TRIM65 promotes vascular smooth muscle cell phenotypic transformation by activating PI3K/Akt/mTOR signaling during atherogenesis.

BACKGROUND AND AIMS: Tripartite motif (TRIM65) is an important member of the TRIM protein family, which is a newly discovered E3 ligase that interacts with and ubiquitinates various substrates and is involved in diverse pathological processes. However, the function of TRIM65 in atherosclerosis remains unarticulated. In this study, we investigated the role of TRIM65 in the pathogenesis of atherosclerosis, specifically in vascular smooth muscle cells (VSMCs) phenotype transformation, which plays a crucial role in formation of atherosclerotic lesions. METHODS AND RESULTS: Both non-atherosclerotic and atherosclerotic lesions during autopsy were collected singly or pairwise from each individual (n = 16) to investigate the relationship between TRIM65 and the development of atherosclerosis. In vivo, Western diet-fed ApoE-/- mice overexpressing or lacking TRIM65 were used to assess the physiological function of TRIM65 on VSMCs phenotype, proliferation and atherosclerotic lesion formation. In vitro, VSMCs phenotypic transformation was induced by platelet-derived growth factor-BB (PDGF-BB). TRIM65-overexpressing or TRIM65-abrogated primary mouse aortic smooth muscle cells (MOASMCs) and human aortic smooth muscle cells (HASMCs) were used to investigate the mechanisms underlying the progression of VSMCs phenotypic transformation, proliferation and migration. Increased TRIM65 expression was detected in α-SMA-positive cells in the medial and atherosclerotic lesions of autopsy specimens. TRIM65 overexpression increased, whereas genetic knockdown of TRIM65 remarkably inhibited, atherosclerotic plaque development. Mechanistically, TRIM65 overexpression activated PI3K/Akt/mTOR signaling, resulting in the loss of the VSMCs contractile phenotype, including calponin, α-SMA, and SM22α, as well as cell proliferation and migration. However, opposite phenomena were observed when TRIM65 was deficient in vivo or in vitro. Moreover, in cultured PDGF-BB-induced TRIM65-overexpressing VSMCs, inhibition of PI3K by treatment with the inhibitor LY-294002 for 24 h markedly attenuated PI3K/Akt/mTOR activation, regained the VSMCs contractile phenotype, and blocked the progression of cell proliferation and migration. CONCLUSIONS: TRIM65 overexpression enhances atherosclerosis development by promoting phenotypic transformation of VSMCs from contractile to synthetic state through activation of the PI3K/Akt/mTOR signal pathway.

TRIM65 Suppresses oxLDL-induced Endothelial Inflammation by Interaction with VCAM-1 in Atherogenesis.

BACKGROUND AND OBJECTIVE: Endothelial cell activation, characterized by increased levels of vascular cell adhesion molecule 1 (VCAM-1), plays a crucial role in the development of atherosclerosis (AS). Therefore, inhibition of VCAM-1-mediated inflammatory response is of great significance in the prevention and treatment of AS. The tripartite motif (TRIM) protein-TRIM65 is involved in the regulation of cancer development, antivirals and inflammation. We aimed to study the functions of TRIM65 in regulating endothelial inflammation by interacting with VCAM-1 in atherogenesis. METHODS AND RESULTS: In vitro, we report that human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (oxLDL) significantly upregulate the expression of TRIM65 in a time- and dose-dependent manner. Overexpression of TRIM65 reduces oxLDL-triggered VCAM-1 protein expression, decreases monocyte adhesion to HUVECs and inhibits the production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α as well as endothelial oxLDL transcytosis. In contrast, siRNA-mediated knockdown of TRIM65 promotes the expression of VCAM-1, resulting in increased adhesion of monocytes and the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α and enhances endothelial oxLDL transcytosis. In vivo, we measured the high expression of TRIM65 in ApoE-/- mouse aortic plaques compared to C57BL/6J mouse aortic plaques. Then, we examined whether the blood levels of VCAM-1 were higher in TRIM65 knockout ApoE-/- mice than in control mice induced by a Western diet. Furthermore, Western blot results showed that the protein expression of VCAM-1 was markedly enhanced in TRIM65 knockout ApoE-/- mouse aortic tissues compared to that of the controls. Immunofluorescence staining revealed that the expression of VCAM-1 was significantly increased in atherosclerotic plaques of TRIM65-/-/ApoE-/- aortic vessels compared to ApoE-/- controls. Mechanistically, TRIM65 specifically interacts with VCAM-1 and targets it for K48-linked ubiquitination. CONCLUSION: Our studies indicate that TRIM65 attenuates the endothelial inflammatory response by targeting VCAM-1 for ubiquitination and provides a potential therapeutic target for the inhibition of endothelial inflammation in AS.

