Evolutionary Events Promoted Polymerase Activity of H13N8 Avian Influenza Virus.

Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

Bone metastasis is a late-onset and unfavorable event in survivors of gastric cancer after radical gastrectomy: Results from a clinical observational cohort.

Background: The timing and incidence of recurrent bone metastasis (BM) after radical gastrectomy in patients with gastric cancer (GC) as well as the survival of these patients were not fully understood. The aim of this study was to analyze the data of an observational GC cohort and identify patients who underwent curative gastrectomy and had recurrent BM to describe and clarify the pattern and profile of BM evolution after surgery. Methods: Data were retrieved from a hospital-based GC cohort, and patients who underwent upfront radical gastrectomy were selected. The time points of specific organ metastatic events were recorded, and the person-year incidence rate of metastatic events was calculated. The latency period of BM events after gastrectomy was measured and compared with that of the other two most common metastatic events, liver metastasis (LM) and distant lymph node metastasis (LNM), using analysis of variance. Propensity score matching and subgroup analysis were used for sensitivity analysis. Results: A total of 1324 GC cases underwent radical gastrectomy between January 2011 and December 2021. Of these, 67 BM, 218 LM, and 248 LNM occurred before the last follow-up. The incidence of BM events was 1.7/100 person-years, which was approximately 3-fold lower than that of LM and distant LNM events (5.5 and 6.3 per 100 person-years, respectively). BM events had a significantly longer latency (median time, 16.5 months) than LM and LNM events (11.1 and 12.0 months, respectively). Recurrent BM led to a worse prognosis (median survival, 4.5 months) than those of LM and LNM events (median survival, 7.7 and 7.1 months, respectively). However, no difference in overall survival after gastrectomy was observed among the groups. Conclusions: Compared with other common metastatic events, BM in GC after gastrectomy is a late-onset event indicating poor survival. Trial registration: No. ChiCTR1800019978; http://www.chictr.org.cn/.

Isoquercitrin attenuates the osteoclast-mediated bone loss in rheumatoid arthritis via the Nrf2/ROS/NF-κB pathway.

An excess of osteoclastogenesis significantly contributes to the development of rheumatoid arthritis (RA). Activation of the nuclear factor erythroid-2 related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS)-to-NF-κB signaling cascade are important mechanisms regulating osteoclastogenesis; however, whether Nrf2 is involved in RANKL-induced NF-κB activation is controversial. Isoquercitrin, a natural flavonoid compound, has been shown to have Nrf2-dependent antioxidant effects inprevious studies. We sought to verify whether isoquercitrin could modulate RANKL-induced NF-κB activation by activating Nrf2, thereby affecting osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay suggested that isoquercitrin significantly inhibited osteoclastogenesis and osteolytic function. Mitosox staining showed that RANKL-induced ROS generation was significantly inhibited by isoquercitrin from day 3 of the osteoclast differentiation cycle. Quantitative real-time PCR, Western blot, and immunofluorescence indicated that isoquercitrin activated the Nrf2 signaling pathway and inhibited NF-κB expression. And when we used the Nrf2-specific inhibitor ML385, the inhibition of NF-κB by isoquercitrin disappeared. Moreover, we found that Nrf2 is not uninvolved in RANKL-induced NF-κB activation and may be related to the timing of ROS regulation. When we limited isoquercitrin administration to 2 days, Nrf2 remained activated and the inhibition of NF-κB disappeared. In vivo experiments suggested that isoquercitrin attenuated RA modeling-induced bone loss. Overall, isoquercitrin-activated Nrf2 blocked the RANKL-induced ROS-to-NF-κB signaling cascade response, thereby inhibiting osteoclastogenesis and bone loss. These findings provide new ideas for the treatment of RA.

The prognostic value of the preoperative albumin/globulin and monocyte ratio in resected early-stage non-small cell lung cancer.

