Correlation of PD-L1 expression with CD8+ T cells and oxidative stress-related molecules NRF2 and NQO1 in esophageal squamous cell carcinoma.

Oxidative stress and the immune microenvironment both contribute to the pathogenesis of esophageal squamous cell carcinoma (ESCC). However, their interrelationships remain poorly understood. We aimed to examine the status of key molecules involved in oxidative stress and the immune microenvironment, as well as their relationships with each other and with clinicopathological features and prognosis in ESCC. The expression of programmed death-ligand 1 (PD-L1), CD8, nuclear factor erythroid-2 related factor-2 (NRF2), and NAD(P)H quinone oxidoreductase 1 (NQO1) was detected using immunohistochemistry in tissue samples from 176 patients with ESCC. We employed both combined positive score (CPS) and tumor proportion score (TPS) to evaluate PD-L1 expression and found a positive correlation between CPS and TPS. Notably, PD-L1 expression, as assessed by either CPS or TPS, was positively correlated with both NRF2 nuclear score and NQO1 score in stage II-IV ESCC. We also observed a positive correlation between the density of CD8+ T cells and PD-L1 expression. Furthermore, high levels of PD-L1 CPS, but not TPS, were associated with advanced TNM stage and lymph node metastases. Moreover, both PD-L1 CPS and the nuclear expression of NRF2 were found to be predictive of shorter overall survival in stage II-IV ESCC. By using the Mandard-tumor regression grading (TRG) system to evaluate the pathological response of tumors to neoadjuvant chemotherapy (NACT), we found that the TRG-5 group had higher NRF2 nuclear score, PD-L1 CPS, and TPS in pre-NACT biopsy samples compared with the TRG-3 + 4 group. The NQO1 scores of post-NACT surgical specimens were significantly higher in the TRG-5 group than in the TRG 3 + 4 group. In conclusion, the expression of PD-L1 is associated with aberrant NRF2 signaling pathway, advanced TNM stage, lymph node metastases, and unfavorable prognosis. The dysregulation of PD-L1 and aberrant activation of the NRF2 signaling pathway are implicated in resistance to NACT. Our findings shed light on the complex interrelationships between oxidative stress and the immune microenvironment in ESCC, which may have implications for personalized therapies and improved patient outcomes.

Exploring the Effect of Pain Sensitive Questionnaire on Guiding Intravenous Analgesia After Cesarean Section: A Randomised Double Blind Controlled Trial.

Objective: This study investigates the effect of the Pain Sensitivity Questionnaire (PSQ) in guiding patient controlled intravenous analgesia (PCIA) on postoperative analgesia in women undergoing cesarean section. Methods: A total of 160 women who were to undergo a cesarean section under combined spinal and epidural anaesthesia were included in this study. Women with a preoperative PSQ <4 were randomly divided into a low pain-sensitive control group (LC group), and a low pain-sensitive observation group (LO group), and women with preoperative PSQ >6 were randomly divided into a high pain-sensitive control group (HC group) and a high pain-sensitive observation group (HO group). After the surgery, patients received the pump butorphanol concentration was 3.5 µg·kg-1·h-1 in the LC and HC groups, 3.0 µg·kg-1·h-1 in the LO group and 4.0 µg·kg-1·h-1 in the HO group.To compare the analgesic effects of postoperative PCIA and postoperative recovery in women. Results: Wound pain and uterine contraction pain VAS scores at rest and activity were significantly lower in the LC group than in the LO group at 4 and 8 h postoperatively (P<0.05). Similarly, wound pain and uterine contraction pain VAS scores at rest and activity were significantly lower in the HO group than in the HC group at 8, 12, and 24 h postoperatively (P<0.05). The Ramsay scores were significantly higher in the LC than in the LO groups at 4, 8, 12, 24, and 48 h postoperatively (P<0.05), but there was no statistically significant difference between the Ramsay scores in the HC group and the HO group. There was no statistical difference in any of the post-operative recoveries (P>0.05). Conclusion: Compared to the weight-based postoperative PCIA, the PSQ-based postoperative PCIA has better analgesic effects and can improve maternal satisfaction with postoperative analgesia.

Real-world data of EGFR mutation testing in Chinese non-small cell carcinoma: Low tumor cell number and tumor cellularity can be accepted.