Metformin regulates expression of DNA methyltransferases through the miR-148/-152 family in non-small lung cancer cells.

BACKGROUND: To understand the molecular mechanisms involved in regulation of DNA methyltransferases (DNMTs) by metformin in non-small cell lung cancer (NSCLC) cells. METHODS: Expression levels of DNMTs in response to metformin were analyzed in NSCLC cells. MicroRNAs regulating expression of DNMTs at the post-transcriptional level were searched using miRNA-target databases (miRDB and miRTarBase), TCGA RNASeqV2 lung cancer data, and miRNA-seq. RESULTS: Metformin dose-dependently downregulated expression of DNMT1 and DNMT3a at the post-transcriptional level and expression of DNMT3b at the transcriptional level in A549 lung cancer cells. Activity of DNMTs was reduced by about 2.6-fold in A549 cells treated with 10 mM metformin for 72 h. miR-148/-152 family members (miR-148a, miR-148b, and miR-152) targeting the 3'UTR of DNMTs were associated with post-transcriptional regulation of DNMTs by metformin. Metformin upregulated expression of miR-148a, miR-148b, and miR-152 in A549 and H1650 cells. Transfection with an miR-148b plasmid or a mimic suppressed expression of DNMT1 and DNMT3b in A549 cells. Transfection with the miR-148a mimic in A549 and H1650 cells decreased the luciferase activity of DNMT1 3'UTR. A combination of metformin and cisplatin synergistically increased expression levels of miR-148/-152 family members but decreased expression of DNMTs in A549 cells. Low expression of miR-148b was associated with poor overall survival (HR = 2.56, 95% CI 1.09-6.47; P = 0.04) but not with recurrence-free survival. CONCLUSIONS: The present study suggests that metformin inhibits expression of DNMTs by upregulating miR-148/-152 family members in NSCLC cells.

Aberrant Methylation of SLIT2 Gene in Plasma Cell-Free DNA of Non-Small Cell Lung Cancer Patients.

This study aimed to understand aberrant methylation of SLITs genes as a biomarker for the early detection and prognosis prediction of non-small cell lung cancer (NSCLC). Methylation levels of SLITs were determined using the Infinium HumanMethylation450 BeadChip or pyrosequencing. Five CpGs at the CpG island of SLIT1, SLIT2 or SLIT3 genes were significantly (Bonferroni corrected p < 0.05) hypermethylated in tumor tissues obtained from 42 NSCLC patients than in matched normal tissues. Methylation levels of these CpGs did not differ significantly between bronchial washings obtained from 76 NSCLC patients and 60 cancer-free patients. However, methylation levels of SLIT2 gene were significantly higher in plasma cell-free DNA of 72 NSCLC patients than in that of 61 cancer-free patients (p = 0.001, Wilcoxon rank sum test). Prediction of NSCLC using SLIT2 methylation was achieved with a sensitivity of 73.7% and a specificity of 61.9% in a plasma test dataset (N = 40). A Cox proportional hazards model showed that SLIT2 hypermethylation in plasma cell-free DNA was significantly associated with poor recurrence-free survival (hazards ratio = 2.19, 95% confidence interval = 1.21-4.36, p = 0.01). The present study suggests that aberrant methylation of SLIT2 in plasma cell-free DNA is a valuable biomarker for the early detection of NSCLC and prediction of recurrence-free survival. However, further research is needed with larger sample size to confirm results.

A Versatile Nanoplatform for Broad-Spectrum Immunotherapy by Reversing the Tumor Microenvironment.

Immunotherapy is currently an important adjuvant therapy for malignant tumors besides surgical treatment. However, the heterogeneity and low immunogenicity of the tumor are two main challenges of the immunotherapy. Here, we have constructed a nanoplatform (CP@mRBC-PpIX) to realize reversion of the tumor acidosis and hypoxia through alkali and oxygen generation triggered by tumor acidosis. By targeting tumor universal features other than endogenous biomarkers, it was found that CP@mRBC-PpIX could polarize tumor-associated macrophages to anti-tumor M1 phenotype macrophages to enhance tumor immune response. Furthermore, under regional light irradiation, the reactive oxygen species produced by photosensitizers located in CP@mRBC-PpIX could increase the immunogenicity of tumors, so that tumor changes from an immunosuppressive "cold tumor" to an immunogenic "hot tumor," thereby increasing the infiltration and response of T cells, further amplifying the effect of immunotherapy. This strategy circumvented the problem of tumor heterogeneity to realize a kind of broad-spectrum immunotherapy, which could effectively prevent tumor metastasis and recurrence.

Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells.

This study aimed at understanding the effect of metformin on histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin significantly reduced H3K4me3 level at the promoters of positive cell cycle regulatory genes such as CCNB2, CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. Metformin suppressed the expression of H3K4 methyltransferases MLL1, MLL2, and WDR82. The siRNA-mediated knockdown of MLL2 significantly downregulated global H3K4me3 level and inhibited lung cancer cell proliferation. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, and a Cox proportional hazards analysis showed that recurrence-free survival of lung adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08-4.72; p = 0.02) times poorer than in those without it. Metformin showed little effect on DNA methylation and chromatin accessibility at the promoter regions of cell cycle regulatory genes. The present study suggests that metformin reduces H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. Additionally, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.

Negative Effect of Reduced NME1 Expression on Recurrence-Free Survival in Early Stage Non-Small Cell Lung Cancer.

This study aimed to understand whether the effect of non-metastatic cells 1 (NME1) on recurrence-free survival (RFS) in early stage non-small cell lung cancer (NSCLC) can be modified by ß-catenin overexpression and cisplatin-based adjuvant chemotherapy. Expression levels of NME1 and ß-catenin were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 425 early stage NSCLC patients. Reduced NME1 expression was found in 39% of samples. The median duration of follow-up was 56 months, and recurrence was found in 186 (44%) of 425 patients. The negative effect of reduced NME1 expression on RFS was worsened by cisplatin-based adjuvant chemotherapy (adjusted hazard ratio = 3.26, 95% CI = 1.16-9.17, p = 0.03). ß-catenin overexpression exacerbated the effect of reduced NME1 expression on RFS and the negative effect was greater when receiving cisplatin-based adjuvant chemotherapy: among patients treated with cisplatin-based adjuvant chemotherapy, hazard ratios of patients with reduced NME1 expression increased from 5.59 (95% confidence interval (CI) = 0.62-50.91, p = 0.13) to 15.52 (95% CI = 2.94-82.38, p = 0.001) by ß-catenin overexpression, after adjusting for confounding factors. In conclusion, the present study suggests that cisplatin-based adjuvant chemotherapy needs to be carefully applied to early stage NSCLC patients with overexpressed ß-catenin in combination with reduced NME1 expression.

Clinicopathological Significance of RUNX1 in Non-Small Cell Lung Cancer.

This study aimed to understand the clinicopathological significance of runt-related transcription factor 1 (RUNX1) in non-small cell lung cancer (NSCLC). The methylation and mRNA levels of RUNX1 in NSCLC were determined using the Infinium HumanMethylation450 BeadChip and the HumanHT-12 expression BeadChip. RUNX1 protein levels were analyzed using immunohistochemistry of formalin-fixed paraffin-embedded tissues from 409 NSCLC patients. Three CpGs (cg04228935, cg11498607, and cg05000748) in the CpG island of RUNX1 showed significantly different methylation levels (Bonferroni corrected p < 0.05) between tumor and matched normal tissues obtained from 42 NSCLC patients. Methylation levels of the CpGs in the tumor tissues were inversely related to mRNA levels of RUNX1. A logistic regression model based on cg04228935 showed the best performance in predicting NSCLCs in a test dataset (N = 28) with the area under the receiver operating characteristic (ROC) curve (AUC) of 0.96 (95% confidence interval (CI) = 0.81-0.99). The expression of RUNX1 was reduced in 125 (31%) of 409 patients. Adenocarcinoma patients with reduced RUNX1 expression showed 1.97-fold (95% confidence interval = 1.16-3.44, p = 0.01) higher hazard ratio for death than those without. In conclusion, the present study suggests that abnormal methylation of RUNX1 may be a valuable biomarker for detection of NSCLC regardless of race. And, reduced RUNX1 expression may be a prognostic indicator of poor overall survival in lung adenocarcinoma.

Curative effect of methotrexate combined with teniposide in the treatment of primary central nervous system lymphoma.