OBJECTIVE: This study investigated the prognostic value of the preoperative albumin/globulin to monocyte ratio (AGMR) in patients with resected non-small cell lung cancer (NSCLC). METHODS: The study retrospectively enrolled patients with resected NSCLC from the Department of Thoracic Surgery, China-Japan Union Hospital of Jilin University from January 2016 to December 2017. Baseline demographic and clinicopathological data were collected. The preoperative AGMR was calculated. Propensity score matching (PSM) analysis was applied. The receiver operating characteristic curve was used to determine the optimal AGMR cut-off value. The Kaplan-Meier method was used to calculate the overall survival (OS) and disease-free survival (DFS). The Cox proportional hazards regression model was used to evaluate the prognostic value of the AGMR. RESULTS: A total of 305 NSCLC patients were included. The optimal AGMR value was 2.80. Before PSM. The high AGMR (>2.80) group had a significantly longer OS (41.34 + 11.32 vs. 32.03 + 17.01 months; P < 0.01) and DFS (39.00 + 14.49 vs. 28.78 + 19.13 months; P < 0.01) compared with the low AGMR (≤2.80) group. Multivariate analyses showed that AGMR (P < 0.01) in addition to sex (P < 0.05), body mass index (P < 0.01), history of respiratory diseases (P < 0.01), lymph node metastasis (P < 0.01), and tumor size (P < 0.01) were associated with OS and DFS. After PSM, AGMR remained as an independent prognostic factor for OS (hazard ratio [HR] 2.572, 95% confidence interval [CI]: 1.470-4.502; P = 0.001) and DFS (HR 2.110, 95% CI: 1.228-3.626; P = 0.007). CONCLUSION: The preoperative AGMR is a potential prognostic indicator for OS and DFS in resected early-stage NSCLC.

Correction to "Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression".

[This corrects the article DOI: 10.1021/acsomega.3c01289.].

Regulation of post-translational modification of PD-L1 and advances in tumor immunotherapy.

The immune checkpoint molecules programmed cell death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) are one of the most promising targets for tumor immunotherapy. PD-L1 is overexpressed on the surface of tumor cells and inhibits T cell activation upon binding to PD⁃1 on the surface of T cells, resulting in tumor immune escape. The therapeutic strategy of targeting PD-1/PD-L1 involves blocking this binding and restoring the tumor-killing effect of immune cells. However, in clinical settings, a relatively low proportion of cancer patients have responded well to PD-1/PD-L1 blockade, and clinical outcomes have reached a bottleneck and no substantial progress has been made. In recent years, PD-L1 post-translation modifications (PTMs) have gradually become a hot topic in the field of PD-L1 research, which will provide new insights to improve the efficacy of current anti-PD-1/PD-L1 therapies. Here, we summarized and discussed multiple PTMs of PD-L1, including glycosylation, ubiquitination, phosphorylation, acetylation and palmitoylation, with a major emphasis on mechanism-based therapeutic strategies (including relevant enzymes and targets that are already in clinical use and that may become drugs in the future). We also summarized the latest research progress of PTMs of PD-L1/PD-1 in regulating immunotherapy. The review provided novel strategies and directions for tumor immunotherapy research based on the PTMs of PD-L1/PD-1.

MFAP2 promotes the progression of oral squamous cell carcinoma by activating the Wnt/ß-catenin signaling pathway through autophagy.

Microfibrillar-associated protein 2 (MFAP2) is a small glycoprotein that is involved in vascular development and metabolic disease. The present study aims to explore the regulatory role of MFAP2 in the development and progression of oral squamous cell carcinoma (OSCC), including the underlying mechanisms. MFAP2 expression and its association with the progression of OSCC are explored using bioinformatics. MFAP2 expression in OSCC tissues is detected by immunohistochemical staining. SCC15 cell migration, invasion, apoptosis, proliferation, and viability are detected by wound healing, Transwell, flow cytometry, colony formation, and cell counting kit-8 assays. An in vivo experiment is used to detect tumor formation. Western blot analysis is used to determine MFAP2's regulatory role in autophagy and the Wnt/ß-catenin signaling pathway. MFAP2 is highly expressed in SCC15 cells and OSCC tissues, which correlates positively with the poor prognosis of patients with OSCCs. Functionally, MFAP2 promotes oncogenic autophagy to increase cell invasion, migration, and proliferation but inhibits apoptosis in SCC15 cells and promotes tumor growth in vivo. Mechanistically, MFAP2 upregulates autophagy and Wnt/ß-catenin signaling to stimulate OSCC development. Intriguingly, regulation of Wnt/ß-catenin signaling dependent on autophagy contributes to the malignant behaviors of SCC15 cells. MFAP2 could serve as a novel biomarker for OSCC and could affect OSCC tumorigenesis and development via autophagic regulation of Wnt/ß-catenin signaling.