INTRODUCTION: Molecular testing on advanced non-small cell lung cancer (NSCLC) often confront of limited specimen.The aim of the study is to compare the mutation frequency in adenocarcinoma samples with poor tumor cell content and the optimal samples, making the optimal strategy of mutation analysis. METHODS: In this retrospective study, mutation status of EGFR, ALK, ROS1, BRAF, KRAS, RET, HER2, CMET, NRAS and PIK3CA in 1594 NSCLCs were tested by ARMS-PCR and qRT-PCR, consists of 790 cases of surgical specimens, 741 cases of small biopsies, 63 cases of cytology cell blocks. We analyzed the discrepancies in mutation frequency with optimal specimens and the suboptimal ones. RESULTS: Comparing the gene mutation frequency in optimal and suboptimal samples, only the EGFR mutation rates of surgical samples (12.5 %, 1 out of 8) with < 10 % tumor cellularity was lower than in those with ≥ 10 % (57.1 %, 385 out of 674， p = 0.015). However, surgical specimens with low tumor cellularity (<20 %) were comparable to the qualified samples. The mutation frequency of EGFR in biopsy specimens with poor specimen adequacy( <20 %, <10 %, <5 %, <200, <100, <50) were comparable to the qualified samples. Low tumor cellularity (<20 %, <10 %, <5 %) and low tumor cell number (<200, <100, <50) was not associated with mutational rate for ALK, ROS1, BRAF, KRAS, RET, HER2, CMET, NRAS and PIK3CA mutations in small biopsy samples. CONCLUSIONS: In clinical practice, specimen with low adequacy could attempt in gene mutation testing, including biopsy and surgical specimen. However, the amount of tumor for molecular testing should be reported and suboptimal samples with a negative EGFR mutation result should be considered for combination using of other mutation test method or repeat testing of an alternate tumor sample, especially for the surgical samples.

High expression of nuclear NRF2 combined with NFE2L2 alterations predicts poor prognosis in esophageal squamous cell carcinoma patients.

Nuclear factor erythroid-2 related factor-2 (NFE2L2 or NRF2) is a frequently mutated gene in esophageal squamous cell carcinoma (ESCC). However, the roles of NFE2L2 alterations in ESCC remain elusive. In order to elucidate this issue, 130 ESCC patients who underwent esophagectomy were enrolled. The majority of tumor tissues were positive for NRF2, which was significantly enriched in the nucleus of the primary tumor tissues compared with the noncancerous mucosae. Primary ESCC tumors positive for NRF2 tended to be positive for NAD(P)H quinone oxidoreductase 1 (NQO1) as the downstream target of NRF2. There was a positive correlation between NRF2 and NQO1 expression level in primary tumors. NQO1 staining in primary tumors with NRF2 nuclear expression was significantly stronger than that with NRF2 cytoplasmic expression. In addition, high concordance for the status of NRF2 expression between primary tumors and corresponding metastatic lesions was observed. Next, we found high expression of nuclear NRF2 (the proportion of nuclear NRF2 expression >20% or nuclear NRF2 immunohistochemistry score >20) predicted shorter overall survival in patients with dual-positive expression of NRF2 and NQO1. Captured-based targeted sequencing revealed that NFE2L2 somatic alterations were observed in 52.8% of ESCC patients with dual-positive expression of NRF2 and NQO1. NFE2L2 amplification and mutations within the DLG/ETGE motifs were seen more frequently in ESCC tumors with nuclear or nucleocytoplasmic expression of NRF2 compared with those with cytoplasmic expression of NRF2. We also found high expression of nuclear NRF2 plus the status of NFE2L2 alteration exhibited high performance in predicting prognosis of ESCC patients. Our study demonstrated that high nuclear NRF2 expression and NFE2L2 alterations were associated with poor prognosis of ESCC patients. These findings suggest that NRF2 signaling pathway might play vital roles in ESCC malignancy and the aberrant activation of NRF2 pathway predicts unfavorable prognosis in ESCC.

Serum exosomal microRNA-146a as a novel diagnostic biomarker for acute coronary syndrome.