The present study aimed to investigate the curative effect of high-dose methotrexate (HD-MTX) combined with teniposide (Vm26) vs. HD-MTX alone in the treatment of primary central nervous system lymphoma (PCNSL), in order to provide data for assisting decisions associated with clinical treatment. Data from 56 patients with PCNSL admitted in Shanghai Huashan Hospital (Shanghai, China) from January 2009 to December 2014 were included into the present study. Clinical data, curative effects and prognosis of patients in these two groups were retrospectively analyzed using SPSS 20 statistical software. In the HD-MTX+Vm26 group, 12 patients (42.85%) achieved complete remission (CR) and 10 patients (35.71%) achieved partial remission (PR), while in the HD-MTX group 7 patients (25%) achieved CR and 11 patients (39.29%) achieved PR (P=0.158). The median progression-free survival (PFS) time was 22 months in the HD-MTX+Vm26 group and 12 months in the HD-MTX group (P=0.019). The median overall survival time was 57 months in the HD-MTX+Vm26 group, and 28 months in the HD-MTX group (P=0.013). Compared with HD-MTX alone, the combined treatment of HD-MTX+Vm26 had an improved curative effect in the treatment of PCNSL, effectively controlled tumor progression in patients, prolonged survival time and improved prognosis. Age was an independent prognostic factor in patients with PCNSL. Patients with an age of ≤60 years exhibited longer PFS compared with patients with an age of >60 years.

B-scan ultrasound and cytology of the vitreous in primary central nervous system lymphoma with vitreoretinal involvement.

AIM: To evaluate the diagnostic value of B-scan ultrasound and explore the cytological characteristics of patients with vitreoretinal lymphoma (VRL) and primary central nervous system lymphoma (PCNSL). METHODS: The clinical data and pathologic specimens from patients with VRL diagnosed at the North Huashan Hospital from 2016 to 2017 were retrospectively reviewed. The patients were diagnosed by slit lamp ophthalmoscopy, B-scan ultrasound, cytology of the vitreous, which was obtained by vitrectomy, and cytokine measurements of interleukin (IL)-10 and IL-6. RESULTS: Twenty-six eyes (19.4%) out of 134 eyes of 67 patients (47 men and 20 women) with PCNSL were diagnosed with VRL by B-scan ultrasound, and 14 eyes (10.4%) were diagnosed by slit lamp ophthalmoscopy. Twenty-four eyes (17.9%) of 17 patients were confirmed as having VRL with cytology. No difference in the association between intracranial lesion location and ocular involvement was found. VRL patients had higher levels of vitreous IL-10 and IL-10/IL-6 when compared with macular hole cases, but the difference was not statistically significant. CONCLUSION: A total of 25.4% of the PCNSL patients had VRL, B-scan ultrasound examination had characteristic features and is recommended over slit lamp ophthalmoscopy for the screening diagnosis of PCNSL with intraocular involvement. Moreover, the cytological and immunohistochemical analyses performed after 25-gauge diagnostic vitrectomy were accurate diagnostic techniques.

Efficient Production of Murine Uterine Damage Model.

BACKGROUND: Thin or damaged endometrium causes uterine factor-derived infertility resulting in a failure of embryonic implantation. Regeneration of endometrium is a major issue in gynecology and reproductive medicine. Various types of cells and scaffolds were studied to establish an effective therapeutic strategy. For this type of investigations, production of optimal animal models is indispensable. In this study, we tried to establish various murine uterine damage models and compared their features. METHODS: Three to ten-week-old C57BL/6 female mice were anesthetized using isoflurane. Chemical and mechanical methods using ethanol (EtOH) at 70 or 100% and copper scraper were compared to determine the most efficient condition. Damage of uterine tissue was induced either by vaginal or dorsal surgical approach. After 7-10 days, gross and microscopic morphology, safety and efficiency were compared among the groups. RESULTS: Both chemical and mechanical methods resulted in thinner endometrium and reduced number of glands. Gross morphology assessment revealed that the damaged regions of uteri showed various shapes including shrinkage or cystic dilatation of uterine horns. The duration of anesthesia significantly affected recovery after procedure. Uterine damage was most effectively induced by dorsal approach using 100% EtOH treatment compared to mechanical methods. CONCLUSION: Taken together, murine uterine damage models were most successfully established by chemical treatment. This production protocols could be applied further to larger animals such as non-human primate.