Microplastic biofilm: An important microniche that may accelerate the spread of antibiotic resistance genes via natural transformation.

Microplastic (MP) biofilms provide a specific microniche for microbial life and are a potential hotspot for the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Nevertheless, the acquisition of ARGs in MP biofilms via natural transformation mediated by extracellular DNA (eDNA) has been rarely explored. This study demonstrated that MP biofilms promoted the natural transformation of extracellular ARGs at the single-cell and multi-species levels, compared to natural substrate (NS) biofilms and bacterioplankton. The transformation frequency on MP biofilms was up to 1000-fold compare to that on NS. The small MPs and aged MPs enhanced the ARG transformation frequencies up to 77.16-fold and 32.05-fold, respectively, compared with the large MPs and pristine MPs. The transformation frequencies on MP biofilms were significantly positively correlated with the bacterial density and extracellular polymeric substance (EPS) content (P < 0.05). Furthermore, MPs significantly increased the expression of the biofilm formation related genes (motA and pgaA) and DNA uptake related genes (pilX and comA) compared to NS and bacterioplankton. The more transformants colonized on MPs contributed to the enhanced transformation frequencies at the community-wide level. Overall, eDNA-mediated transformation in MP biofilms may be an important path of ARG spread, which was promoted by heterogeneous biofilm.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral.

Background: Oral squamous cell carcinoma (OSCC) is a common malignant tumor. Recently, Laminin Gamma 2 (LAMC2) has been shown to be abnormally expressed in OSCC; however, how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored. This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC. Methods: To explore the mechanism by which LAMC2 is highly expressed in OSCC, we used small interfering RNA (siRNA) to knock down LAMC2 to further observe the changes in the signaling pathway. Furthermore, we used cell proliferation assays, Transwell invasion assays, and wound-healing assays to observe the changes in OSCC proliferation, invasion, and metastasis. RFP-LC3 was used to detect the level of autophagy intensity. A cell line-derived xenograft (CDX) model was used to detect the effect of LAMC2 on tumor growth in vivo. Results: This study found that the level of autophagy was correlated with the biological behavior of OSCC. The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation, invasion, and metastasis via inhibiting the PI3K/AKT/mTOR pathway. Moreover, autophagy has a dual effect on OSCC, and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. Conclusions: LAMC2 interacts with autophagy to regulate OSCC metastasis, invasion, and proliferation via the PI3K/AKT/mTOR pathway. LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration, invasion, and proliferation.

Rapamycin Inhibits Osteoclastogenesis and Prevents LPS-Induced Alveolar Bone Loss by Oxidative Stress Suppression.

Periodontitis is a progressive inflammatory skeletal disease characterized by periodontal tissue destruction, alveolar bone resorption, and tooth loss. Chronic inflammatory response and excessive osteoclastogenesis play essential roles in periodontitis progression. Unfortunately, the pathogenesis that contributes to periodontitis remains unclear. As a specific inhibitor of the mTOR (mammalian/mechanistic target of rapamycin) signaling pathway and the most common autophagy activator, rapamycin plays a vital role in regulating various cellular processes. The present study investigated the effects of rapamycin on osteoclast (OC) formation in vitro and its effects on the rat periodontitis model. The results showed that rapamycin inhibited OC formation in a dose-dependent manner by up-regulating the Nrf2/GCLC signaling pathway, thus suppressing the intracellular redox status, as measured by 2',7'-dichlorofluorescein diacetate and MitoSOX. In addition, rather than simply increasing the autophagosome formation, rapamycin increased the autophagy flux during OC formation. Importantly, the anti-oxidative effect of rapamycin was regulated by an increase in autophagy flux, which could be attenuated by blocking autophagy with bafilomycin A1. In line with the in vitro results, rapamycin treatment attenuated alveolar bone resorption in rats with lipopolysaccharide-induced periodontitis in a dose-dependent manner, as assessed by micro-computed tomography, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase staining. Besides, high-dose rapamycin treatment could reduce the serum levels of proinflammatory factors and oxidative stress in periodontitis rats. In conclusion, this study expanded our understanding of rapamycin's role in OC formation and protection from inflammatory bone diseases.