BACKGROUND: Circulating microRNAs (miRNAs) have emerged as potential biomarkers for cardiovascular diseases. However, few studies have focused on the role of exosomal miRNAs in acute coronary syndrome (ACS). The purpose of this study was to explore weather serum exosomal microRNA-146a (exo-miR-146a) could be used as a novel diagnostic biomarker for ACS and to investigate its relationship with inflammatory response. METHODS: A total of 63 ACS patients and 25 patients with normal coronary arteries (Control) were enrolled respectively. The serum exosomes were isolated and then identified by transmission electron microscopy (TEM), western blot, and nanoparticle tracking analysis (NTA). The expression levels of exo-miR-146a in serum were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and the expression levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in serum were assessed by enzyme-linked immunosorbent assay (ELISA). Spearman's correlation analysis was used to appraise the potential factors related to serum exo-miR-146a and receiver operating characteristic (ROC) curve analysis was applied for predicting the accuracy of ACS via the area under curve (AUC). RESULTS: Exosomes isolated from serum were of typical cup-like shape, with 50-150 nm diameter, and expressed CD9, CD63, CD81, and HSP70. The expression levels of serum exo-miR-146a, IL-1ß, IL-6, and TNF-α were significantly increased in ACS patients compared with the control group, Spearman's correlation analysis indicated that exo-miR-146a expression was markedly positively correlated with IL-1ß, IL-6, and TNF-α. The ROC curve analyses revealed that exo-miR-146a could distinguish ACS patients from their normal controls. CONCLUSIONS: The serum exo-miR-146a may be used as a novel diagnostic biomarker for ACS patients, and it is also associated with inflammatory response.

Homologous recombination repair gene mutations in Chinese localized and locally advanced prostate cancer patients.

BACKGROUND: Homologous recombination repair gene (HRR) mutations have been proven to be effective biomarkers for PARP inhibitor therapy for metastatic castration resistant prostate cancer. However, the frequency of HRR mutations in patients with localized and locally advanced prostate cancer is still unclear. This study investigated the profile of HRR gene mutations in Chinese localized and locally advanced prostate cancer patients. MATERIALS AND METHODS: 74 patients with localized and locally advanced prostate cancer patients in Beijing Chaoyang Hospital between May 2018 and September 2019 were retrospectively included. Matched prostate cancer and histologically normal tissues were subjected to next-generation sequencing. Pathogenic alterations of 19 HRR genes were examined. RESULTS: Ten deleterious and suspected deleterious mutations (4 germline and 6 somatic mutations) were detected in 9 of 74 (12.16 %) patients, occurred in seven HRR-related genes, including CDK12, NBN, ATM, ATR, BRCA2, PALB2 and RAD51C. The mutation frequency of HRR genes in this study (12.16 %) was higher than TCGA cohort (7.29 %), and the mutation sites in 7 HRR genes detected in this cohort were different from those of TCGA data. Patients with HRR gene mutations had higher Gleason grade (≥ 3) (P = 0.03) and risk level (very-high) (P = 0.03). Postoperative prostate specific antigen level and positive surgical margin rate was not associated with HRR gene mutation status. CONCLUSIONS: This study illustrated the mutation patterns of HRR genes in Chinese population with localized and locally advanced prostate cancer. These results provide further evidence that HRR gene mutations were more prevalent in patients with higher Gleason grade, or with very-high-risk level. Patients with these clinicopathologic characteristics may need more precise stratification through molecular detection.

Genetic features and application value of next generation sequencing in the diagnosis of synchronous multifocal lung adenocarcinoma.

The present study aimed to elucidate the genetic features of multiple lung cancer (MLC) and identify effective molecular markers for diagnosis using next generation sequencing (NGS). The present data may also inform patient treatment and prognosis. A total of 35 lesions were obtained from 17 patients with MLC. Based on lesion histology and NGS, 13 cases of multiple primary lung cancer (MPLC) were identified and 4 cases were classified as intrapulmonary metastasis (IPM). All 4 patients with IPM exhibited an epidermal growth factor receptor (EGFR) mutation and synchronous mutation of at least one tumor suppressor gene. The frequency and percentage of EGFR mutations, accompanied with tumor suppressor genes, were significantly higher in patients with IPM compared with MPLC. Furthermore, a high EGFR-heterogeneity score and male sex were risk factors of IPM occurrence. There were significant differences in mean EGFR mutation abundance alone, mutations of tumor suppressor genes and mutations of EGFR combined with tumor suppressor genes between patients with adenocarcinoma (ADC) and adenocarcinoma in situ (AIS). In conclusion, histological characteristics combined with genetic alterations may be an effective method for the diagnosis of MPLC and IPM, and NGS may serve as a useful diagnostic tool. MLC exhibited unique molecular characteristics, including higher rates of EGFR mutations, EGFR driver mutations accompanied with tumor suppressor gene mutations and the absence of anaplastic lymphoma kinase mutations, which may help distinguish between patients with MPLC or IPM. The present study hypothesized that the mean frequency of EGFR mutations, mutations of tumor suppressor genes and mutations of both EGFR and tumor suppressor genes may serve an important role in the development of AIS to ADC. The results of the present study highlight the potential underlying mechanisms of lung ADC development, which may assist with future elucidation of effective treatments to prevent the progression of lung cancer.