Bioinformatics Analysis and Identification of Genes and Molecular Pathways Involved in Synovial Inflammation in Rheumatoid Arthritis.

BACKGROUND Rheumatoid arthritis (RA) has a high prevalence in the elderly population. The genes and pathways in the inflamed synovium in patients with RA are poorly understood. This study aimed to identify differentially expressed genes (DEGs) linked to the progression of synovial inflammation in RA using bioinformatics analysis. MATERIAL AND METHODS Gene expression profiles of datasets GSE55235 and GSE55457 were acquired from the Gene Expression Omnibus (GEO) database. DEGs were identified using Morpheus software, and co-expressed DEGs were identified with Venn diagrams. Protein-protein interaction (PPI) networks were assembled with Cytoscape software and separated into subnetworks using the Molecular Complex Detection (MCODE) algorithm. The functions of the top module were assessed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. RESULTS DEGs that were upregulated were significantly enhanced in protein binding, the cell cytosol, organization of the extracellular matrix (ECM), regulation of RNA transcription, and cell adhesion. DEGs that were downregulated were associated with control of the immune response, B-cell and T-cell receptor signaling pathway regulation. KEGG pathway analysis showed that upregulated DEGs enhanced pathways associated with the cell adherens junction, osteoclast differentiation, and hereditary cardiomyopathies. Downregulated DEGs were enriched in primary immunodeficiency, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction, and hematopoietic cell lineages. CONCLUSIONS The findings from this bioinformatics network analysis study identified molecular mechanisms and the key hub genes that may contribute to synovial inflammation in patients with RA.

Treatment of Anterior Sternoclavicular Joint Dislocation with Acromioclavicular Joint Hook Plate.

OBJECTIVE: To evaluate the safety and efficacy of using acromioclavicular joint hook plates for the treatment of anterior sternoclavicular joint dislocation. METHODS: Ten patients who suffered anterior sternoclavicular joint dislocation were retrospectively analyzed, and underwent acromioclavicular joint hook plate surgeries from January 2015 to May 2017. There were 7 male and 3 female patients, with a mean age of 43.6 years. According to the American Shoulder and Elbow Society (ASES) scoring system, the preoperative physical function had a mean of 83.5. RESULTS: Reduction and fixation were performed with hook plates in all 10 patients. All patients were followed up, with a mean duration of 16.9 months. There were no complications, no wound infections, and no plate or screw breakages. Movement of the shoulder girdle was improved in all patients. According to the ASES scoring system, the postoperative physical function had a mean of 94.8. CONCLUSION: The acromioclavicular joint hook plate demonstrates safety and efficacy for the treatment of anterior sternoclavicular joint dislocation. However, there are still some deficiencies that need to be improved.

Metformin and tenovin-6 synergistically induces apoptosis through LKB1-independent SIRT1 down-regulation in non-small cell lung cancer cells.

Sirtuin 1 (SIRT1) is known to play a role in a variety of tumorigenesis processes by deacetylating histone and non-histone proteins; however, antitumour effects by suppressing SIRT1 activity in non-small cell lung cancer (NSCLC) remain unclear. This study was designed to scrutinize clinicopathological significance of SIRT1 in NSCLC and investigate effects of metformin on SIRT1 inhibition. This study also evaluated new possibilities of drug combination using a SIRT1 inhibitor, tenovin-6, in NSCLC cell lines. It was found that SIRT1 was overexpressed in 300 (62%) of 485 formalin-fixed paraffin-embedded NSCLC tissues. Its overexpression was significantly associated with reduced overall survival and poor recurrence-free survival after adjusted for histology and pathologic stage. Thus, suppression of SIRT1 expression may be a reasonable therapeutic strategy for NSCLC. Metformin in combination with tenovin-6 was found to be more effective in inhibiting cell growth than either agent alone in NSCLC cell lines with different liver kinase B1 (LKB1) status. In addition, metformin and tenovin-6 synergistically suppressed SIRT1 expression in NSCLC cells regardless of LKB1 status. The marked reduction in SIRT1 expression by combination of metformin and tenovin-6 increased acetylation of p53 at lysine 382 and enhanced p53 stability in LKB1-deficient A549 cells. The combination suppressed SIRT1 promoter activity more effectively than either agent alone by up-regulating hypermethylation in cancer 1 (HIC1) binding at SIRT1 promoter. Also, suppressed SIRT1 expression by the combination synergistically induced caspase-3-dependent apoptosis. The study concluded that metformin with tenovin-6 may enhance antitumour effects through LKB1-independent SIRT1 down-regulation in NSCLC cells.

