BACKGROUND: L-arginine (L-arg) can reduce apoptosis in a variety of cells. Cementoblast apoptosis is related to root resorption during orthodontic treatment. In the present study, we aimed to study the regulatory effect and potential mechanism of L-arg on cementoblast apoptosis and root resorption. METHODS: The apoptosis-related mRNA and protein expression of murine cementoblast (OCCM-30) was assessed after L-arg treatment. To investigate the role of Sirtuin 1 (Sirt1) and autophagy in L-arg resistance to cementoblast apoptosis and root absorption, resveratrol, and EX527 were used to activate or inhibit Sirt1, and chloroquine (CQ) was used to inhibit autophagy. RESULTS: In vitro, L-arg inhibited hypoxia-induced apoptosis in OCCM-30. Further, L-arg increased Sirt1 expression whereas Sirt1 suppression by EX527 reversed the inhibitory effect of L-arg on cell apoptosis. Sirt1 activator resveratrol increased the ratio of microtubule-associated protein light chain 3 (LC3) II/I and decreased the expression of SQSTM1/p62 (p62), suggesting autophagy activation. Autophagy enhancement could reduce apoptosis. Caspase-3 and Bax expression was decreased, and Bcl-2 expression was increased. When autophagy was inhibited by CQ, the positive effects of Sirt1 were attenuated. In vivo, L-arg application reduced root resorption in rats, as demonstrated by decreased root absorption volume. Similarly, L-arg upregulated Sirt1, which activated autophagy in the root resorption model, and less root resorption was observed in the Sirt1 activation group. CONCLUSION: L-arg reduced cementoblast apoptosis in hypoxia and reduced root resorption induced by loading force in rats, which may be partly mediated by Sirt1-enhanced autophagy.

Overloading stress-induced progressive degeneration and self-repair in condylar cartilage.

Overloading stress-induced condylar cartilage degeneration acts as the main pathologic change in temporomandibular joint osteoarthritis (TMJ-OA). However, the progression of degeneration and the ability for self-repair remain poorly understood. Here, we explored the progression of cartilage degeneration by dividing pathological stages using a steady mouth-opening mouse model. Then, we observed changes of cartilage by removing the loading at different stages to test the potential self-repair after degeneration induced. Three-dimensional confocal microscopy combined with histology and micro-CT scanning was applied to examine TMJ at different stages of degeneration before and after self-repair. We found the cartilage underwent progressive and thorough degeneration as the overloading stress developed. During the initial adaptation stage, robust proliferation of posteromedial cartilage began at the area of direct loading. Subsequently, widespread chondrocyte apoptosis was found, followed by new chondrocyte proliferation in aggregates with matrix degradation and subchondral bone catabolism. Finally, with cartilage surface damage, the degeneration reached a point where the lesion could not be reversed by self-repair. While the cartilage nearly returned to normal when the interference was removed within 5 days. These results suggested overloading force induces a pathological process of successive degeneration in TMJ cartilage, which can be reversed by self-repair at early stages.

Chemerin/ChemR23 regulates cementoblast function and tooth resorption in mice via inflammatory factors.

BACKGROUND: Periodontitis and orthodontic treatment can lead to inflammatory root resorption (IRR) through an unclear mechanism. Chemerin, a novel chemoattractant protein, is closely associated with inflammation, affects osteoblast and osteoclast differentiation, and may play a role in IRR. We aimed to explore possible roles of the chemerin/ChemR23 interaction in cementoblast function and IRR and reveal a new IRR therapeutic target. METHODS: Cementoblast function-related gene and protein expression in the immortalized murine cementoblast cell line OCCM-30 after treatment with chemerin and siChemR23 was examined by qRT-PCR and Western blotting. The roles of the MAPK and PI3K-Akt signaling pathways were studied using specific inhibitors. Cementoblast cytokine production under different treatment conditions was measured by enzyme-linked immunosorbent assay and qRT-PCR. Additionally, we modeled IRR in wild-type and chemerin-overexpressing mice and injected transgenic mice with anti-ChemR23 antibody to block ChemR23. We then calculated the root resorption volume and examined periodontal tissue cathepsin K, Runx2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression. RESULT: Chemerin suppressed cementoblast differentiation and mineralization and exerted a proinflammatory effect on cementoblasts. These effects were partially reversed by siChemR23 and reversed to different extents by p38, Erk1/2 and PI3K-Akt pathway inhibition, suggesting p38, Erk1/2 and PI3K-Akt pathways as signaling pathways downstream of chemerin/ChemR23. In vivo, chemerin overexpression worsened IRR. Moreover, chemerin expression was positively correlated with TNF-α, IL-6, and cathepsin K expression and negatively correlated with Runx2 expression. ChemR23 downregulation reversed these effects. CONCLUSION: Chemerin/ChemR23 induced TNF-α and IL-6 expression dependent on Erk1/2, p38 MAPK, and PI3K-Akt signaling pathway activation, thereby regulating cementoblast function and affecting IRR.

Characterization of the complete chloroplast genome sequence of Hemsleya zhejiangensis (cucurbitaceae), a rare and endangered wild plant species in Zhejiang province, China.

Hemsleya zhejiangensis is a rare and endangered plant species which is listed as a key protected wild plant in Zhejiang province, China. In our present study, we assembled the complete chloroplast (CP) genome of H. zhejiangensis using high-throughput sequencing data. The whole genome sequence of H. zhejiangensis is 157,289 bp in size, with a GC content of 37.1%. Sequencing analyses reveal that the CP genome encodes 133 genes, including 84 protein-coding genes, 8 rRNA genes, 37 tRNA genes, and four pseudogenes. Phylogenetic analysis results indicate that H. zhejiangensis is clustered with H. lijiangensis, with a support value of 100%, and they are sister to the three Gynostemma species.