The CEACAM1-derived peptide QLSN impairs collagen-induced human platelet activation through glycoprotein VI.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates collagen-mediated platelet activation through its cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). However, the function of CEACAM1's extracellular cleavage fragments is currently unknown. In the present study, we used mass spectrometry (MS) to identify 9 cleavage fragments shed by matrix metallopeptidase 12 (MMP-12), and then we synthesized peptides with sequences corresponding to the fragments. QLSNGNRTLT (QLSN), a peptide from the A1-domain of CEACAM1, significantly attenuated collagen-induced platelet aggregation. QLSN also attenuated platelet static adhesion to collagen. Additionally, QLSN reduced human platelet secretion and integrin αIIbß3 activation in response to glycoprotein VI (GPVI)-selective agonist, convulxin. Correspondingly, QLSN treatment significantly decreased convulxin-mediated phosphorylation of Src, protein kinase B (Akt), spleen tyrosine kinase (Syk) and phospholipase Cγ2 (PLCγ2) in human platelets. These data indicate that the CEACAM1-derived peptide QLSN inhibits GPVI-mediated human platelet activation. QLSN could potentially be developed as a novel antiplatelet agent.

Platelet Carcinoembryonic Antigen Cell Adhesion Molecule 5 (CEACAM5) as a Possible Novel Diagnostic Tool for Evaluation of Acute Coronary Syndrome.

BACKGROUND Acute coronary syndrome (ACS) occurs approximately every 40 seconds, and was an underlying cause of death in 1 out of every 7 deaths. More accurate indicators are needed to distinguish patients with ACS from patients manifesting negative changes in electrocardiogram (ECG) and myocardial enzymes. This study aimed to investigate whether the expression of platelet carcinoembryonic antigen cell adhesion molecule-5 (CEACAM5/CEA/CD66e) could help predict ACS. MATERIAL AND METHODS We enrolled 82 participants (mean age 60 years, 33 females and 49 males). The expression of CEA on washed human platelets was assessed using two-color flow cytometry. The CEA levels on platelets and in serum of these 82 consecutive patients were detected using two-color whole-blood flow cytometry analysis and a custom-made Luminex multiplex assay, respectively. RESULTS CEA was expressed on the surface of human platelets. The expression of platelet CEA (P<0.01), but not serum CEA (P=0.30), was significantly higher in patients with ACS compared to patients with normal coronary artery. Increased platelet CEA levels could serve as a new independent indicator for ACS (P=0.0003). Platelet CEA testing (P=0.000002), as well as cardiac troponin I (cTnI) (P=0.0005), can diagnose ACS with high sensitivity and specificity, and, combined with cTnI (P<0.0001), can improve the diagnostic value. CONCLUSIONS Platelet CEA expression was higher in individuals presenting with ACS. Hence, platelet CEA might be a novel and reliable biomarker for ACS. Large-scale studies are needed to confirm this hypothesis.

Xinmailong Modulates Platelet Function and Inhibits Thrombus Formation via the Platelet αIIbß3-Mediated Signaling Pathway.