Application of the Guiding Template Designed by Three-dimensional Printing Data for the Insertion of Sacroiliac Screws: a New Clinical Technique.

This study is aimed to explore the clinical application of the guiding template designed by three-dimensional printing data for the insertion of sacroiliac screws. A retrospective study of 7 cases (from July 2016 to December 2016), in which the guiding template printed by the threedimensional printing technique was used for the insertion of sacroiliac screws of patients with posterior ring injuries of pelvis, was performed. Totally, 4 males and 3 females were included in template group, aged from 38 to 65 years old (mean 50.86±8.90). Of them, 5 had sacral fractures (3 with Denis type I and 2 with type II) and 2 the separation of sacroiliac joint. Guiding templates were firstly made by the three-dimensional printing technique based on the pre-operative CT data. Surgical operations for the stabilization of pelvic ring by applying the guiding templates were carried out. A group of 8 patients with sacroiliac injuries treated by percutaneous sacroiliac screws were analyzed as a control group retrospectively. The time of each screw insertion, volume of intra-operative blood loss, and the exposure to X ray were analyzed and the Matta's radiological criteria were used to evaluate the reduction quality. The Majeed score was used to evaluate postoperative living quality. The visual analogue scale (VAS) was applied at different time points to judge pain relief of coccydynia. All the 7 patients in the template group were closely followed up radiographically and clinically for 14 to 20 months, mean (16.57±2.44) months. Totally 9 sacroiliac screws for the S1 and S2 vertebra were inserted in the 7 patients. The time length for each screw insertion ranged from 450 to 870 s, mean (690.56±135.68) s, and the number of times of exposure to X ray were 4 to 8, mean (5.78±1.20). The intra-operative blood loss ranged from 45 to 120 mL, mean (75±23.32) mL. According to Matta's radiology criteria, the fracture and dislocation reduction were excellent in 6 cases and good in 1. The pre-operative VAS score ranged from 5.2 to 8.1, mean (7.13±1.00). The average one-week/six-month post-operative VAS was (5.33±0.78) and (1.33±0.66), respectively (P<0.05 when compared with pre-operative VAS). The 12-month postoperative Majeed score ranged from 86 to 92, mean (90.29±2.21). The three-dimensional printed guiding template for sacroiliac screw insertion, which could significantly shorten the operation time, provide a satisfied outcome of the stabilization of the pelvic ring, and protect doctors and patients from X-ray exposure, might be a practical and valuable new clinical technique.

Genome-wide analysis of DNA methylation in bronchial washings.

Background: The objective of this study was to discover DNA methylation biomarkers for detecting non-small lung cancer (NSCLC) in bronchial washings and understanding the association between DNA methylation and smoking cessation. Methods: DNA methylation was analyzed in bronchial washing samples from 70 NSCLCs and 53 hospital-based controls using Illumina HumanMethylation450K BeadChip. Methylation levels in these bronchial washings were compared to those in 897 primary lung tissues of The Cancer Genome Atlas (TCGA) data. Results: Twenty-four CpGs (p < 1.03E-07) were significantly methylated in bronchial washings from 70 NSCLC patients compared to those from 53 controls. The CpGs also had significant methylation in the TCGA cohort. The 123 participants were divided into a training set (N = 82) and a test set (N = 41) to build a classification model. Logistic regression model showed the best performance for classification of lung cancer in bronchial washing samples: the sensitivity and specificity of a marker panel consisting of seven CpGs in TFAP2A, TBX15, PHF11, TOX2, PRR15, PDGFRA, and HOXA11 genes were 87.0 and 83.3% in the test set, respectively. The area under the curve (AUC) was equal to 0.87 (95% confidence interval = 0.73-0.96, p < 0.001). Methylation levels of two CpGs in RUNX3 and MIR196A1 genes were inversely associated with duration of smoking cessation in the controls, but not in NSCLCs, after adjusting for pack-years of smoking. Conclusions: The present study suggests that NSCLC may be detected by analyzing methylation changes of seven CpGs in bronchial washings. Furthermore, smoking cessation may lead to decreased DNA methylation in nonmalignant bronchial epithelial cells in a gene-specific manner.

Bronchial biopsy specimen as a surrogate for DNA methylation analysis in inoperable lung cancer.