Effect of single-port video-assisted thoracoscopy on surgical site wound infection and healing in patients with lung cancer: A meta-analysis.

We performed a meta-analysis to comprehensively assess the effect of single-port video-assisted thoracoscopy on surgical site wound infection and healing in patients with lung cancer. A computerised search for studies on single-port video-assisted thoracoscopy treatment of lung cancer was conducted from the time of database creation through February 2023 using the PubMed, EMBASE, the Cochrane Library, China National Knowledge Infrastructure, and Wanfang databases. Two investigators independently screened the literature, extracted information, and evaluated the quality of studies according to inclusion and exclusion criteria. Either a fixed or random-effects model was used in calculating the relative risk (RR) with 95% confidence intervals (CIs). Meta-analysis was performed using RevMan 5.4 software. The results showed that, compared with multi-port video-assisted thoracoscopy, single-port video-assisted thoracoscopy significantly reduced surgical site wound infection (RR: 0.38, 95% CI: 0.19-0.77, P = .007) and significantly promoted wound healing (RR: 0.37, 95% CI: 0.22-0.64, P < .001). Compared with multi-port video-assisted thoracoscopy, single-port video-assisted thoracoscopy significantly reduced surgical site wound infections and also promoted wound healing. However, because of large variations in study sample sizes, some of the literature reported methods of inferior quality. Additional high-quality studies containing large sample sizes are needed to further validate these results.

Emergence of a novel reassortant H3N6 canine influenza virus.

Although the natural hosts of avian influenza viruses (AIVs) are wild birds, multiple subtypes of AIVs have established epidemics in numerous mammals due to their cross-species spillover. Replication and evolution in intermedia mammalian hosts may facilitate AIV adaptation in humans. Because of their large population and intimacy with humans, dogs could act as such an intermedia host. To monitor the epidemiology of canine influenza viruses (CIVs) in Liaoning, China, we performed three surveillances in November 2018, March 2019, and April 2019. Five H3N2 and seven novel H3N6 CIVs had been isolated. Since the N6 neuraminidase (NA) genes were clustered with the H5N6 AIV, there is a high possibility that these H3N6 CIVs were generated from a H3N2 CIVs and H5N6 AIVs reassortment case. In addition, the H3N6 CIV showed increased mammalian adaptation ability compared to all the H3N2 strains in both in vitro and in vivo studies. Even though isolated 3 months later, the March 2019 isolated H3N2 viruses replicated more efficiently than the November 2018 isolated viruses. Our study indicated that H3 CIVs were undergoing an evolution process, through both genetic mutations and gene reassortment, at an incredible speed.

Clinical application of VATS combined with 3D-CTBA in anatomical basal segmentectomy.

Objective: This study aimed to summarize the clinical application experience of video-assisted thoracic surgery (VATS) combined with three-dimensional computed tomography-bronchography and angiography (3D-CTBA) in anatomical basal segmentectomy. Methods: Clinical data of 42 patients who underwent bilateral lower sub-basal segmentectomy by VATS combined with 3D-CTBA in our hospital from January 2020 to June 2022 were retrospectively analyzed; the patients included 20 males and 22 females, with a median age of 48 (30-65) years. Combined with the preoperative enhanced CT and 3D-CTBA techniques to identify the altered bronchi, arteries, and veins during the operation, the anatomical resection of each basal segment of both lower lungs was completed through the fissure approach or inferior pulmonary vein approach. Results: All operations were successfully completed without conversion to thoracotomy or lobectomy. The median operation time was 125 (90-176) min, the median intraoperative blood loss was 15 (10-50) mL, the median postoperative thoracic drainage time was 3 (2-17) days, and the median postoperative hospital stay was 5 (3-20) days. The median number of resected lymph nodes was 6 (5-8). There was no in-hospital death. Postoperative pulmonary infection occurred in 1 case, lower extremity deep vein thrombosis (DVT) in 3 cases, pulmonary embolism in 1 case, and persistent air leakage in the chest in 5 cases, all of which were improved by conservative treatment. Two cases of pleural effusion after discharge were improved after ultrasound guided drainage. Postoperative pathology showed 31 cases of minimally invasive adenocarcinoma, 6 cases of adenocarcinoma in situ (AIS), 3 cases of severe atypical adenomatous hyperplasia (AAH), and 2 cases of other benign nodules. All cases were lymph node-negative. Conclusion: VATS combined with 3D-CTBA is safe and feasible in anatomical basal segmentectomy; consequently, this approach should be promoted and applied in clinical work.