Background: Xinmailong (XML), a bioactive composite extracted from Periplaneta americana, has been widely used to treat cardiovascular diseases such as congestive heart failure. However, it is unclear whether XML has antiplatelet and antithrombotic effects. Methods: The effects of XML on agonist-induced platelet aggregation, adhesion and spreading, granule secretion, integrin α II bß3 activation, and thrombus formation were evaluated. Phosphorylation of Syk, PLCγ2, Akt, GSK3ß, and MAPK signaling molecules was also studied on agonist-induced platelets. In addition, the antithrombotic effects of XML were observed in vivo using an acute pulmonary thrombosis mouse model. Results: XML dose-dependently inhibited in vitro platelet aggregation and granule secretion induced by thrombin, collagen, and arachidonic acid (AA). XML also greatly reduced platelet adhesion and spreading on both collagen- and fibrinogen-coated surfaces. Biochemical analysis revealed that XML inhibited thrombin-, collagen-, and AA-induced phosphorylation of Syk, PLCγ2, Akt, GSK3ß, and MAPK. Additionally, XML significantly inhibited in vivo thrombus formation in a collagen-epinephrine-induced acute pulmonary thrombosis mouse model. Conclusions and General Significance: Here, we provide the first report showing that XML inhibits platelet function and that it possesses antithrombotic activity. This suggests that XML could be a potential therapeutic candidate to prevent or treat platelet-related cardiovascular diseases.

Celastrus Orbiculatus Extracts Inhibit the Metastasis through Attenuating PI3K/Akt/mTOR Signaling Pathway in Human Gastric Cancer.

BACKGROUND: Rapamycin receptor inhibitors have been applied in the clinic and achieved satisfactory therapeutic effect recently. The mechanisms did not clearly show how the Celastrus Orbiculatus Extracts (COE) inhibited the expression of the mammalian Target of Rapamycin (mTOR) in human gastric cancer cells. The aim of this study was to investigate whether the COE inhibited the metastasis through the mTOR signaling pathway in human gastric cancer MGC-803 cells. METHODS: The abnormal expression level of mTOR protein was detected by immunohistochemistry in human gastric cancer tissue. The MGC-803/mTOR- cells were constructed by knockdown of mTOR using lentivirus infection technique. The human gastric cancer MGC-803/mTOR- cells were treated with different concentrations (20, 40, 80 µg/ml) of COE for 24 hours. The ability of cell metastasis was analyzed by the cell invasion and migration assay. The expression levels of PI3K/Akt/mTOR signaling pathway were detected by Western Blotting. RESULTS: COE inhibited the proliferation, invasion and migration of MGC-803/mTOR- cells in a concentrationdependent manner. The expression of E-cadherin protein increased, and the expression of N-cadherin and Vimentin decreased simultaneously in the MGC-803/mTOR- cells. 4EBP1, p-4EBP1, P70S6k, p-P70S6k, mTOR, p-mTOR, PI3K and Akt proteins in MGC-803/mTOR- cells were reduced in a dose-dependent manner. CONCLUSION: COE could not only inhibit cell growth, invasion and migration, but also inhibit the epithelialmesenchymal transition of gastric cancer cells. The molecular mechanism of COE inhibited the metastasis which may be related to the PI3K/Akt/mTOR signal pathway. This study provides ideas for the development of new anti-gastric cancer drugs.

Mmu-miR-185 depletion promotes osteogenic differentiation and suppresses bone loss in osteoporosis through the Bgn-mediated BMP/Smad pathway.

MicroRNAs (miRs) play an essential role in the regulation of bone formation and homeostasis. miR-185 has been reported to negatively regulate osteogenesis in vitro. However, whether it has an impact on in vivo bone homeostasis remains unknown. Here, we demonstrated that primary osteoblasts and mesenchymal stem cells derived from miR-185-knockout (KO) mice exhibited enhanced osteogenesis. Further, we constructed an ovariectomized mouse model to investigate the role of miR-185 during osteoporosis. Micro-computed tomography revealed an increased bone volume in KO compared to wild-type mice 6 weeks after surgery, indicating redundant bone formation after miR-185 depletion. Dual-luciferase reporter assays identified biglycan (Bgn), which promotes bone formation through the BMP/Smad pathway, as the direct target of miR-185. Taken together, these findings indicate that blocking miR-185 expression increases bone formation during osteoporosis, which may partly occur through the regulation of Bgn expression and BMP/Smad signaling.

GXYLT2 accelerates cell growth and migration by regulating the Notch pathway in human cancer cells.