Background: This study was aimed at understanding whether bronchial biopsy specimen can be used as a surrogate for DNA methylation analysis in surgically resected lung cancer. Methods: A genome-wide methylation was analyzed in 42 surgically resected tumor tissues, 136 bronchial washing, 12 sputum, and 8 bronchial biopsy specimens using the Infinium HumanMethylation450 BeadChip, and models for prediction of lung cancer were evaluated using TCGA lung cancer data. Results: Four thousand seven hundred and twenty-six CpGs (P < 1.0E-07) that were highly methylated in tumor tissues were identified from 42 lung cancer patients. Ten CpGs were selected for prediction of lung cancer. Genes including the 10 CpGs were classified into three categories: (i) transcription (HOXA9, SOX17, ZNF154, HOXD13); (ii) cell signaling (HBP1, SFRP1, VIPR2); and (iii) adhesion (PCDH17, ITGA5, CD34). Three logistic regression models based on the 10 CpGs classified 897 TCGA primary lung tissues with a sensitivity of 95.0~97.8% and a specificity of 97.4~98.7%. However, the classification performance of the models was very poor in bronchial washing samples: the area under the curve (AUC) was equal to 0.72~0.78. The methylation levels of the 10 CpGs in bronchial biopsy were not significantly different from those in surgically resected tumor tissues (P > 0.05, Wilcoxon rank-sum test). However, their methylation levels were significantly different between paired bronchial biopsy and washing (P < 0.05, Wilcoxon signed-rank test). Conclusions: The present study suggests that bronchial biopsy specimen may be used as a surrogate for DNA methylation analysis in patient with inoperable lung cancer.

Metformin induces cell cycle arrest at the G1 phase through E2F8 suppression in lung cancer cells.

A target molecule responsible for cell cycle arrest by metformin was discovered using a gene chip array in lung cancer cells and the effect of metformin on E2F8 was assessed. The siRNA-mediated knockdown of E2F8 significantly suppressed G1-S progression while ectopic expression of E2F8 relieved metformin-induced G1 arrest. The mRNA levels of p21 were found to be inversely related to those of E2F8 in lung cancer cells while siRNA-mediated knockdown of p21 partly rescued siE2F8-induced arrest of the cell cycle. Metformin had no effect on degradation of E2F8 mRNA. Activation and inhibition of AMPK by AICAR and Dorsomorphin, respectively, did not affect E2F8 suppression by metformin. The clinical significance of E2F8 was analyzed in The Cancer Genome Atlas (TCGA) data. One hundred six (13%) of 848 TCGA lung cancers overexpressed E2F8 mRNA. The overexpression of E2F8 was associated with poor overall survival (adjusted hazard ratio = 1.58, 95% confidence interval = 1.13-2.22; P = 0.008). The present study suggests that metformin may induce cell cycle arrest at the G1 phase by suppressing E2F8 expression in lung cancer cells. In addition, E2F8 may be associated with poor overall survival in lung cancer patients irrespective of histology.

Hemodynamic effects of intraoperative anesthetics administration in photothrombotic stroke model: a study using laser speckle imaging.

BACKGROUND: Previous neuroimaging studies have shown the hemodynamic effect of either preconditioning or postconditioning anesthesia in ischemic stroke model. However, the anesthetic effect in hemodynamics during and immediately after the stroke modeling surgery remains unknown due to the lack of appropriate anesthesia-free stroke model and intraoperative imaging technology. In the present study, we utilized our recently developed photothrombotic model of focal cerebral ischemia in conscious and freely moving rats, and investigated transient hemodynamic changes with or without isoflurane administration. Laser speckle imaging was applied to acquire real-time two-dimensional full-field cerebral blood flow (CBF) information throughout the surgical operations and early after. RESULTS: Significantly larger CBF reduction area was observed in conscious rats from 8 min immediately after the onset of stroke modeling, compared with anesthetized rats. Stroke rats without isoflurane administration also showed larger lesion volume identified by magnetic resonance imaging 3 h post occlusion (58.9%), higher neurological severity score 24 h post occlusion (28.3%), and larger infarct volume from 2,3,5-triphenyltetrazolium chloride staining 24 h post occlusion (46.9%). CONCLUSIONS: Our results demonstrated that the hemodynamic features were affected by anesthetics at as early as during the stroke induction. Also, our findings about the neuroprotection of intraoperative anesthetics administration bring additional insights into understanding the translational difficulty in stroke research.