A novel EDAR variant identified in non-syndromic tooth agenesis: Insights from molecular dynamics.

OBJECTIVE: This study aims to investigate a novel pathogenic variant in a Chinese family of non-syndromic tooth agenesis (NSTA) and study the impact of the variant on related protein and pathway. DESIGN: One NSTA family was collected. Whole exome sequencing and Sanger sequencing were performed on the proband with NSTA and his 5 family members. The pathogenic influence of the mutant is evaluated by bioinformatics analyses including evolutionary conservation analysis and secondary structure prediction. Molecular dynamics (MD) simulations and binding free energy calculations were then performed to explore changes in the tertiary structure and binding ability of the protein. RESULTS: We found a novel missense ectodysplasin A receptor (EDAR) variant (c .1292 T > G; p.Ile431Arg) in all affected family members. The results of bioinformatics analyses revealed that the EDAR had harmful changes after mutation. MD simulations and the binding free energy calculations results showed that the mutant EDAR protein and EDAR/ectodysplasin-A receptor-associated adapter (EDARADD) complex displayed tertiary structural change, and EDAR possessed a lower affinity to EDARADD after mutation. CONCLUSIONS: We found a novel EDAR variant (c.1292 T > G; p.Ile431Arg) in one NSTA family, which affects the binding of EDAR and EDARADD.

Incorporation of liver chemistry score in predicting survival of liver-involved advanced gastric cancer patients who received palliative chemotherapy.

BACKGROUND: Gastric cancer liver metastasis (GCLM) patients usually accompany by abnormal serum liver function tests (LFTs) more or less; however, the prognostic value of LFTs is not fully understood. This study aimed to develop a liver chemistry score (LCS) based on LFTs and incorporate it into prognosis determination for GCLM patients who received palliative chemotherapy. METHODS: Data were derived from hospitalized GCLM patients in two general hospitals in China. LCS was generated based on the results of LFTs by LASSO regression. Cutoff value of the score was determined by restricted cubic spline. The score was then incorporated into Cox regression analysis to construct a predictive nomogram; the model was then evaluated internally and externally by AUC of time-dependent receiver operating characteristic curves (ROC) and calibration curves. RESULTS: Three hundred and thirty-six and 72 patients were included in development and validation cohort, respectively. LASSO regression analysis in development cohort finally reached a two-parametric LCS calculated on AST and ALP levels as 0.03343515 × ln (AST, U/L) + 0.02687997 × ln (ALP, U/L), and 0.232 was set as optimal cutoff value. Patients in low (LCS < 0.232) or high (LCS ≥ 0.232) score group experienced different survival times; median OS was 13.54 (95% CI: 11.1-15.6) months in the low LCS group and 7.3 (6.6-9.3) months in the high LCS group (p < 0.001). A nomogram including LCS and other clinical parameters was constructed and showed superior performance than model not including LCS. AUC of 6-month ROC improved from 0.647 (95% CI: 0.584-0.711) to 0.699 (0.638-0.759) in internal validation, and 0.837 (0.734-0.940) to 0.875 (0.784-0.966) in external validation. CONCLUSIONS: Liver chemistry score is useful in determining the prognosis of gastric cancer patients with liver metastasis and may be helpful to clinicians in decision-making.