Glucoside xylosyltransferase2 (GXYLT2), a member of the human α-1,3-D-xylosyltransferases, functions to modify the first xylose to the O-Glucose residue on epidermal growth factor (EGF) repeats of Notch receptors. It is well-established that the Notch signaling pathway plays a critical role in proper development and homeostasis. However, the regulatory role of EGF xylosylation in Notch signaling and different cell activities in human cells remains unknown. In this study, we showed that knockdown of GXYLT2 suppressed human cell proliferation and induced G1/S phase cell cycle arrest. GXYLT2 downregulation also inhibited cell migration and invasion, whereas the overexpression of GXYLT2 had the opposite effects. Additionally, GXYLT2 activated Notch signaling and promoted the phosphorylation of MAPKs but not PI3K and Akt. Taken together, our findings indicated that GXYLT2 plays an important role in cell activities via regulation of the Notch signaling.

Young and old adipocytes have differential influence on the development of osteoblasts.

OBJECTIVE: The aim of the current study was to investigate the effect of adipocytes on the differentiation of osteoblasts at different stages of adipocyte development. METHODS: BMSCs were isolated from 4-week-old male wistar rat femurs and tibias, and flow cytometry was performed. Adipocytes were derived from BMSCs, cell morphology was continually observed from day 21 to day 50. Adipocyte medium was collected once every 2days (d) and ELISA kits were used for detection of triglycerides (TG), tumor necrosis factor-α (TNF-α), and interleukin-6(IL-6) expression level. 21d and 40d old adipocyte and osteoblast cells were co-cultured, and alizarin red staining was performed after 21d. After co-culture, the adherent cells were collected, and the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) was detected by real time PCR. RESULT: Results of cell characterisation showed that the cells had positive expression of CD29 (97.92%) and CD44 (89.32%). With the increase of the induction time of mature adipocytes, the number of adipocyte on 21thd was significantly higher than 40thd, while the volume of adipocyte was significantly lower than 40thd (P<0.05). The levels of TG(2.6±0.83mmol/l VS 3.8±0.66mmol/l), TNF-α(30.5±2.53pg/ml VS 57.6±5.1pg/ml), and IL-6(32.5±1.42pg/ml VS 55.1±5.97pg/ml) secreted by adipocytes increased with induction time: 40thd was significantly higher than 21thd (P<0.01). When 21thd adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly increased over that of the positive control group, When 40thd adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly decreased over that of the positive control group (P<0.05). The OPG(68.9±5.39 VS 1.00±0.36) expression was significantly increased, and the expression of RANKL (2.0±0.84 VS 34.4±2.01) was significantly decreased from the 21thd adipocytes co-cultured group compared with the 40thd adipocytes co-cultured group (P<0.001). CONCLUSION: The differential size of adipocytes in the bone marrow can affect bone metabolism by regulating the expression of OPG/RANKL.

The Effect and Mechanism of TLR9/KLF4 in FFA-Induced Adipocyte Inflammation.

OBJECTIVE: Current research has reported that obesity is a chronic inflammatory state, which is closely related with excessive accumulation of free fatty acid, while the specific mechanism that high level of FFA causes inflammation is not very clear. Thus, our research intended to observe the high FFA effects on TLR9/KLF4 expression and the downstream inflammatory factors, to explore the mechanism of inflammatory response suppressed by TLR9/KLF4. METHODS: qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of TLR9, KLF4, and key inflammation-related factors. ELISA was used to detect the release level of inflammatory cytokines. The triglyceride (TG) and glucose (GLU) testing cassettes were used to detect the TG and GLU levels in culture medium. RESULTS: In the omental tissue of obese individuals (OB), we found that TLR9, KLF4, mRNA, and the protein expression levels were lower than those of the normal weight control (NC) group. Similarly, in the omental tissue of high-fat diet (HFD) rats, we found that the mRNA expression levels of TLR9 and KLF4 were lower than those of the normal diet control group. In mature adipocytes, we found that KLF4 played an important anti-inflammatory role; moreover, PA can promote the development of inflammation by inhibiting KLF4 expression; TLR9 has a positive regulation function on KLF4 expression, but unrelated to PA. CONCLUSIONS: TLR9/KLF4 is involved in regulating FFA-induced adipocyte inflammation.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Identification of four novel XPC mutations in two xeroderma pigmentosum complementation group C patients and functional study of XPC Q320X mutant.

Xeroderma pigmentosum (XP) is a rare, recessive hereditary disease characterized by sunlight hypersensitivity and high incidence of skin cancer with clinical and genetic heterogeneity. We collected two unrelated Chinese patients showing typical symptoms of XPC without neurologic symptoms. Direct sequencing of XPC gene revealed that patient 1 carried IVS1+1G>A and c.958 C>T mutations, and patient 2 carried c.545_546delTA and c.2257_2258insC mutations. All these four mutations introduced premature terminal codons (PTCs) in XPC gene. The nonsense mutation c.958 C>T yielded truncated mutant Q320X, and we studied its function for global genome repair kinetics. Overexpressed Q320X mutant can localize to site of DNA damage, but it is defective in CPD and 6-4PP repair. Readthrough of PTCs is a new approach to treatment of genetic diseases. We found that aminoglycosides could significantly increase the full length protein expression of Q320X mutant, but NER defects were not rescued in vitro.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats.

Estrogen affects the generation and transmission of neuropathic pain, but the specific regulatory mechanism is still unclear. Activation of the N-methyl-D-aspartate acid receptor 1 (NMDAR1) plays an important role in the production and maintenance of hyperalgesia and allodynia. The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain. A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats. These rats were then subcutaneously injected with 17ß-estradiol, the NMDAR1 antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP-5), or both once daily for 15 days. Compared with injured drug naïve rats, rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency, indicating increased sensitivity to mechanical and thermal pain. Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity (as assessed by immunohistochemistry) and protein (as determined by western blot assay) in spinal dorsal root ganglia. This 17ß-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5, whereas AP-5 alone did not affect NMDAR1 expression. These results suggest that 17ß-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve, and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

The effects of fat-induced obesity on bone metabolism in rats.

OBJECTIVE: The aim of the current study was to investigate the effects of obesity, induced via a high-fat diet, on bone metabolism in rats. METHODS: Two hundred healthy Wistar male rats aged 4 weeks were fed a standard diet and a high-fat diet. At specific time points (week 0, 4, 6, 8, and 10), plasma was collected to determine the levels of glucose and lipid metabolism. Additionally, enzyme-linked immunoassays were performed to determine the plasma levels of adipocyte and bone metabolism factors. Micro-CT imaging was used to determine the parameters of bone metabolism. At 10th week, immunohistochemistry evaluation of femoral bone samples was performed to determine the expression of adipocyte factors. RESULT: Receptor activator of nuclear factor kB ligand (RANKL) was positively correlated with levels of triglyceride (TG), free fatty acids (FFA), and tumor necrosis factor alpha (TNF-α) (P<0.05), while receptor activator of the NF-κB (RANK) showed a positive correlation with TG, FFA, TNF-α and leptin (LPT) (P<0.05). CT imaging demonstrated that bone mineral density and trabecular thickness were elevated compared to controls before 6 weeks, but these values were found to be lower in rats fed a high fat diet in the following weeks (P<0.05). Immunohistochemistry showed that the expression of TNF-α, Interleukin- 6 (IL-6) and peroxisome proliferator activated receptor-γ (PPAR-γ) were increased and the expression of adiponectin (APN) were diminished in rats fed a high-fat diet compared to controls at 10 weeks (P<0.05). CONCLUSION: With obesity intensifies, the release of FFA cause inflammation factor increase, resulting in bone parameters decreased.

Novel mutations in the lipase H gene lead to secretion defects of LIPH in Chinese patients with autosomal recessive woolly hair/hypotrichosis (ARWH/HT).

Autosomal recessive woolly hair/hypotrichosis (ARWH/HT: OMIM #278150/604379) is a rare hereditary hair disease characterized by tightly curled hair at birth which can lead to sparse hair later in life. The mutations in both LIPH and LPAR6/P2RY5 are responsible for autosomal recessive woolly hair with or without hypotrichosis (ARWH/HT). To conduct clinical and genetic investigations in four patients from three unrelated Chinese Han families with ARWH/HT, we performed mutation screening of LIPH and LPAR6/P2RY5 gene and identified four mutations in LIPH: c.454G>A, c.614A>G, c.736T>A, c.742C>A. c.736T>A and c.742C>A mutations were reported in previous studies, and c.454G>A, c.614A>G were identified for the first time. We carried out functional studies of the two mutants with c.454G>A (p.Gly152Arg, G152R) or c.614A>G (p.His205Arg, H205R). Interestingly, both of them lead to secretion defects of LIPH, which are involved in the pathogenesis of ARWH/HT.