Machine learning models for predicting one-year survival in patients with metastatic gastric cancer who experienced upfront radical gastrectomy.

Tumor metastasis is a common event in patients with gastric cancer (GC) who previously underwent curative gastrectomy. It is meaningful to employ high-volume clinical data for predicting the survival of metastatic GC patients. We aim to establish an improved machine learning (ML) classifier for predicting if a patient with metastatic GC would die within 12 months. Eligible patients were enrolled from a Chinese GC cohort, and the complete detailed information from medical records was extracted to generate a high-dimensional dataset. Appropriate feature engineering and feature filter were conducted before modeling with eight algorithms. A 10-fold cross validation (CV) nested in a holdout CV (8:2) was employed for hyperparameter tuning and model evaluation. Model selection was based on the area under the receiver operating characteristic (AUROC) curve, recall, and precision. The selected model was globally explained using interpretable surrogate models. Of the total 399 cases (median survival of 8.2 months), 242 patients survived less than 12 months. The linear discriminant analysis (LDA), support vector machine (SVM), and random forest (RF) model had the highest AUROC (0.78 ± 0.021), recall (0.93 ± 0.031), and precision (0.80 ± 0.026), respectively. The LDA model created a new function that generally separated the two classes. The predicted probability of the SVM model was interpreted using a linear regression model visualized by a nomogram. The predicted class of the RF model was explained using a decision tree model. In summary, analyzing high-volume medical data by ML is helpful to produce an improved model for predicting the survival in patients with metastatic GC. The algorithm should be carefully selected in different practical scenarios.

The Role of HSP90 Inhibitors in the Treatment of Cardiovascular Diseases.

Cardiovascular disease is the result of complicated pathophysiological processes in the tissues that make up the blood vessels and heart. Heat shock protein 90 (HSP90) can interact with 10% of the proteome and is the most widely studied molecular chaperone in recent years. HSP90 is extensively involved in the regulation of protein folding and intracellular protein stability, making HSP90 a hopeful target for the treatment of multiple cardiovascular diseases. Numerous client proteins of HSP90 have been identified in known cardiac disease pathways, including MAPK signaling, PI3K/AKT (PKB)/mTOR, and TNF-α signaling. Therefore, these pathways can be controlled by regulating HSP90. Among them, the activity of HSP90 can be regulated via numerous inhibitors. In this review, first, we will discuss the function of HSP90 and its role in pathological pathways. In addition, HSP90 plays a significant role in most cardiovascular diseases, including hypertension, pulmonary venous hypertension, atherosclerosis, and heart failure; next we will focus on this part. Finally, we will summarize the currently known HSP90 inhibitors and their potential in the treatment of heart disease.

Effect of Lemon Essential Oil Microemulsion on the Cariogenic Virulence Factor of Streptococcus mutans via the Glycolytic Pathway.

PURPOSE: To investigate the effects and mechanisms of lemon essential oil products on dental caries prevention. MATERIALS AND METHODS: Lemon essential oil microemulsions (LEOM) with concentrations of 1/8 minimum inhibitory concentration (MIC), 1/4 MIC, and 1/2 MIC were applied to S. mutans at concentrations of 0.2%, 1%, and 5% glucose, respectively. Changes in acid production capacity of S. mutans were measured based on changes in pH. The effect of the reductive coenzyme I oxidation method on LDH activity was examined. The effect of lemon essential oil microemulsion on the expression of the lactate dehydrogenase gene (ldh) was detected by a quantitative real-time polymerase chain reaction. RESULTS: Lemon essential oil microemulsion at 1/2 MIC concentration reduced the environmental pH value at different glucose concentrations, compared to those observed in the control group (p < 0.05). LDH activity of S. mutans was decreased at three subinhibitory concentrations of lemon essential oil microemulsions (p < 0.05). The effect of lemon essential oil microemulsions on S. mutans LDH activity and bacterial acid production were positively correlated (r = 0.825, p < 0.05). Lemon essential oil microemulsion at 1/2 MIC concentration downregulated the expression of the ldh gene of S. mutans at different glucose concentrations (p < 0.05). In different glucose environments, lemon essential oil microemulsions at subminimum inhibitory concentrations can inhibit the acid production of S. mutans by reducing ldh expression and LDH activity in the glycolytic pathway, proving its anti-caries potential. CONCLUSIONS: LEOM can effectively prevent dental caries and maintain the microecological balance of the oral environment.

Bioinformatics-Guided Identification of Ethyl Acetate Extract of Citri Reticulatae Pericarpium as a Functional Food Ingredient with Anti-Inflammatory Potential.

Citri Reticulatae Pericarpium (CRP) is one of the most commonly used food supplements and folk medicines worldwide, and possesses cardiovascular, digestive, and respiratory protective effects partially through its antioxidant and anti-inflammatory functions. The unique aromatic flavor and mild side effects make CRP a promising candidate for the development of anti-inflammatory functional food. However, recent studies show that the crude alcoholic extract and some isolated compounds of CRP show compromised anti-inflammatory activity, which became the main factor hindering its further development. To identify the bioactive compounds with anti-inflammatory potential, and improve the anti-inflammatory effects of the extract, a bioinformatics-guided extraction protocol was employed in this study. The potential bioactive candidates were identified by combing network pharmacology analysis, molecular docking, principal components analysis, k-means clustering, and in vitro testing of reference compounds. Our results demonstrated that 66 compounds in CRP could be grouped into four clusters according to their docking score profile against 24 receptors, while the cluster containing flavonoids and phenols might possess a more promising anti-inflammatory function. In addition, in vitro anti-inflammatory tests of the seven reference compounds demonstrated that hesperitin, naringenin, and gardenin B, which were grouped into a cluster containing flavonoids and phenols, significantly decreased LPS-induced NO, TNF-α, and IL-6 production of macrophages. While the compounds outside of that cluster, such as neohesperidin, naringin, hesperidin, and sinensetin showed little effect on alleviating LPS-induced NO and proinflammatory cytokine production. Based on the chemical properties of selected compounds, ethyl acetate (EtOAc) was selected as the solvent for extraction, because of its promising solubility of flavonoids and phenols. Furthermore, the ethanol alcoholic extract was used as a reference. The chemical profiling of EtOAc and crude alcoholic extract by HPLC/MS/MS also demonstrated the decreased abundance of flavonoid glycosides in EtOAc extract but increased abundance of phenols, phenolic acid, and aglycones. In accordance with the prediction, the EtOAc extract of CRP, but not the crude alcoholic extract, significantly decreased the NO, IL-6, and TNF-α production. Taken together, the results suggested selective extraction of phenols and flavonoids rich extract was able to increase the anti-inflammatory potential of CRP partially because of the synergistic effects between flavonoids, phenols, and enriched polymethoxyflavones. Our study might pave the road for the development of ethyl acetate extract of CRP as a novel functional food with anti-inflammatory function.

Design and Fabrication of a Liver-on-a-chip Reconstructing Tissue-tissue Interfaces.

Despite the rapid advances in the liver-on-a-chip platforms, it remains a daunting challenge to construct a biomimetic liver-on-a-chip for in vitro research. This study aimed to reconstruct the tissue-tissue interfaces based on bilayer microspheres and form vascularized liver tissue. Firstly, we designed a tri-vascular liver-on-a-chip (TVLOC) comprising a hepatic artery, a portal vein and a central vein, and theoretically analyzed the distribution of velocity and concentration fields in the culture area. Secondly, we designed a bilayer microsphere generating microsystem based on the coaxial confocal principle, which is primarily used to produce bilayer microspheres containing different kinds of cells. Finally, the bilayer microspheres were co-cultured with endothelial cells in the cell culture area of the TVLOC to form vascularized liver tissue, and the cell viability and vascular network growth were analyzed. The results revealed that the TVLOC designed in this study can provide a substance concentration gradient similar to that of the liver microenvironment, and the bilayer microspheres can form a three-dimensional (3D) orderly liver structure with endothelial cells. Such a liver-on-a-chip is capable of maintaining the function of hepatocytes (HCs) pretty well. This work provides full insights into further simulation of the liver-on-a-chip.